CN116568822A - Methods and systems for improving the signal-to-noise ratio of DNA methylation partition assays - Google Patents

Abstract

In one aspect, the present disclosure provides a method for determining methylation status, the method comprising: providing a biological sample of nucleic acid molecules; partitioning at least a subset of the nucleic acid molecules in the biological sample into more than one partition group based on the methylation state of the nucleic acid molecules; digesting at least a subset of one or more of the more than one partition groups with at least one methylation-sensitive restriction enzyme; enriching at least a subset of the nucleic acid molecules in more than one partition group for a genomic region of interest, wherein at least a subset of the nucleic acid molecules comprises digested nucleic acid molecules in one or more of the partition groups; and determining the methylation state at one or more genetic loci of the nucleic acid molecules in at least one of the partition groups.

Description

Methods and systems for improving the signal-to-noise ratio of DNA methylation partitioning assays

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/086,000, filed on September 30, 2020, and U.S. Provisional Patent Application No. 63/105,183, filed on October 23, 2020, each of which is incorporated herein by reference in its entirety for all purposes.

Field of the Invention

The present disclosure provides compositions and methods related to analyzing nucleic acids such as DNA (such as cell-free DNA). In some embodiments, the cell-free DNA is from a subject suffering from or suspected of having cancer and/or the cell-free DNA includes DNA from cancer cells. In some embodiments, the DNA is partitioned into more than one partition group based on the methylation state of the nucleic acid molecule, and at least one subset of at least one partition group is digested with at least one methylation-sensitive restriction endonuclease.

background

Current cancer diagnostic methods for measuring cell-free nucleic acids (e.g., cell-free DNA or cell-free RNA) can focus on detecting tumor-associated somatic variations, including single nucleotide variations (SNVs), copy number variations (CNVs), fusions, and insertions/deletions (indels) (i.e., insertions or deletions), which are all mainstream targets for liquid biopsies. Increasing evidence suggests that non-sequence modifications, such as methylation status and fragmentomic signals in cell-free DNA, can provide information about the source of cell-free DNA and disease levels. Non-sequence modifications of cell-free DNA, when combined with somatic mutation calls, can produce a more comprehensive assessment of tumor status than either method alone.

However, due to the low concentration and heterogeneity of cell-free DNA, it has been challenging to develop accurate and sensitive methods for analyzing liquid biopsy materials that provide detailed information about nucleobase modifications. Separating and processing fractions of cell-free DNA that can be used for further analysis in liquid biopsy procedures is an important part of these methods. Therefore, there is a need for improved methods and compositions for analyzing cell-free DNA (e.g., in liquid biopsies).

Overview

The present disclosure is intended to meet the need for improved analysis of cell-free DNA and/or to provide other benefits. The present disclosure provides methods, compositions and systems for analyzing nucleic acids. Therefore, the following exemplary embodiments are provided. Embodiment 1 is a method for analyzing nucleic acid molecules in a biological sample, the method comprising:

a) partitioning at least a subset of nucleic acid molecules in a biological sample into more than one partitioning group based on the methylation status of the nucleic acid molecules, wherein the biological sample comprises methylated nucleic acid molecules and unmethylated nucleic acid molecules;

b) digesting at least a subset of one or more of the more than one partition groups with at least one methylation-sensitive restriction endonuclease; and

c) determining the methylation status at one or more genetic loci of the nucleic acid molecules in at least one of the partitioning groups.

Embodiment 2 is a method for determining the methylation state of a nucleic acid molecule, the method comprising:

a) providing a biological sample of nucleic acid molecules, wherein the nucleic acid molecules include methylated nucleic acid molecules and unmethylated nucleic acid molecules;

b) partitioning at least a subset of nucleic acid molecules in the biological sample into more than one partitioning group based on the methylation status of the nucleic acid molecules;

c) digesting at least a subset of one or more of the more than one partition groups with at least one methylation-sensitive restriction endonuclease;

d) enriching at least a subset of nucleic acid molecules in more than one partitioning group for a genomic region of interest, wherein at least a subset of nucleic acid molecules comprises digested nucleic acid molecules in one or more partitioning groups; and

e) determining the methylation status at one or more genetic loci of the nucleic acid molecules in at least one of the partitioning groups.

Embodiment 3 is a method for analyzing nucleic acid molecules in a biological sample, the method comprising:

a) partitioning at least a subset of nucleic acid molecules in a biological sample into more than one partitioning group based on the methylation status of the nucleic acid molecules, wherein the biological sample comprises methylated nucleic acid molecules and unmethylated nucleic acid molecules, and the more than one partitioning group comprises a first partitioning group and a second partitioning group, wherein methylated nucleic acid molecules are over-represented in the first partitioning group relative to the second partitioning group;

b) digesting at least a subset of a first partition of the more than one partitions with at least one methylation-sensitive restriction endonuclease; and

c) capturing a first set of target regions comprising the epigenetic target region from at least a portion of the first partitioning set, and capturing a second set of target regions comprising the epigenetic target region from at least a portion of the second partitioning set.

Embodiment 4 is a method according to embodiment 3, wherein capturing the first target region set comprises contacting the DNA in the first partition set with a first target-specific probe set, and capturing the second target region set comprises contacting the DNA in the second partition set with a second target-specific probe set.

Embodiment 5 is a method according to embodiment 3 or 4, further comprising determining the methylation status at one or more genetic loci of the nucleic acid molecules in at least one of the partitioning group or the target group.

Embodiment 6 is a method according to any one of the above embodiments, wherein the genomic region of interest, the first target region group and/or the second target region group comprises sequence variable target regions.

Embodiment 7 is a method according to any one of the above embodiments, further comprising attaching one or more adaptors to at least one end of at least a portion of the nucleic acid molecules in more than one partitioning group before the digestion step.

Embodiment 8 is a method for determining the methylation state of a nucleic acid molecule, the method comprising:

a) providing a biological sample of nucleic acid molecules, wherein the nucleic acid molecules include methylated nucleic acid molecules and unmethylated nucleic acid molecules;

b) partitioning at least a subset of nucleic acid molecules in the biological sample into more than one partitioning group based on the methylation status of the nucleic acid molecules;

c) attaching one or more adaptors to at least one end of the nucleic acid molecules in more than one partitioning group;

d) digesting at least a subset of one or more of the more than one partition groups with at least one methylation-sensitive restriction endonuclease;

e) enriching at least a subset of nucleic acid molecules in more than one partition grouping for a genomic region of interest; wherein at least a subset of nucleic acid molecules comprises digested nucleic acid molecules in one or more partition groups; and

f) determining the methylation status at one or more genetic loci of the nucleic acid molecules in at least one of the partitioning groups.

Embodiment 9 is a method according to embodiment 7 or 8, wherein adaptors are attached to both ends of at least a portion of the nucleic acid molecules in more than one partitioning group.

Embodiment 10 is a method according to embodiment 1, which further comprises, before c), enriching at least one subset of nucleic acid molecules in more than one partition group for the genomic region of interest, wherein at least one subset of nucleic acid molecules comprises digested nucleic acid molecules in one or more partition groups.

Embodiment 11 is a method according to any of the preceding embodiments, further comprising detecting the presence or absence of cancer in the biological sample.

Embodiment 12 is a method according to any of the above embodiments, further comprising determining the level of cancer in the biological sample.

Embodiment 13 is a method according to any of the above embodiments, wherein determining the methylation status comprises sequencing at least a subset of the digested nucleic acid molecules.

Embodiment 14 is a method according to any one of embodiments 7-13, wherein one or more adaptors comprise at least one tag.

Embodiment 15 is a method according to any one of the above embodiments, wherein the methylation-sensitive restriction endonuclease selectively digests nucleic acid molecules that are unmethylated at the recognition site of the methylation-sensitive restriction endonuclease.

Embodiment 16 is a method according to any one of the above embodiments, wherein at least a portion of the nucleic acid molecules are amplified and/or sequenced after the digestion step, and the nucleic acid molecules digested by the methylation-sensitive restriction endonuclease are not amplified and/or sequenced.

Embodiment 17 is a method according to any one of the above embodiments, comprising digesting at least a subset of one or more of the more than one partition groups with at least two methylation-sensitive restriction endonucleases.

Embodiment 18 is a method according to embodiment 17, wherein the at least two methylation-sensitive restriction endonucleases consist of two methylation-sensitive restriction endonucleases.

Embodiment 19 is a method according to embodiment 17 or 18, wherein the methylation-sensitive restriction endonuclease comprises or consists of BstUI and HpaII.

Embodiment 20 is a method according to embodiment 17 or 18, wherein the methylation-sensitive restriction endonuclease comprises or consists of HhaI and AccII.

Embodiment 21 is a method according to embodiment 17 or 18, wherein the at least two methylation-sensitive restriction endonucleases include or consist of three methylation-sensitive restriction endonucleases.

Embodiment 22 is a method according to embodiment 17 or 21, wherein the methylation-sensitive restriction endonuclease comprises or consists of BstUI, HpaII and Hin6I.

Embodiment 23 is a method according to any one of the above embodiments, wherein the methylation-sensitive restriction endonuclease is selected from the group consisting of: AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI, Cfr10I, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, MspI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI and SnaBI.

Embodiment 24 is a method according to any one of embodiments 7-23, wherein one or more adaptors are resistant to digestion by a methylation-sensitive restriction endonuclease.

Embodiment 25 is a method according to embodiment 24, wherein one or more tolerant adaptors comprise one or more methylated nucleotides, optionally wherein the methylated nucleotides comprise 5-methylcytosine and/or 5-hydroxymethylcytosine.

Embodiment 26 is a method according to embodiment 24, wherein one or more tolerant adaptors comprise one or more nucleotide analogs that are resistant to a methylation-sensitive restriction endonuclease.

Embodiment 27 is a method according to embodiment 24, wherein one or more tolerant adaptors comprise a nucleotide sequence that is not recognized by a methylation-sensitive restriction endonuclease.

Embodiment 28 is a method according to any one of embodiments 14-27, wherein the tag comprises a molecular barcode.

Embodiment 29 is a method according to embodiment 28, wherein the molecular barcode attached to the nucleic acid molecules in a first one of the more than one partitions is different from the molecular barcode attached to the nucleic acid molecules in a second one of the more than one partitions.

Embodiment 30 is a method according to embodiments 1-29, wherein a first partition group among the more than one partition groups and a second partition group among the more than one partition groups are differentially labeled.

Embodiment 31 is a method according to embodiment 30, wherein a first partition tag is attached to the nucleic acid molecules in the first partition grouping, and a second partition tag is attached to the nucleic acid molecules in the second partition grouping.

Embodiment 32 is a method according to any one of the above embodiments, wherein the methylated nucleic acid molecule comprises 5-methylcytosine and/or 5-hydroxymethylcytosine.

Embodiment 33 is a method according to any one of embodiments 13-32, wherein sequencing is performed by a next generation sequencer.

Embodiment 34 is a method according to any one of the preceding embodiments, wherein the biological sample is selected from the group consisting of a DNA sample, an RNA sample, a polynucleotide sample, a cell-free DNA sample, and a cell-free RNA sample.

Embodiment 35 is a method according to any one of the preceding embodiments, wherein the biological sample is a cell-free DNA sample.

Embodiment 36 is a method according to embodiment 35, wherein the cell-free DNA is between 1 ng and 500 ng.

Embodiment 37 is a method according to any one of the preceding embodiments, wherein partitioning comprises partitioning the nucleic acid molecules based on their different binding affinities to a binding agent that preferentially binds to nucleic acid molecules comprising methylated nucleotides.

Embodiment 38 is a method according to embodiment 37, wherein the binding agent is a methyl binding domain (MBD) protein.

Embodiment 39 is a method according to embodiment 37, wherein the binding agent is an antibody specific for one or more methylated nucleotide bases.

Embodiment 40 is a method according to any one of embodiments 2-39, wherein the genomic region or epigenetic target region of interest comprises a differentially methylated region for cancer detection.

Embodiment 41 is a method according to any one of embodiments 13-40, further comprising amplifying at least a portion of the nucleic acid molecule prior to sequencing.

Embodiment 42 is a method according to embodiment 41, wherein the primers used in amplification comprise at least one sample index.

Embodiment 43 is a method according to any of the above embodiments, wherein the one or more genetic loci include more than one genetic locus.

Embodiment 44 is a method according to embodiment 43, wherein the more than one genetic loci comprise one or more genomic regions.

In any of the foregoing embodiments, epigenetic target regions can be captured from one or more or each partition group. Any of the methods can also include quantifying the captured epigenetic target regions, for example by sequencing or quantitative PCR. In some embodiments, the method includes capturing a first target region group comprising an epigenetic target region from at least a portion of the first partition group, and capturing a second target region group comprising an epigenetic target region from at least a portion of the second partition group. The first and second target region groups can be the same or different.

The epigenetic target regions may include a set of hypermethylated variable target regions, for example, including regions having a higher degree of methylation in at least one type of tissue than in cell-free DNA from healthy subjects. Any of the methods may also include determining the presence, absence, or likelihood of cancer based at least in part on the sequence or amount of the regions in the set of hypermethylated variable target regions. Any of the methods may also include quantifying tumor DNA in a sample based at least in part on the sequence or amount of the regions in the set of hypermethylated variable target regions.

The epigenetic target regions may include a set of hypomethylated variable target regions, for example, including regions having a lower degree of methylation in at least one type of tissue than in cell-free DNA from healthy subjects. Any of the methods may also include determining the presence, absence, or likelihood of cancer based at least in part on the sequence or amount of the regions in the set of hypomethylated variable target regions. Any of the methods may also include quantifying tumor DNA in the sample based at least in part on the sequence or amount of the regions in the set of hypomethylated variable target regions.

In any of the foregoing embodiments, sequence variable target regions can be captured from one or more or each partition group. Any of the methods can also include quantifying the captured epigenetic target regions, for example, by sequencing or quantitative PCR. The DNA molecules corresponding to the sequence variable target region group can be sequenced to a deeper sequencing depth than the DNA molecules corresponding to the epigenetic target region group.

In any of the foregoing embodiments, capturing the target region group may include contacting the DNA to be captured with the target-specific probe group, thereby forming a complex of the target-specific probe and the DNA. Capturing may also include separating the complex from the DNA not bound to the target-specific probe, thereby providing captured DNA.

In any of the foregoing embodiments, the DNA may be amplified prior to the sequencing step, or the DNA may be amplified prior to the capture step.

In any of the foregoing embodiments, the DNA may include DNA obtained from a body fluid, optionally wherein the body fluid is plasma, urine, lymph, or spinal fluid. For example, the DNA may include cell-free DNA (cfDNA) obtained from a test subject.

In any one of the aforementioned embodiments, the methylation-sensitive restriction endonuclease can cleave unmethylated CpG sequences. In any one of the aforementioned embodiments, the methylation-sensitive restriction endonuclease can be one or more of the following: AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI, Cfr10I, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, and SnaBI.

In any of the foregoing embodiments, the method may further include determining the likelihood that the subject has cancer. For example, wherein sequencing may generate more than one sequencing read; and the method may further include mapping the more than one sequence read to one or more reference sequences to generate mapped sequence reads, and processing the mapped sequence reads corresponding to the sequence variable target region group and the epigenetic target region group to determine the likelihood that the subject has cancer.

In any of the foregoing embodiments, the test subject may have been previously diagnosed with cancer and received one or more previous cancer treatments, optionally wherein cfDNA is obtained at one or more preselected time points after one or more previous cancer treatments, and the capture group of cfDNA molecules is sequenced, thereby generating a sequence information group. Such a method may also include using the sequence information group to detect the presence or absence of DNA derived from or derived from tumor cells at a preselected time point. Such a method may also include determining a cancer recurrence score, the cancer recurrence score indicating the presence or absence of DNA derived from or derived from tumor cells of the test subject, the method optionally also includes determining a cancer recurrence status based on the cancer recurrence score, wherein when the cancer recurrence score is determined to be at or above a predetermined threshold, the cancer recurrence status of the test subject is determined to be at risk of cancer recurrence, or when the cancer recurrence score is below a predetermined threshold, the cancer recurrence status of the test subject is determined to be at a lower risk of cancer recurrence. Such a method may further include comparing the test subject's cancer recurrence score to a predetermined cancer recurrence threshold, wherein when the cancer recurrence score is above the cancer recurrence threshold, classifying the test subject as a candidate for subsequent cancer treatment, or when the cancer recurrence score is below the cancer recurrence threshold, classifying the test subject as a non-candidate for subsequent cancer treatment.

In another aspect, the present disclosure provides a system comprising a controller comprising or having access to a computer-readable medium comprising non-transitory computer-executable instructions, which, when executed by at least one electronic processor, perform a method comprising: (a) partitioning at least one subset of nucleic acid molecules in a biological sample into more than one partition group based on the methylation state of the nucleic acid molecules, wherein the biological sample comprises methylated nucleic acid molecules and unmethylated nucleic acid molecules; (b) digesting at least one subset of one or more of the more than one partition groups with at least one methylation-sensitive restriction endonuclease; and (c) determining the methylation state of one or more genetic loci of the nucleic acid molecules in at least one of the partition groups. In some embodiments, the method further comprises, prior to (c), enriching at least one subset of the nucleic acid molecules in more than one partition group for a genomic region of interest, wherein at least one subset of the nucleic acid molecules comprises digested nucleic acid molecules in one or more partition groups. In some embodiments, the method further comprises, prior to (b), attaching one or more adapters to at least one end of the nucleic acid molecules in more than one partition group. In some embodiments, the method further comprises, prior to determining the methylation status, enriching at least a portion of the nucleic acid molecules in more than one partitioning group; wherein the at least a portion of the nucleic acid molecules comprises digested nucleic acid molecules in one or more partitioning groups.

In another aspect, the present disclosure provides a system comprising a controller comprising or having access to a computer-readable medium comprising non-transitory computer-executable instructions, which, when executed by at least one electronic processor, perform a method comprising: a) providing a biological sample of nucleic acid molecules, wherein the nucleic acid molecules include methylated nucleic acid molecules and unmethylated nucleic acid molecules; (b) partitioning at least one subset of nucleic acid molecules in the biological sample into more than one partition group based on the methylation state of the nucleic acid molecules; (c) digesting at least one subset of one or more of the partition groups with at least one methylation-sensitive restriction endonuclease; (d) enriching at least one subset of nucleic acid molecules in more than one partition group for a genomic region of interest, wherein at least one subset of nucleic acid molecules includes digested nucleic acid molecules in one or more partition groups; and (e) determining the methylation state of one or more genetic loci of nucleic acid molecules in at least one of the partition groups. In some embodiments, the method further comprises, prior to (b), attaching one or more adapters to at least one end of the nucleic acid molecules in more than one partition group.

In another aspect, the present disclosure provides a system comprising a controller comprising or having access to a computer-readable medium comprising non-transitory computer-executable instructions which, when executed by at least one electronic processor, perform a method comprising: (a) providing a biological sample of nucleic acid molecules, wherein the nucleic acid molecules include methylated nucleic acid molecules and unmethylated nucleic acid molecules; (b) partitioning at least a subset of the nucleic acid molecules in the biological sample into more than one partition group based on the methylation status of the nucleic acid molecules; (c) attaching one or more adapters to at least one end of the nucleic acid molecules in more than one partition group; (d) digesting at least one subset of one or more of the more than one partition groups with at least one methylation-sensitive restriction endonuclease; (e) enriching at least one subset of the nucleic acid molecules in more than one partition group for a genomic region of interest; wherein at least one subset of the nucleic acid molecules includes digested nucleic acid molecules in one or more partition groups; and (f) determining the methylation status at one or more genetic loci of the nucleic acid molecules in at least one of the partition groups.

In another aspect, the present disclosure provides a method for determining the methylation state of a nucleic acid molecule, the method comprising: (a) providing a biological sample of nucleic acid molecules, wherein the nucleic acid molecules include methylated nucleic acid molecules and unmethylated nucleic acid molecules; (b) partitioning at least one subset of the nucleic acid molecules in the biological sample into more than one partition group based on the methylation state of the nucleic acid molecules; (c) attaching one or more adapters to at least one end of the nucleic acid molecules in more than one partition group; (d) digesting at least one subset of one or more of the more than one partition groups with at least one methylation-sensitive restriction endonuclease; (e) enriching at least one subset of the nucleic acid molecules in more than one partition group for a genomic region of interest; wherein at least one subset of the nucleic acid molecules comprises digested nucleic acid molecules in one or more partition groups; and (f) determining the methylation state at one or more genetic loci of the nucleic acid molecules in at least one of the partition groups.

On the other hand, the present disclosure provides a method for determining the methylation state of a nucleic acid molecule, the method comprising: (a) providing a biological sample of nucleic acid molecules, wherein the nucleic acid molecules include methylated nucleic acid molecules and unmethylated nucleic acid molecules; (b) partitioning at least one subset of nucleic acid molecules in the biological sample into more than one partition group based on the methylation state of the nucleic acid molecules; (c) digesting at least one subset of one or more of the partition groups with at least one methylation-sensitive restriction endonuclease; (d) enriching at least one subset of nucleic acid molecules in more than one partition group for a genomic region of interest, wherein at least one subset of nucleic acid molecules comprises digested nucleic acid molecules in one or more partition groups; and (e) determining the methylation state of one or more genetic loci of nucleic acid molecules in at least one of the partition groups. In some embodiments, the method further comprises, prior to (b), attaching one or more adapters to at least one end of the nucleic acid molecules in more than one partition group.

In some embodiments, the method also includes detecting the presence or absence of cancer in the biological sample. In some embodiments, the method also includes, for example, determining the level of cancer in the biological sample by determining the level of DNA from cancer cells in the biological sample. In some embodiments, determining the methylation state includes sequencing at least one subset of the digested nucleic acid molecules. In some embodiments, sequencing is performed by a next-generation sequencer. In some embodiments, one or more adapters include at least one tag. In some embodiments, the adapter tolerates digestion of a methylation-sensitive restriction endonuclease. In some embodiments, the adapter includes one or more methylated nucleotides (e.g., nucleotides comprising methylated bases). In some embodiments, the adapter includes one or more nucleotide analogs (e.g., nucleotide analogs with linkage modifications (such as thiophosphates)) that tolerate methylation-sensitive restriction endonucleases. In some embodiments, the adapter includes a nucleotide sequence that is not recognized by a methylation-sensitive restriction endonuclease. In some embodiments, the adapter does not include any sequence recognized by a methylation-sensitive restriction endonuclease used in the method. In some embodiments, the tag includes a molecular barcode. In some embodiments, the molecular barcode attached to the nucleic acid molecules in the first partition is different from the molecular barcode attached to the nucleic acid molecules in the second partition. In some embodiments, the first partition is differentially labeled from the second partition. In some embodiments, a first partition tag is attached to the nucleic acid molecules in the first partition, and a second partition tag is attached to the nucleic acid molecules in the second partition.

In some embodiments, the method includes digesting at least one subset of one or more partition groups in more than one partition group with at least two methylation sensitivity restriction endonucleases (MSRE). As used herein, the reference to two (or more) MSREs means using two (or more) different MSREs with different properties (e.g., different recognition sequences). In some embodiments, at least two methylation sensitivity restriction endonucleases are composed of two methylation sensitivity restriction endonucleases. In some embodiments, two methylation sensitivity restriction endonucleases include BstUI and HpaII. In some embodiments, two methylation sensitivity restriction endonucleases include HhaI and AccII. In some embodiments, at least two methylation sensitivity restriction endonucleases include three methylation sensitivity restriction endonucleases. In some embodiments, three methylation sensitivity restriction endonucleases include BstUI, HpaII and Hin6I. In some embodiments, the methylation-sensitive restriction endonuclease is selected from the group consisting of: AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI, Cfr10I, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, MspI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI and SnaBI. In some embodiments, at least one MSRE selectively digests unmethylated nucleic acid molecules. In some embodiments, at least one MSRE selectively digests methylated nucleic acid molecules.

In some embodiments, the methylated nucleotides include 5-methylcytosine and/or 5-hydroxymethylcytosine. In some embodiments, the biological sample is selected from the group consisting of: a DNA sample, an RNA sample, a polynucleotide sample, a cell-free DNA sample, and a cell-free RNA sample. In some embodiments, the biological sample is a cell-free DNA sample. In some embodiments, the cell-free DNA is between 1 ng and 500 ng.

In some embodiments, partitioning includes partitioning nucleic acid molecules based on different binding affinities of binding agents that preferentially bind to nucleic acid molecules comprising methylated nucleotides (e.g., nucleotides comprising methylated bases). In some embodiments, the binding agent is a methyl binding domain (MBD) protein. In some embodiments, the binding agent is an antibody specific to one or more methylated nucleotide bases. In some embodiments, the genomic region of interest includes a differentially methylated region for cancer detection.

In some embodiments, method also comprises before order-checking at least a portion of nucleic acid molecules is amplified (for example, after digestion step, or after enrichment or capture step).In some embodiments, the primer used in amplification comprises at least a sample index.In some embodiments, nucleic acid molecules digested by MSRE are not amplified.In some such embodiments, except nucleic acid molecules digested by MSRE, substantially all nucleic acid molecules are amplified in the sample.

In some embodiments, the one or more genetic loci include more than one genetic loci. In some embodiments, the more than one genetic loci include one or more genomic regions.

In some embodiments, the method comprises digesting at least one subset of one or more partition groups in more than one partition group with at least two methylation-sensitive restriction endonucleases. In some embodiments, the at least two methylation-sensitive restriction endonucleases consist of two methylation-sensitive restriction endonucleases. In some embodiments, the two methylation-sensitive restriction endonucleases comprise BstUI and HpaII. In some embodiments, the two methylation-sensitive restriction endonucleases comprise HhaI and AccII. In some embodiments, at least two methylation-sensitive restriction endonucleases comprise three methylation-sensitive restriction endonucleases. In some embodiments, three methylation-sensitive restriction endonucleases comprise BstUI, HpaII and Hin6I. In some embodiments, the methylation-sensitive restriction endonuclease is selected from the group consisting of: AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI, Cfr10I, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, MspI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI and SnaBI. In some embodiments, at least one MSRE selectively digests unmethylated nucleic acid molecules. In some embodiments, at least one MSRE selectively digests methylated nucleic acid molecules.

In some embodiments of various aspects of the present invention, the results of the system and/or method disclosed herein are used as input to generate a report. The report can be in paper or electronic format. For example, information about the presence or absence of cancer as determined by the method or system disclosed herein can be shown in such a report. Alternatively or additionally, the report can include information related to the epigenetic rate of epigenetic features, such as whether they are higher or lower than the adjusted epigenetic rate threshold. The method or system disclosed herein can also include the step of transmitting the report to a third party, such as a subject or a health care practitioner in the sample source.

The steps of the methods disclosed herein, or the steps performed by the systems disclosed herein, can be performed at the same time or at different times and/or at the same geographic location or at different geographic locations such as countries. The steps of the methods disclosed herein can be performed by the same person or by different people.

Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure can have other and different embodiments, and its several details can be modified in various obvious aspects, all without departing from the present disclosure. Accordingly, the drawings and descriptions should be considered to be illustrative and non-restrictive in nature.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate certain embodiments and are used together with the written description to explain certain principles of the methods, computer-readable media, and systems disclosed herein. The description provided herein is better understood when read in conjunction with the accompanying drawings, which are included by way of example and not by way of limitation. It should be understood that, unless the context otherwise indicates, the same reference numerals represent the same parts in all the drawings. It should also be understood that some or all of the drawings may be schematic diagrams for illustrative purposes and do not necessarily depict the actual relative sizes or positions of the elements shown.

Fig. 1 is when restriction endonuclease (RE) recognition site comprises unmethylated nucleotide, the schematic diagram (upper figure) of methylation sensitivity restriction endonuclease (MSRE) digestion/cracking DNA；And when restriction endonuclease (RE) recognition site comprises methylated nucleotide, the schematic diagram (lower figure) of methylation sensitivity restriction endonuclease (MSRE) non-cracking DNA.Therefore, Fig. 1 shows a type of MSRE, which selectively digests the recognition site comprising unmethylated nucleotide, and does not digest the recognition site comprising methylated nucleotide usually.

2 is a flow chart representation of a method for determining the methylation status of nucleic acid molecules in a polynucleotide sample obtained from a subject, according to an embodiment of the present disclosure.

3 is a flow chart representation of a method for detecting the presence or absence of cancer in a subject according to an embodiment of the present disclosure.

4 is a schematic diagram of a method for detecting the presence or absence of cancer in a subject according to certain embodiments of the present disclosure.

5 is a schematic diagram of an example of a system suitable for use with some embodiments of the present disclosure.

FIG. 6 shows the molecular counts in three partitions with and without MSRE treatment in normal samples and diluted CRC samples.

Figure 7 shows the CpG methylation quantification results obtained from three samples of a subject with early colorectal cancer ("early CRC") and three healthy subjects ("normal") as described in Example 3. For the early CRC graph, MAF indicates the mutant allele fraction.

Figures 8A-8D show counts of positive and negative control molecules with FspEI palindromic sites under the indicated enzyme and buffer conditions, as described in Example 4. Figures 8A and 8C correspond to the first donor, and Figures 8B and 8D correspond to the second donor. For readability, the data points are distributed along the horizontal axis.

9A-9D show digestion efficiency and positive control molecule counts, as described in Example 4.

Figures 10A-10J show the low methylated variable target region ("Low VTR") molecule counts (10A-E) or the Low VTR/negative control molecule ratios (10F-J) under the indicated conditions, as described in Example 5. For readability, the data points are distributed along the horizontal axis. The triangle, circle, plus sign, and square indicate that the source of the normal cfDNA is the first, second, third, or fourth of four healthy donors, respectively.

definition

In order to more easily understand the present disclosure, some terms are first defined below. The following terms and other definitions of other terms can be set forth through this specification. If the definition of the term set forth below is inconsistent with the definition in the application or patent incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods and/or steps of the type described herein and/or which will become apparent upon reading this disclosure and so forth.

It should also be understood that the terms used herein are intended to be restrictive only for the purpose of describing specific embodiments and are not intended to be limiting. In addition, unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present disclosure belongs. In describing and claiming methods, computer-readable media, and systems, the following terms and their grammatical variations will be used according to the definitions set forth below.

About: As used herein, "about" or "approximately" applied to one or more values or elements of interest refers to a value or element similar to the reference value or element. In certain embodiments, the term "about" or "approximately" refers to a series of values or elements that fall within (greater than or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value or element in either direction, unless otherwise specified or obvious from the context (unless such a number exceeds 100% of the possible value or element).

Adaptor: As used herein, "adaptor" refers to a short nucleic acid (e.g., less than about 500 nucleotides, less than about 100 nucleotides, or less than about 50 nucleotides in length) that is typically at least partially double-stranded and is attached to either end or both ends of a given sample nucleic acid molecule (i.e., two adaptors are attached to both ends of a nucleic acid-one adaptor at one end of a nucleic acid). An adaptor may include a nucleic acid primer binding site and/or a sequencing primer binding site, which allows amplification of nucleic acid molecules flanking the adaptor at both ends, and a sequencing primer binding site includes a primer binding site for sequencing applications such as various next generation sequencing (NGS) applications. An adaptor may also include a binding site for a capture probe such as an oligonucleotide attached to a flow cell support. An adaptor may also include a nucleic acid tag as described herein. Nucleic acid tags are typically positioned relative to amplification primer binding sites and sequencing primer binding sites so that the nucleic acid tag is included in the amplicon and sequence reads of a specific nucleic acid molecule. Adaptors of the same or different sequences may be connected to the corresponding ends of a nucleic acid molecule. In some embodiments, adapters of the same sequence except that the nucleic acid tags are different are connected to the corresponding ends of the nucleic acid molecules. In some embodiments, the adapter is a Y-shaped adapter, one of which is flat-ended or tailed as described herein, for connecting nucleic acid molecules that are also flat-ended or tailed with one or more complementary nucleotides, and the other end of the Y-shaped adapter comprises a non-complementary sequence that does not hybridize to form a double strand. In yet other exemplary embodiments, the adapter is a bell-shaped adapter, which comprises a flat end or tailed end for connecting to the nucleic acid molecule to be analyzed. Other examples of adapters include adapters with T tails and C tails.

Amplification: As used herein, "amplify" or "amplification" in the context of nucleic acids refers to the production of multiple copies of a polynucleotide or a portion of a polynucleotide, typically starting from a small amount of the polynucleotide (e.g., a single polynucleotide molecule), wherein the amplification product or amplicon is typically detectable. Amplification of polynucleotides includes a variety of chemical and enzymatic processes. Amplification includes, but is not limited to, the polymerase chain reaction (PCR).

Barcode: As used herein, "barcode" in the context of nucleic acids refers to a nucleic acid molecule comprising a sequence that can be used as an identifier. For example, a barcode can be used as an identifier of a molecule (i.e., a molecular barcode), an identifier of a sample (i.e., a sample barcode), or an identifier of a partition (i.e., a partition barcode). During next-generation sequencing (NGS) library preparation, individual "barcode" sequences are typically added to each DNA fragment, enabling identification and sorting of each read prior to final data analysis.

Cancer type: As used herein, "cancer type" refers to a type or subtype of cancer defined, for example, by histopathology. Cancer type can be defined by any conventional criteria, such as based on occurrence in a particular tissue (e.g., blood cancer, central nervous system (CNS) cancer, brain cancer, lung cancer (small cell and non-small cell), skin cancer, nose cancer, laryngeal cancer, liver cancer, bone cancer, lymphoma, pancreatic cancer, intestinal cancer, rectal cancer, thyroid cancer, bladder cancer, kidney cancer, oral cancer, stomach cancer, breast cancer, prostate cancer, ovarian cancer, lung cancer, small intestine cancer, soft tissue cancer, neuroendocrine cancer, gastroesophageal cancer, head and neck cancer, gynecological cancer, colorectal cancer, urothelial cancer, solid state cancer, heterogeneous cancer, homogeneous cancer Cancer)), primary origin unknown, etc., and/or may have the same cell lineage (e.g., carcinoma, sarcoma, lymphoma, cholangiocarcinoma, leukemia, mesothelioma, melanoma, or glioblastoma) and/or may be a cancer that exhibits cancer markers (such as, but not limited to Her2, CA15-3, CA19-9, CA-125, CEA, AFP, PSA, HCG, hormone receptors, and NMP-22). Cancer may also be classified according to stage (e.g., stage 1, stage 2, stage 3, or stage 4) and whether it is primary or secondary.

Capture set: As used herein, a "capture set" of nucleic acids refers to nucleic acids that have undergone capture.

Capture: As used herein, "capturing" or "enriching" one or more target nucleic acids refers to preferentially isolating or separating one or more target nucleic acids from non-target nucleic acids.

Cell-free nucleic acid: As used herein, "cell-free nucleic acid" refers to nucleic acids that are not contained in cells or are not originally associated with cells, or in some embodiments, refers to nucleic acids that remain in a sample after the complete cells are removed. Cell-free nucleic acids may include, for example, all unencapsulated nucleic acids derived from body fluids from a subject (e.g., blood, plasma, serum, urine, cerebrospinal fluid (CSF), etc.). Cell-free nucleic acids include DNA (cfDNA), RNA (cfRNA), and their hybrids, including genomic DNA, mitochondrial DNA, circulating DNA, siRNA, miRNA, circulating RNA (cRNA), tRNA, rRNA, small nucleolar RNA (snoRNA), Piwi interacting RNA (piRNA), long non-coding RNA (long ncRNA), and/or fragments of any of these. Cell-free nucleic acids may be double-stranded, single-stranded, or hybrids thereof. Cell-free nucleic acids may be released into body fluids by secretion or cell death processes (e.g., cell necrosis, apoptosis, etc.). Some cell-free nucleic acids are released into body fluids from cancer cells, for example, circulating tumor DNA (ctDNA). Others are released from healthy cells. ctDNA can be fragmented DNA of tumor origin that is not encapsulated. Cell-free nucleic acid can have one or more epigenetic modifications, for example, cell-free nucleic acid can be acetylated, 5-methylated and/or hydroxymethylated.

Cellular nucleic acid: As used herein, "cellular nucleic acid" refers to nucleic acid that is within one or more cells that produce the nucleic acid at least at the time the sample is obtained or collected from the subject, even if the nucleic acid is subsequently removed (e.g., by cell lysis) as part of a particular analytical procedure.

Corresponding to a target region group: As used herein, "corresponding to a target region group" means that a nucleic acid, such as cfDNA, is derived from a locus in the target region group or specifically binds to one or more probes for the target region group.

Coverage: As used herein, the terms "coverage", "total molecule counts" or "total allele counts" are used interchangeably. They refer to the total number of DNA molecules at a specific genomic location in a specific sample.

Deoxyribonucleic acid or ribonucleic acid: As used herein, "deoxyribonucleic acid" or "DNA" refers to a natural or modified nucleotide having a hydrogen group at the 2'-position of the sugar portion. DNA generally includes a nucleotide chain containing the following four types of nucleotide bases: adenine (A), thymine (T), cytosine (C) and guanine (G). As used herein, "ribonucleic acid" or "RNA" refers to a natural or modified nucleotide having a hydroxyl group at the 2'-position of the sugar portion. RNA generally includes a nucleotide chain containing the following four types of nucleotide bases: A, uracil (U), G and C. As used herein, the term "nucleotide" refers to a natural nucleotide or a modified nucleotide. Certain nucleotide pairs specifically bind to each other in a complementary manner (referred to as complementary base pairing). In DNA, adenine (A) pairs with thymine (T) and cytosine (C) pairs with guanine (G). In RNA, adenine (A) pairs with uracil (U) and cytosine (C) pairs with guanine (G). When the first nucleic acid chain is combined with a second nucleic acid chain consisting of nucleotides complementary to those in the first chain, the two chains combine to form a double strand. As used herein, "sequencing data", "nucleic acid sequencing information", "sequence information", "nucleic acid sequence", "nucleotide sequence", "genomic sequence", "sequence reads" or "sequencing reads" refer to any information or data indicating the order and identity of nucleotide bases (e.g., adenine, guanine, cytosine, and thymine or uracil) in a molecule (e.g., a whole genome, a whole transcriptome, an exome, an oligonucleotide, a polynucleotide, or a fragment) of a nucleic acid such as DNA or RNA. It should be understood that the present teachings contemplate sequence information obtained using all available various techniques, platforms or technologies, including but not limited to: capillary electrophoresis, microarrays, ligation-based systems, polymerase-based systems, hybridization-based systems, direct or indirect nucleotide identification systems, pyrophosphate sequencing, ion-based or pH-based detection systems, and electronic signal-based systems.

Digestion efficiency: As used herein, "digestion efficiency" or "cutting efficiency" refers to the efficiency of restriction endonuclease digestion. Digestion efficiency can be calculated based on the number of control molecules observed when digesting with restriction endonucleases and the number of control molecules observed in the absence of restriction endonucleases. MSRE digestion efficiency can be calculated by the following: efficiency = 1-(number of negative control molecules _[MSRE] / number of negative control molecules _[simulation] ). MDRE (MSRE that preferentially cleaves methylated DNA, also known as methylation-dependent restriction endonucleases) digestion efficiency can be calculated by the following: efficiency = 1-(number of positive control molecules _[MDRE] / number of positive control molecules _[simulation] ).

DNA sequence: As used herein, "DNA sequence" or "sequence" refers to "raw sequence reads" and/or "consensus sequences". Raw sequence reads are the output of a DNA sequencer and typically include redundant sequences of the same parent molecule, such as after amplification. A "consensus sequence" is a sequence derived from a redundant sequence of a parent molecule intended to represent the sequence of the original parent molecule. The consensus sequence includes base identities at a single position. In some embodiments, a consensus sequence may represent a single nucleotide base at a specific genomic position. In some embodiments, a consensus sequence may represent a string of nucleotide bases at more than one genomic position. The consensus sequence can be generated by voting (wherein each major nucleotide, for example, the nucleotide most frequently observed at a specific base position in the sequence is a consensus nucleotide) or other methods (such as comparison with a reference genome). The consensus sequence can be generated by tagging the original parent molecule with a unique or non-unique molecular tag, which allows tracing back subsequences (e.g., after amplification) by tracing back tags and/or using sequence read internal information. Examples of labeling or barcoding, and the use of labels or barcodes, are provided in, for example, U.S. Patent Publication Nos. 2015/0368708, 2015/0299812, 2016/0040229, and 2016/0046986, each of which is incorporated herein by reference in its entirety.

Enriched sample: As used herein, "enriched sample" refers to a sample that has been enriched for a particular region of interest. This can be accomplished by amplifying the region of interest or by using single-stranded DNA/RNA probes or double-stranded DNA probes that can hybridize to nucleic acid molecules of interest (e.g., In some embodiments, the enriched sample refers to a subset or portion of the processed sample that is enriched, wherein the subset or portion of the processed sample that is enriched comprises nucleic acid molecules from the cell-free polynucleotide or polynucleotide sample.

Epigenetic characterization: As used herein, "epigenetic characterization" refers to any directly observable measure of a DNA molecule that can be used to analyze the epigenetic characteristics of a DNA molecule. For example, if the epigenetic characteristic is methylation, the epigenetic characterization of the DNA molecule may refer to, but is not limited to, the partitioning of the DNA molecule, the number of CpG residues in the DNA molecule, and the position (or offset) of the DNA molecule. For example, if the epigenetic characteristic is a fragment group signal, the epigenetic characterization may be, but is not limited to, the length of the cfDNA molecule, the position (or offset) of the cfDNA molecule-the starting and/or ending position of the cfDNA molecule.

Epigenetic signature: As used herein, "epigenetic signature" refers to any parameter that can manifest non-sequence modifications of nucleic acids and also includes chromatin modifications. These modifications do not change the sequence of the DNA. Epigenetic signatures can include, but are not limited to, methylation status; fragment group signal; location/distribution of nucleosomes, CTCF protein, transcription start site, regulatory proteins, and any other protein that can bind to DNA.

Epigenetic target set: As used herein, "epigenetic target set" refers to a set of target regions that can exhibit non-sequence modifications in neoplastic cells (e.g., tumor cells and cancer cells) and non-tumor cells (e.g., immune cells, cells from the tumor microenvironment). These modifications do not change the sequence of the DNA. Examples of non-sequence modification changes include, but are not limited to, changes in methylation (increase or decrease), nucleosome distribution, CTCF binding, transcription start site, regulatory protein binding region, and any other protein that may bind to DNA. For the purposes of the present invention, loci that are prone to focused amplification and/or gene fusion associated with neoplasms, tumors or cancers can also be included in the epigenetic target set, because detection of copy number changes by sequencing or detection of fusion sequences mapped to more than one locus in a reference genome tends to be more similar to detection of the exemplary epigenetic changes discussed above than detection of nucleotide substitutions, insertions or deletions, for example, in the following aspects: focused amplification and/or gene fusions can be detected with relatively shallow sequencing depth because their detection does not rely on the accuracy of base calls at one or a few individual positions. For example, the epigenetic target group may include a target group for analyzing the fragment length or fragment end position distribution. In some embodiments, the epigenetic target group includes one or more genomic regions, wherein the epigenetic state (e.g., methylation state) of the cfDNA molecules in these regions is unchanged in cancer, but their presence/amount in the blood indicates the abnormal presentation of the increase of cfDNA from certain tissues (e.g., cancer source) to the circulation. The terms "epigenetics" and "epigenomics" are used interchangeably herein.

Fragment group signal: As used herein, "fragment group signal" refers to the distribution of cfDNA fragment sizes and cfDNA fragment positions at specific genomic regions. Fragment group signals may include, but are not limited to, cfDNA fragment lengths, starting and/or ending positions of cfDNA molecules (size coverage of fragments). Fragment group signals may also include the frequency at which DNA molecule endpoints appear at genomic locations (regions of interest at or around specific locations). Fragment group signals may also include nucleosome positioning of DNA molecules. In some embodiments, the fragment group signal includes endpoint information of the DNA molecule, but does not necessarily include length parameters of the DNA molecule).

Genomic region: As used herein, "genomic region" refers to any region (e.g., base pair position range) of a genome, such as a chromosome, a chromosome arm, a gene, or an exon. A genomic region can be a continuous or discontinuous region. A "genetic locus" (or "locus") can be part or all of a genomic region (e.g., a gene, a portion of a gene, or a single nucleotide of a gene). In some embodiments, the size of a genomic region includes a length of up to a chromosome/chromosome arm or a topologically associated domain (TAD). In some embodiments, the size of a genomic region may be limited to the biological activity of the region (e.g., a transcription unit or a regulatory unit).

Hypermethylation: As used herein, "hypermethylation" refers to an increased level or degree of methylation of a nucleic acid molecule relative to other nucleic acid molecules within a nucleic acid molecule population (e.g., a sample). In some embodiments, hypermethylation refers to an increased level or degree of methylation of a nucleic acid molecule from a specific genomic region in a tumor sample relative to the degree of methylation of a nucleic acid molecule from the same genomic region in a non-tumor sample. In some embodiments, hypermethylated DNA may include a DNA molecule comprising at least 1 methylated residue, at least 2 methylated residues, at least 3 methylated residues, at least 5 methylated residues, at least 10 methylated residues, at least 20 methylated residues, at least 25 methylated residues, or at least 30 methylated residues.

Hypomethylation: As used herein, "hypomethylation" refers to a reduced methylation level or degree of a nucleic acid molecule relative to other nucleic acid molecules within a nucleic acid molecule population (e.g., a sample). In some embodiments, hypomethylated DNA includes unmethylated DNA molecules. In some embodiments, hypomethylation refers to a reduced methylation level or degree of a nucleic acid molecule from the same genomic region in a tumor sample relative to the degree of methylation of a nucleic acid molecule from a specific genomic region in a non-tumor sample. In some embodiments, hypomethylated DNA can include a DNA molecule comprising 0 methylated residues, at most 1 methylated residue, at most 2 methylated residues, at most 3 methylated residues, at most 4 methylated residues, or at most 5 methylated residues.

Methylation: As used herein, "methylation" or "DNA methylation" may refer to the presence of a methyl group on a cytosine at a CpG site (cytosine-phosphate-guanine site, i.e., a guanine following a cytosine in the 5'->3' direction of a nucleic acid sequence). In some embodiments, DNA methylation comprises adding a methyl group to an adenine, such as in N ⁶ -methyladenine. In some embodiments, DNA methylation is 5-methylation (modification of the 5th carbon of the 6-carbon ring of cytosine). In some embodiments, 5-methylation comprises adding a methyl group to the 5C position of cytosine to produce 5-methylcytosine (m5c). In some embodiments, methylation comprises derivatives of m5c. Derivatives of m5c include, but are not limited to, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC). In some embodiments, DNA methylation is 3C methylation (modification of the 3rd carbon of the 6-carbon ring of cytosine). In some embodiments, 3C methylation includes adding a methyl group to the 3C position of cytosine to produce 3-methylcytosine (3mC). Methylation can also occur at non-CpG sites, for example, methylation can occur at CpA, CpT or CpC sites. DNA methylation can change the activity of methylated DNA regions. For example, when the DNA in the promoter region is methylated, the transcription of the gene may be suppressed. DNA methylation is crucial for normal development, and the abnormality of methylation can destroy epigenetic regulation. The destruction of epigenetic regulation, such as suppression, can cause diseases, such as cancer. Promoter methylation in DNA can indicate cancer.

Methylation-sensitive restriction endonuclease (MSRE): As used herein, "methylation-sensitive restriction endonuclease" or "MSRE" refers to a restriction endonuclease that is sensitive to the methylation state of DNA (e.g., cytosine methylation), i.e., the presence or absence of a methyl group in a nucleotide base changes the rate at which the enzyme cleaves the target DNA. In some embodiments, if a specific nucleotide base at the recognition sequence is methylated, the methylation-sensitive restriction endonuclease does not cleave DNA. For example, HpaII is a methylation-sensitive restriction endonuclease with a recognition sequence of "CCGG", and if the second cytosine in the recognition sequence is methylated, HpaII does not cleave DNA. In some embodiments, if a specific nucleotide base at the recognition sequence is methylated, the methylation-sensitive restriction endonuclease cleaves DNA. For example, Sgel is a methylation-sensitive restriction endonuclease with a recognition sequence of " ^5mCNNG (N) ₉ ", and if the cytosine in the recognition sequence is methylated ( ^5mC ), Sgel cleaves DNA. As another example, FspEI is a methylation-sensitive restriction endonuclease with a recognition sequence of "C ^5m C(N) ₁₂ ", and if the cytosine in the recognition sequence is methylated ( ^5m C), FspEI cleaves DNA. Figure 1 is a schematic diagram (upper figure) of methylation-sensitive restriction endonuclease (MSRE) digestion/cleavage of DNA when the restriction endonuclease (RE) recognition site contains unmethylated nucleotides; and a schematic diagram (lower figure) of methylation-sensitive restriction endonuclease (MSRE) not cleaving DNA when the restriction endonuclease (RE) recognition site (dashed box) contains methylated nucleotides at a position that affects the activity of MSRE. In some embodiments, the enzymatic activity of MSRE to methylated recognition sites is at least 10 times, 20 times, 50 times, or 100 times higher relative to the unmethylated form of the same recognition site. In some embodiments, the enzymatic activity of MSRE to unmethylated recognition sites is at least 10 times, 20 times, 50 times, or 100 times higher relative to the methylated form of the same recognition site.

Methylation state: As used herein, "methylation state" can refer to the presence or absence of a methyl group on a DNA base (e.g., cytosine) at a particular genomic position in a nucleic acid molecule. It can also refer to the degree of methylation in a nucleic acid sequence (e.g., a highly methylated, hypomethylated, moderately methylated, or unmethylated nucleic acid molecule). Methylation state can also refer to the number of methylated nucleotides in a particular nucleic acid molecule.

Mutation: As used herein, "mutation" refers to a variation from a known reference sequence, and includes mutations such as, for example, single nucleotide variations (SNVs) and insertions/deletions. Mutations can be germline mutations or somatic mutations. In some embodiments, the reference sequence for comparison purposes is the wild-type genomic sequence of the species of the subject providing the test sample, typically the human genome.

Neoplasm: As used herein, the terms "neoplasm" and "tumor" are used interchangeably. They refer to an abnormal growth of cells of a subject. A neoplasm or tumor may be benign, potentially malignant, or malignant. A malignant tumor refers to a cancer or cancerous tumor.

Next Generation Sequencing: As used herein, "Next Generation Sequencing" or "NGS" refers to sequencing technologies with increased throughput compared to traditional Sanger and capillary electrophoresis-based methods, e.g., the ability to generate hundreds of thousands of relatively small sequence reads at a time. Some examples of next generation sequencing technologies include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization. In some embodiments, next generation sequencing includes the use of an instrument capable of sequencing a single molecule. Examples of commercially available instruments for performing next generation sequencing include, but are not limited to, NextSeq, HiSeq, NovaSeq, MiSeq, IonPGM, and Ion GeneStudio S5.

Nucleic acid tag: As used herein, "nucleic acid tag" refers to a short nucleic acid (e.g., less than about 500 nucleotides, about 100 nucleotides, about 50 nucleotides, or about 10 nucleotides in length) used to distinguish nucleic acids from different samples (e.g., presented as a sample index), nucleic acids from different partitions (e.g., presented as a partition tag), or different types of nucleic acid molecules or nucleic acid molecules that have undergone different treatments in the same sample (e.g., presented as a molecular barcode). Nucleic acid tags contain predetermined, fixed, non-random, random, or semi-random oligonucleotide sequences. Such nucleic acid tags can be used to label different nucleic acid molecules or different nucleic acid samples or subsamples. Nucleic acid tags can be single-stranded, double-stranded, or at least partially double-stranded. Nucleic acid tags optionally have the same length or different lengths. Nucleic acid tags can also include double-stranded molecules with one or more blunt ends, including 5' or 3' single-stranded regions (e.g., overhangs), and/or include one or more other single-stranded regions at other positions within a particular molecule. Nucleic acid tags can be attached to one or both ends of other nucleic acids (e.g., sample nucleic acids to be amplified and/or sequenced). Nucleic acid tags can be decoded to reveal information such as sample sources, forms or processing of specific nucleic acids. For example, nucleic acid tags can also be used to make it possible to collect and/or process in parallel a plurality of samples containing nucleic acids with different molecular barcodes and/or sample indexes, wherein nucleic acids are subsequently deconvoluted by detecting (e.g., reading) nucleic acid tags. Nucleic acid tags can also be referred to as identifiers (e.g., molecular identifiers, sample identifiers). Additionally or alternatively, nucleic acid tags can be used as molecular identifiers (e.g., for distinguishing between different molecules or amplicons of different parental molecules in the same sample or subsample). This includes, for example, uniquely labeling different nucleic acid molecules in a particular sample, or non-uniquely labeling such molecules. In the case of non-unique tagging applications, each nucleic acid molecule can be tagged with a limited number of tags (i.e., molecular barcodes) so that different molecules can be distinguished based on a combination of their endogenous sequence information (e.g., the start and/or end position mapped to a selected reference genome, a subsequence at one or both ends of the sequence, and/or the length of the sequence) and at least one molecular barcode. Typically, a sufficient number of different molecular barcodes are used so that any two molecules may have the same endogenous sequence information (e.g., start and/or end position, a subsequence at one or both ends of the sequence, and/or length) and also have a low probability of having the same molecular barcode (e.g., a probability of less than about 10%, less than about 5%, less than about 1%, or less than about 0.1%).

Partition: As used herein, "partition" refers to physical separation or classification of a mixture of nucleic acid molecules in a sample based on the characteristics of nucleic acid molecules. Partitions can be physical partitions of molecules. Partitions can include dividing nucleic acid molecules into groups or groups based on epigenetic features (e.g., methylation) levels. For example, nucleic acid molecules can be partitioned based on the methylation levels of nucleic acid molecules. In some embodiments, methods and systems for partitioning can be found in PCT patent application No. PCT/US2017/068329, which is incorporated herein by reference in its entirety. After partitioning, groups or groups of separated or graded nucleic acid molecules are also referred to herein as fractions, partitions, or partition groups.

Partition group: As used herein, "partition group" or "partition" refers to a group of nucleic acid molecules that are partitioned into a set or group based on the different binding affinities of nucleic acid molecules or proteins associated with nucleic acid molecules and binding agents. The binding agent preferentially binds to nucleic acid molecules containing nucleotides with epigenetic modifications. For example, if the epigenetic modification is methylation, the binding agent can be a methyl binding domain (MBD) protein. In some embodiments, the partition group can include nucleic acid molecules belonging to epigenetic features (e.g., methylation) of a specific level or degree. For example, nucleic acid molecules can be partitioned into three groups-one group is a highly methylated nucleic acid molecule (high partition group or high methylation partition group), a second group is a low methylated nucleic acid molecule (low partition group or low methylation partition group), and a third group is a medium methylated nucleic acid molecule (medium partition group or medium methylation partition group). In another example, nucleic acid molecules can be partitioned based on the number of methylated nucleotides-one partition group can have a nucleic acid molecule with 9 methylated nucleotides, and another partition group can have an unmethylated nucleic acid molecule (zero methylated nucleotide).

Polynucleotide: As used herein, "polynucleotide", "nucleic acid", "nucleic acid molecule" or "oligonucleotide" refers to a linear polymer of nucleosides (including deoxyribonucleosides, ribonucleosides or their analogs) linked by internucleoside bonds. Typically, a polynucleotide contains at least three nucleosides. The size of an oligonucleotide typically ranges from a few monomer units, such as 3-4, to hundreds of monomer units. When a polynucleotide is represented by a letter sequence, such as "ATGCCTG", the nucleotides are presented from left to right in a 5'→3' order, and in the case of DNA, "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, and "T" represents deoxythymidine, unless otherwise indicated. The letters A, C, G and T can be used to refer to a base itself, a nucleoside, or a nucleotide comprising a base.

Processing: As used herein, "processing" refers to a set of steps used to generate a nucleic acid library suitable for sequencing. This set of steps may include, but is not limited to, partitioning of nucleic acids, end repair, addition of sequencing adapters, tagging, and/or PCR amplification.

Quantitative measure: As used herein, "quantitative measure" refers to an absolute measure or a relative measure. A quantitative measure can be, but is not limited to, a number, a statistical measure (e.g., frequency, mean, median, standard deviation, or quantile) or a degree or relative amount (e.g., high, medium, and low). A quantitative measure can be the ratio of two quantitative measures. A quantitative measure can be a linear combination of quantitative measures. A quantitative measure can be a normalized measure.

Reference sequence: As used herein, a "reference sequence" refers to a known sequence for the purpose of comparison with an experimentally determined sequence. For example, the known sequence can be an entire genome, a chromosome, or any segment thereof. The reference sequence can be aligned to a single continuous sequence of a genome or chromosome or chromosome arm, or can include non-contiguous segments aligned to different regions of a genome or chromosome. Examples of reference sequences include, for example, human genomes, such as hg19 and hg38.

Restriction endonuclease: As used herein, "restriction endonuclease" is an enzyme that recognizes and cleaves DNA at or near a specific recognition site.

Sample: As used herein, "sample" means anything capable of being analyzed by the methods and/or systems disclosed herein.

Sequencing: As used herein, "sequencing" refers to any of a number of techniques for determining the sequence (e.g., identity and order of monomeric units) of a biomolecule, e.g., a nucleic acid such as DNA or RNA. Examples of sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole genome sequencing, hybridization sequencing, pyrophosphate sequencing, duplex sequencing, cycle sequencing, single base extension sequencing, solid phase sequencing, high throughput sequencing, massively parallel signature sequencing, emulsion PCR, low denaturation temperature co-amplification PCR (COLD-PCR), multiplex PCR, reversible dye terminator sequencing, paired end sequencing, near-term sequencing, exonuclease sequencing, ligation sequencing, short read sequencing, single molecule sequencing, sequencing by synthesis, real-time sequencing, reverse terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing, and combinations thereof. In some embodiments, sequencing can be performed by a commercially available genetic analyzer such as, for example, available from Illumina, Inc., Pacific Biosciences, Inc., or Applied Biosystems/Thermo Fisher Scientific, among many others.

Sequence information: "Sequence information" as used herein in the context of a nucleic acid polymer means the order and identity of the monomeric units (eg, nucleotides, etc.) in the polymer.

Sequence variable target region group: As used herein, "sequence variable target region group" refers to a group of target regions that can exhibit sequence changes (such as nucleotide substitutions, insertions, deletions, or gene fusions or transpositions) in neoplastic cells (e.g., tumor cells and cancer cells). In some embodiments, the nucleotide substitution is a single nucleotide variation.

Somatic mutation: As used herein, the terms "somatic mutation" or "somatic variation" are used interchangeably. They refer to mutations in the genome that occur after conception. Somatic mutations can occur in any cell of the body except germ cells and are therefore not passed on to offspring.

Specific binding: As used herein, "specific binding" in the context of a probe or other oligonucleotide and a target sequence means that under appropriate hybridization conditions, the oligonucleotide or probe hybridizes to its target sequence or a copy thereof to form a stable probe: target hybrid, while minimizing the formation of stable probe: non-target hybrids. Thus, the probe hybridizes to a target sequence or a copy thereof to a greater extent than to a non-target sequence to achieve capture or detection of the target sequence. Appropriate hybridization conditions are well known in the art and can be predicted based on sequence composition or can be determined by use of routine testing procedures (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at §§1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, in particular §§9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57, incorporated herein by reference).

Subject: As used herein, "subject" refers to an animal, such as a mammalian species (e.g., human), or an avian (e.g., bird) species, or other organisms, such as plants. More specifically, the subject can be a vertebrate, for example, a mammal, such as a mouse, a primate, an ape, or a human. Animals include farm animals (e.g., production cattle, dairy cows, poultry, horses, pigs, etc.), sports animals, and companion animals (e.g., pets or support animals). The subject can be a healthy individual, an individual suffering from or suspected of having a disease or having a tendency to suffer from the disease, or an individual who needs treatment or is suspected of needing treatment. The term "individual" or "patient" is intended to be interchangeable with "subject". For example, the subject can be an individual who has been diagnosed with cancer, is about to receive cancer treatment, and/or has received at least one cancer treatment. The subject can be in cancer remission. As another example, the subject can be an individual diagnosed with an autoimmune disease. As another example, the subject can be a female individual who is pregnant or planning pregnancy, who may have been diagnosed with or suspected of having a disease, such as cancer, autoimmune disease.

Target region set: As used herein, "target-region set" or "set of target regions" or "target region" or "target region of interest" or "genomic region of interest" refers to more than one genomic locus or more than one genomic region that is targeted for capture and/or targeted by a probe set (e.g., by sequence complementarity).

Tumor fraction: As used herein, "tumor fraction" refers to the proportion of cfDNA molecules that are derived from tumor cells for a given sample or sample-region pair.

As used herein, the terms "or combinations thereof" and "or combinations thereof" refer to any and all permutations and combinations of the terms listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in the particular context, also includes BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. Those skilled in the art will understand that there is generally no limit to the number of items or terms in any combination unless otherwise apparent from the context.

"Or" is used in an inclusive sense, ie, equivalent to "and/or", unless the context requires otherwise.

Detailed Description

Certain embodiments of the present invention are described herein. Although the present invention will be described in conjunction with such embodiments, it should be understood that they are not intended that the present invention is limited to these embodiments. On the contrary, it is intended that the present invention covers all substitutions, modifications and equivalents, which may be included in the present invention limited by the appended claims.

Numerical ranges include the numbers that define the range. Measurements and measurable values are understood to be approximate, taking into account significant figures and errors associated with the measurements. In addition, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It should be understood that both the foregoing general description and the detailed description are exemplary and illustrative only, and are not intended to limit the present teachings.

Unless specifically mentioned in the above description, the embodiments described in the specification that "include/comprise" various components/components are also contemplated as "consisting of the described components/components" or "consisting mainly of the described components/components"; the embodiments described in the specification that "consisting of various components/components" are also contemplated as "consisting of/including" or "consisting mainly of the described components/components"; and the embodiments described in the specification that "consisting mainly of various components/components" are also contemplated as "consisting of the described components/components" or "include/comprise" the described components/components (this interchangeability does not apply to the use of these terms in the claims).

The section headings used herein are for organizational purposes and should not be construed as limiting the disclosed subject matter in any way. In the event that any document or other material incorporated by reference contradicts any explicit content of this specification, including definitions, this specification controls.

I. Overview

The formation and progression of cancer can be caused by both genetic modifications and epigenetic features of deoxyribonucleic acid (DNA). The present disclosure provides methods and systems for analyzing DNA, such as cell-free DNA (cfDNA). The present disclosure provides methods and systems for improving the signal-to-noise ratio of methylation partitioning assays.

Without wishing to be bound by any particular theory, cells in or around cancer or neoplasms may shed more DNA than cells of the same tissue type in healthy subjects. Therefore, the distribution of the source tissue of certain DNA samples (such as cfDNA) may change when cancerous. Therefore, for example, an increase in the level of high methylation variable target regions in healthy cfDNA that shows low methylation than at least one other tissue type can be used as an indicator of cancer presence (or recurrence, depending on the subject's medical history). Similarly, an increase in the level of low methylation variable target regions in a sample can be an indicator of cancer presence (or recurrence, depending on the subject's medical history).

In addition, cancer can be indicated by non-sequence modification (such as methylation).The example of methylation change in cancer includes the local increase of DNA methylation of CpG island at TSS of genes involved in normal growth control, DNA repair, cell cycle regulation and/or cell differentiation.This hypermethylation may be associated with the abnormal loss of transcriptional ability of related genes, and occurs at least as frequently as point mutations and deletions that cause gene expression changes.DNA methylation spectrum analysis (profiling) can be used to detect regions with different degrees of methylation in the genome ("differential methylation regions" or "DMR"), such as during development or by abnormal methylation caused by disease (e.g., cancer or any cancer-related disease).For example, DNA methylation spectrum analysis can be used to detect normal high methylation or low methylation in a given sample type (e.g., cfDNA from bloodstream) but may show abnormal methylation degree related to neoplasm or cancer (e.g., due to abnormal increase in contribution of tissue to sample type (e.g., due to increased DNA shedding in or around neoplasm or cancer) and/or abnormal increase in contribution from degree of methylation) region.

In some embodiments, DNA methylation comprises adding a methyl group to a cytosine residue at a CpG site (cytosine-phosphate-guanine site (i.e., guanine follows cytosine in the 5'->3' direction of the nucleic acid sequence)). In some embodiments, DNA methylation comprises adding a methyl group to an adenine residue, such as in N ⁶ -methyladenine. In some embodiments, DNA methylation is 5-methylation (modification of the 5th carbon of the 6-carbon ring of cytosine). In some embodiments, 5-methylation comprises adding a methyl group to the 5C position of a cytosine residue to produce 5-methylcytosine (m5c or 5-mC or 5mC). In some embodiments, methylation comprises a derivative of m5c. Derivatives of m5c include, but are not limited to, 5-hydroxymethylcytosine (5-hmC or 5hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC). In some embodiments, DNA methylation is 3C methylation (modification of the 3rd carbon of the 6-carbon ring of a cytosine residue). In some embodiments, 3C methylation includes adding a methyl group to the 3C position of a cytosine residue to produce 3-methylcytosine (3mC). Methylation can also occur at non-CpG sites, for example, methylation can occur at CpA, CpT or CpC sites. DNA methylation can change the activity of methylated DNA regions. For example, when the DNA in the promoter region is methylated, transcription of the gene can be inhibited. DNA methylation is critical for normal development, and methylated abnormalities can destroy epigenetic regulation. The destruction of epigenetic regulation, such as inhibition, can cause diseases, such as cancer. Promoter methylation in DNA can indicate cancer.

Methylation spectrum analysis can include determining the methylation pattern throughout different regions of the genome. For example, after the molecules are partitioned and sequenced based on the degree of methylation (e.g., the relative number of methylated nucleotides in each molecule), the sequences of the molecules in the different partitions can be mapped to the reference genome. This can show regions in the genome that are more methylated or less methylated than other regions. In this way, in contrast to a single molecule, the degree of methylation in a genomic region can be different.

Combining the signals obtained from methylation profiling with those obtained from somatic variants (e.g., SNVs, indels, CNVs, and gene fusions) can aid in the detection of cancer.

Nucleic acid molecules in a sample can be graded or partitioned based on the methylation state of nucleic acid molecules. Partitioning nucleic acid molecules in a sample can increase rare signals. For example, genetic variations present in hypermethylated DNA but less present (or absent) in hypomethylated DNA can be easily detected by partitioning the sample into hypermethylated and hypomethylated nucleic acid molecules. By analyzing more than one fraction of a sample, a single molecule can be multidimensionally analyzed, and thereby greater sensitivity can be achieved. Partitioning can include physically partitioning nucleic acid molecules into subgroups or groups based on the presence or absence of one or more methylated nucleotides (e.g., nucleotides comprising methylated bases). Samples can be graded or partitioned into one or more partition groups based on the features indicating differential gene expression or disease states. In the analysis of nucleic acids (e.g., cell-free DNA ("cfDNA"), non-cfDNA, tumor DNA, circulating tumor DNA ("ctDNA"), and cell-free nucleic acids ("cfNA")), samples can be graded based on the features or combinations thereof that provide signal differences between normal states and disease states.

In some embodiments, the sample can be partitioned into two or more partition groups (e.g., at least 3, 4, 5, 6, or 7 partition groups) based on the different binding affinities of the methylated nucleic acid molecules to the binding agent (i.e., a binding agent that binds to a methylated nucleotide (e.g., a nucleotide comprising a methylated base). Examples of binding agents include, but are not limited to, methyl binding domains (MBDs) and methyl binding proteins (MBPs). Examples of MBPs contemplated herein include, but are not limited to:

(a) Proteins MeCP2 and MBD2 that preferentially bind 5-methyl-cytosine over unmodified cytosine.

(b) RPL26, PRP8, and the DNA mismatch repair protein MHS6 that preferentially bind to 5-hydroxymethyl-cytosine over unmodified cytosine;

(c) FOXK1, FOXK2, FOXP1, FOXP4, and FOXI3 that preferentially bind 5-formyl-cytosine over unmodified cytosine (Iurlaro et al., Genome Biol. 14, R119 (2013)); and

(d) Antibodies specific for one or more methylated nucleotide bases.

In such an embodiment, nucleic acids overrepresented in a modification are bound to the agent to a greater extent than nucleic acids underrepresented in the modification. Alternatively, nucleic acids with modifications can be bound in an all-or-nothing manner. However, then, various levels of modifications can be sequentially eluted from the binding agent.

For example, in some embodiments, the partition can be binary or based on the degree/level of methylation. For example, all methylated fragments and unmethylated fragments can be partitioned using a methyl binding domain protein (e.g., MethylMiner methylated DNA enrichment kit (ThermoFisherScientific)). Subsequently, other partitions can include eluting fragments with different methylation levels by adjusting the salt concentration of a solution containing a methyl binding domain and a binding fragment. As the salt concentration increases, fragments with greater methylation levels are eluted.

Compared with standard methylation analysis methods (such as bisulfite sequencing), methylation partitioning methods are highly effective in recovering analyte molecules, and somatic changes can be detected simultaneously. However, when the method identifies the methylation level of a molecule by partitioning, the sensitivity and specificity of the method are challenged by incorrect partitioning of methylated/unmethylated molecules (for example, unmethylated molecules are partitioned into high partition groups). The technical noise of the molecular error partitioning determined from methylation partitioning limits the performance of the assay. In order to increase the signal-to-noise ratio of methylation partitioning assays, specific partitioning groups can be subjected to methylation-sensitive restriction endonucleases (RE) digestion reactions to specifically remove molecules of incorrect partitions. For example, methylation-sensitive restriction endonucleases (MSEs) that only crack unmethylated molecules with RE recognition sites can be applied to high partitioning groups, to selectively crack and remove (from the assay process) unmethylated molecules that are only partitioned by errors. Therefore, by reducing the number of unmethylated molecules in high partitioning groups, the sensitivity and specificity of the assay are improved, which then improves the ability to detect the presence or absence of circulating tumor DNA (ctDNA).

The present disclosure provides methods and systems for improving the sensitivity and specificity of DNA methylation partitioning assays. These methods and systems can be used in various applications, such as predicting the prognosis of cancer, diagnosis, monitoring, recurrence and/or relapse of cancer.

Thus, in one aspect, the present disclosure provides a method for analyzing nucleic acid molecules in a biological sample, the method comprising: (a) partitioning at least a subset of nucleic acid molecules in the biological sample into more than one partitioning group based on the methylation status of the nucleic acid molecules, wherein the biological sample comprises methylated nucleic acid molecules and unmethylated nucleic acid molecules; (b) digesting at least one subset of one or more of the more than one partitioning groups with at least one methylation-sensitive restriction endonuclease; and (c) determining the methylation status at one or more genetic loci of the nucleic acid molecules in at least one of the partitioning groups.

In some embodiments, the method further includes detecting the presence or absence of cancer in the biological sample. In some embodiments, the method includes determining the level of cancer in the biological sample, for example, by determining the level of DNA from cancer cells in the biological sample. In some embodiments, the method further includes attaching one or more adapters to at least one end (i.e., 5' and/or 3' ends) of the nucleic acid molecules in more than one partition group before digestion. In some embodiments, determining the methylation state includes sequencing at least one subset of the digested nucleic acid molecules. In some embodiments, the method further includes, before determining the methylation state, enriching at least one subset of the nucleic acid molecules in more than one partition group for the genomic region of interest, wherein at least one subset of the nucleic acid molecules comprises digested nucleic acid molecules in one or more partition groups. In some such embodiments, the genomic region of interest includes an epigenetic target region group. In some such embodiments, the method includes enriching or capturing a first epigenetic target region group from at least a portion of a first partition group, and enriching or capturing a second epigenetic target region group from at least a portion of a second partition group.

In another aspect, the present disclosure provides a method for determining the methylation state of a nucleic acid molecule, the method comprising: (a) providing a biological sample of nucleic acid molecules, wherein the nucleic acid molecules include methylated nucleic acid molecules and unmethylated nucleic acid molecules; (b) partitioning at least one subset of nucleic acid molecules in the biological sample into more than one partition group based on the methylation state of the nucleic acid molecules; (c) digesting at least one subset of one or more of the more than one partition groups with at least one methylation-sensitive restriction endonuclease; (d) enriching at least one subset of nucleic acid molecules in more than one partition group for a genomic region of interest, wherein at least one subset of nucleic acid molecules comprises digested nucleic acid molecules in one or more partition groups; and (e) determining the methylation state at one or more genetic loci of nucleic acid molecules in at least one of the partition groups. In some such embodiments, the genomic region of interest includes an epigenetic target region group. In some such embodiments, the method includes enriching or capturing a first epigenetic target region group from at least a portion of the first partition group, and enriching or capturing a second epigenetic target region group from at least a portion of the second partition group.

In some embodiments, the method further comprises detecting the presence or absence of cancer in the biological sample. In some embodiments, the method comprises determining the level of cancer in the biological sample, for example, by determining the level of DNA from cancer cells in the biological sample. In some embodiments, the method further comprises attaching one or more adapters to at least one end (i.e., 5' and/or 3' ends) of the nucleic acid molecules in more than one partition group prior to digestion. In some embodiments, determining the methylation state comprises sequencing at least one subset of the digested nucleic acid molecules.

Fig. 2 illustrates an exemplary embodiment of a method 200 for determining the methylation state of nucleic acid molecules in a polynucleotide sample obtained from a subject. At 202, a polynucleotide sample is obtained from a subject. In some embodiments, the polynucleotide sample is a DNA sample obtained from a tumor tissue biopsy. In some embodiments, the polynucleotide sample is a cell-free DNA (cfDNA) sample obtained from blood. At 204, the polynucleotide sample is partitioned into at least two partition groups. In some embodiments, partitioning includes partitioning nucleic acid molecules based on different binding affinities of a binding agent that preferentially binds to a polynucleotide comprising a methylated nucleotide (e.g., a nucleotide comprising a methylated base). Examples of binding agents include, but are not limited to, methyl binding domains (MBDs) and methyl binding proteins (MBPs). Examples of MBPs contemplated herein are listed above.

(e) Antibodies specific for one or more methylated nucleotide bases.

Partitioning can refer to physical separation or classification of nucleic acid molecules based on the characteristics of nucleic acid molecules.Partitioning can be the physical partitioning of molecules.Partitioning can include dividing nucleic acid molecules into groups or groups based on the methylation level of nucleic acid molecules.In some embodiments, the method and system for partitioning can be carried out as described in PCT patent application WO2018/119452, and the patent application is incorporated by reference in its entirety.In these embodiments, nucleic acid is partitioned based on different methylation levels (for example, the number or frequency of different methylated nucleotides (for example, nucleotides comprising methylated bases)).In some embodiments, nucleic acid can be partitioned into two or more partition groups (for example, at least 3, 4, 5, 6 or 7 partition groups).For example, nucleic acid molecules can be partitioned into three groups--one group is a highly methylated nucleic acid molecule (high partition group or high methylation partition group), the second group is a low methylated nucleic acid molecule (low partition group or low methylation partition group), and the third group is a medium methylated nucleic acid molecule (medium partition group or medium methylation partition group). In some embodiments, partition groups represent nucleic acids with varying degrees of methylation (modifications that are overrepresentative or underrepresentative). Overrepresentation and underrepresentation can be defined by the number of methylated nucleotides present in a DNA molecule (e.g., a cfDNA molecule) relative to the median of methylated nucleotides in each chain in a population. For example, if the median of 5-methylcytosine nucleotides in a nucleic acid molecule in a sample is 2, nucleic acid molecules containing more than two 5-methylcytosine residues are overrepresentative, while nucleic acids with 1 or 0 5-methylcytosine residues are underrepresentative. The effect of affinity separation is to enrich for nucleic acids that are overrepresented in the binding phase (i.e., methylation levels) and nucleic acids that are underrepresented in the unbound phase (i.e., in solution). Nucleic acids in the binding phase can be eluted before subsequent treatment.

At 206, the nucleic acid molecules in at least one partition group are digested with at least one methylation-sensitive restriction endonuclease (MSRE). In some embodiments, the nucleic acids in at least one partition group are digested with at least two MSREs. In some embodiments, two MSREs are used to digest the nucleic acid molecules in at least one partition group. In some embodiments, the two MSREs are BstUI and HpaII. In some embodiments, the two MSREs are HhaI and AccII. In some embodiments, three MSREs are used to digest the nucleic acid molecules in at least one partition group. In some embodiments, the three MSREs are BstUI, HpaII and Hin6I. In some embodiments, the MSRE is selected from the group consisting of AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI, Cfr10I, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, MspI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, and SnaBI. In some embodiments, any commercially available MSRE can be used (products from TakaraBio USA Inc., New England Biolabs Inc., or the like can be used). Inc. and/or MSRE provided by Thermo Fisher Scientific Inc.

In some embodiments, FspEI is used to digest the nucleic acid molecules in at least one other partition group (for example, hypomethylation partition). In some embodiments, BstUI, HpaII and Hin6I are used to digest the nucleic acid molecules in at least one other partition group (for example, hypermethylation partition), and FspEI is used to digest the nucleic acid molecules in at least one other partition group (for example, hypomethylation partition). In the embodiment comprising medium methylation partition, nucleic acid molecules therein can be digested with at least one methylation-sensitive restriction endonuclease that preferentially cleaves methylated or unmethylated DNA. In some embodiments, the nucleic acid molecules in the medium methylation partition are digested with the MSRE identical with the hypermethylation partition. For example, the medium methylation partition and the hypermethylation partition can be collected, and then the partition collected can be digested. In some embodiments, the nucleic acid molecules in the medium methylation partition are digested with the MSRE identical with the hypomethylation partition. For example, the medium methylation partition and the hypomethylation partition can be collected, and then the partition collected can be digested.

In some embodiments, before restriction digestion with MSRE, at least one adapter is attached to at least one end of the nucleic acid molecule (i.e., 5' and/or 3' ends of the DNA molecule). In some such embodiments, the adapter is attached to both ends of the nucleic acid molecule. In other embodiments, after digestion but before 208 enrichment, at least one adapter is attached to at least one end of the nucleic acid molecule. In some embodiments, the adapter tolerates digestion of methylation-sensitive restriction endonucleases. In some embodiments, the adapter comprises one or more methylated nucleotides (e.g., nucleotides comprising methylated bases). In some embodiments, the methylated nucleotides can be 5-methylcytosine and/or 5-hydroxymethylcytosine. In some embodiments, the adapter comprises one or more nucleotide analogs that tolerate methylation-sensitive restriction endonucleases. In some embodiments, the adapter comprises a nucleotide sequence that is not recognized by methylation-sensitive restriction endonucleases. In some embodiments, a tag can be provided as a component of an adapter. In some embodiments, the tag comprises a molecular barcode (i.e., a molecular identifier). In some embodiments, the label of the nucleic acid molecule attached to a partition group is different from the label of the nucleic acid molecule attached to other partition groups. In some embodiments, a partition group and other partition groups are differentially labeled. The differential labeling of the partition group helps to keep the nucleic acid molecules belonging to a specific partition group. The nucleic acid molecules in different partition groups receive different labels that can distinguish the members of a partition group from the members of another partition group. The labels connected to the nucleic acid molecules of the same partition group can be the same or different from each other. But if they are different from each other, a part of the label sequence can be shared, so that the molecules to which they are attached are identified as specific partition groups. For example, if the molecules of the sample are partitioned into two partition groups--P1 and P2, then the molecules in P1 can be labeled with A1, A2, A3, etc., and the molecules in P2 can be labeled with B1, B2, B3, etc. Such a labeling system allows distinguishing partition groups and distinguishing molecules in partition groups. In some embodiments, the label includes a partition label (i.e., a partition identifier). In such an embodiment, the nucleic acid molecules in the partition group receive the same partition label, and are different from the partition labels of the nucleic acid molecules attached to other partition groups.

At 208, after the MSRE digestion, the nucleic acid molecules in one or more partition groups can be enriched for the genomic region of interest. In some embodiments, the genomic region of interest can include the region of differential methylation for cancer detection. At 210, at least one subset of the enriched molecules is sequenced with a next-generation sequencer. At 212, the sequencing reads produced by the bioinformatics tool/algorithm analysis sequencer are then used to determine the number of molecules in one or more partition groups, and the number of molecules is then used to determine the methylation state of one or more genetic loci of nucleic acid molecules in at least one partition group. In some embodiments, one or more genetic loci can include more than one genetic loci. In some embodiments, one or more genetic loci can include one or more genomic regions. In some embodiments, the genomic region can be the promoter region of a gene. In some embodiments, before sequencing, nucleic acid molecules can be amplified by pcr amplification. In some embodiments, the primer used in the amplification can include at least one sample index.

In some embodiments, the method may also include, based on the methylation state of one or more genetic loci of nucleic acid molecules in at least one partition group, detecting the presence or absence of cancer in the subject. In some embodiments, the method also includes determining the level of cancer in the polynucleotide sample, for example, by determining the level of DNA from cancer cells in the polynucleotide sample.

In another aspect, the present disclosure provides a method for determining the methylation state of a nucleic acid molecule, the method comprising: (a) providing a biological sample of nucleic acid molecules, wherein the nucleic acid molecules include methylated nucleic acid molecules and unmethylated nucleic acid molecules; (b) partitioning at least one subset of the nucleic acid molecules in the biological sample into more than one partition group based on the methylation state of the nucleic acid molecules; (c) attaching one or more adapters to at least one end of the nucleic acid molecules in more than one partition group; (d) digesting at least one subset of one or more of the partition groups with at least one methylation-sensitive restriction endonuclease; (d) enriching at least one subset of the nucleic acid molecules in more than one partition group for a genomic region of interest; wherein at least one subset of the nucleic acid molecules comprises digested nucleic acid molecules in one or more partition groups; and (e) determining the methylation state at one or more genetic loci of the nucleic acid molecules in at least one of the partition groups. In some such embodiments, the genomic region of interest includes an epigenetic target region group. In some such embodiments, the method includes enriching or capturing a first set of epigenetic target regions from at least a portion of the first partitioning group, and enriching or capturing a second set of epigenetic target regions from at least a portion of the second partitioning group.

In some embodiments, the method further comprises detecting the presence or absence of cancer in the biological sample. In some embodiments, the method comprises determining the level of cancer in the biological sample, for example, by determining the level of DNA from cancer cells in the biological sample. In some embodiments, the method further comprises attaching one or more adapters to at least one end (i.e., 5' and/or 3' ends) of the nucleic acid molecules in more than one partition group prior to digestion. In some embodiments, determining the methylation state comprises sequencing at least one subset of the digested nucleic acid molecules.

Fig. 3 shows an exemplary embodiment of a method 300 for detecting the presence or absence of cancer in a subject according to an embodiment of the present disclosure. At 302, a polynucleotide sample is obtained from a subject. In some embodiments, the polynucleotide sample is a DNA sample obtained from a tumor tissue biopsy. In some embodiments, the polynucleotide sample is a cell-free DNA (cfDNA) sample obtained from blood (e.g., from plasma). At 304, the polynucleotide sample is partitioned into at least two partition groups. In some embodiments, partitioning includes partitioning nucleic acid molecules based on different binding affinities of a binding agent that preferentially binds to a polynucleotide comprising a methylated nucleotide (e.g., a nucleotide comprising a methylated base). Examples of binding agents include, but are not limited to, methyl binding domains (MBDs) and methyl binding proteins (MBPs). Examples of MBPs contemplated herein are listed above.

In some embodiments, nucleic acids can be partitioned into two or more partition groups (e.g., at least 3, 4, 5, 6, or 7 partition groups). In some embodiments, the partition groups represent nucleic acids with varying degrees of methylation (overrepresentation or underrepresentation of modifications). For example, nucleic acid molecules can be partitioned into three groups—one group is highly methylated nucleic acid molecules (high partition group or high methylation partition group), a second group is low methylated nucleic acid molecules (low partition group or low methylation partition group), and a third group is medium methylated nucleic acid molecules (medium partition group or medium methylation partition group).

At 306, the nucleic acid molecules in one or more partition groups are attached with adapters, wherein the adapters include at least one tag and are attached to at least one end of the nucleic acid molecule (i.e., 5' and/or 3' ends of the DNA molecule). In some embodiments, the adapters are tolerant to digestion of methylation-sensitive restriction endonucleases. In some embodiments, the adapters include one or more methylated nucleotides (e.g., nucleotides including methylated bases). In some embodiments, the methylated nucleotides may be 5-methylcytosine and/or 5-hydroxymethylcytosine. In some embodiments, the adapters include one or more nucleotide analogs tolerant to methylation-sensitive restriction endonucleases. In some embodiments, the adapters include nucleotide sequences that are not recognized by methylation-sensitive restriction endonucleases. In some embodiments, the adapters do not include nucleotide sequences recognized by the methylation-sensitive restriction endonucleases used in the method. In some embodiments, the adapters include one or more modifications (e.g., connection modifications, such as thiophosphates) that inhibit cleavage of methylation-sensitive restriction endonucleases. In some embodiments, the tags may be provided as components of the adapters. In some embodiments, the label includes a molecular barcode (i.e., a molecular identifier). In some embodiments, the label of the nucleic acid molecule attached to a partition group is different from the label of the nucleic acid molecule attached to other partition groups. In some embodiments, a partition group is labeled with other partition group differences. Labeling the partition group differences helps to keep tracing the nucleic acid molecules belonging to a specific partition group. The nucleic acid molecules in different partition groups receive different labels that can distinguish the members of a partition group from the members of another partition group. The labels connected to the nucleic acid molecules of the same partition group can be the same or different from each other. But if different from each other, a part of the label sequence can be shared, so that the molecules to which they are attached are identified as specific partition groups. For example, if the molecules of the sample are partitioned into two partition groups--P1 and P2, then the molecules in P1 can be labeled with A1, A2, A3, etc., and the molecules in P2 can be labeled with B1, B2, B3, etc. Such a labeling system allows distinguishing partition groups and distinguishing molecules in partition groups. In some embodiments, the label includes a partition label (i.e., a partition identifier). In such embodiments, nucleic acid molecules within a partitioning group receive the same partitioning tag, and are different from partitioning tags attached to nucleic acid molecules of other partitioning groups.In some embodiments, the tag sequence used does not comprise a nucleotide sequence recognized by a methylation-sensitive restriction endonuclease used in the method.

At 308, the nucleic acid molecules in at least one partition group are digested with at least one methylation-sensitive restriction endonuclease (MSRE). In some embodiments, the nucleic acid in at least one partition group is digested with at least two MSREs. In some embodiments, two MSREs are used to digest the nucleic acid molecules in at least one partition group. In some embodiments, the two MSREs are BstUI and HpaII. In some embodiments, the two MSREs are HhaI and AccII. In some embodiments, three MSREs are used to digest the nucleic acid molecules in at least one partition group. In some embodiments, the three MSREs are BstUI, HpaII and Hin6I. In some embodiments, the MSRE is selected from the group consisting of AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI, Cfr10I, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, MspI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, and SnaBI. In some embodiments, any commercially available MSRE can be used (products from TakaraBio USA Inc., New England Biolabs Inc., or the like can be used). Inc. and/or MSRE provided by Thermo Fisher Scientific Inc.

At 310, after MSRE digestion, the nucleic acid molecules in one or more partition groups can be enriched for the genomic region of interest. In some embodiments, the genomic region of interest can include the region of differential methylation for cancer detection. At 312, at least one subset of the enriched molecules is sequenced with a next-generation sequencer. At 314, the sequencing reads produced by the bioinformatics tool/algorithm analysis sequencer are then used to determine the number of molecules in one or more partition groups, and the number of molecules is then used to determine the methylation state of one or more genetic loci of nucleic acid molecules in at least one partition group. In some embodiments, one or more genetic loci can include more than one genetic loci. In some embodiments, one or more genetic loci can include one or more genomic regions. In some embodiments, the genomic region can be the promoter region of a gene. In some embodiments, before sequencing, nucleic acid molecules can be amplified by PCR amplification. In some embodiments, the primer used in amplification can include at least one sample index. In some embodiments, the nucleic acid molecules digested by MSRE are not amplified. In some such embodiments, except for the nucleic acid molecules digested by MSRE, substantially all nucleic acid molecules in the sample are amplified.

In some embodiments, the method may also include, based on the methylation state of one or more genetic loci of nucleic acid molecules in at least one partition group, detecting the presence or absence of cancer in the subject. In some embodiments, the method also includes determining the level of cancer in the polynucleotide sample, for example, by determining the level of DNA from cancer cells in the polynucleotide sample.

Fig. 4 illustrates an exemplary workflow for detecting the presence or absence of cancer from a cfDNA sample according to certain embodiments of the present disclosure, wherein cfDNA is separated from a blood sample, and the cfDNA sample comprises cfDNA molecules belonging to a cancer hypermethylated DMR region and an unmethylated control region; cfDNA is partitioned into low methylation, residual (i.e., medium methylation) and high methylation partition groups using a methyl binding domain protein (MBD); molecular barcoding is performed on each partition group, so that the DNA from the low partition group, the residual partition group and the high partition group can be distinguishably labeled; the high partition group is digested with two MSREs (HhaI and AccI), and the unmethylated cfDNA molecules are cracked at the RE recognition site; and then the partition group (including the high partition group digested by MSRE) is collected, captured, amplified and sequenced. In some embodiments, the nucleic acid molecules digested by MSRE are not amplified. In some such embodiments, in addition to the nucleic acid molecules digested by MSRE, substantially all nucleic acid molecules in the sample are amplified.

In some embodiments, MSRE is selected to maximize the number of targeted methylation biomarker sequences (i.e., DMRs). In some embodiments, if two or more MSREs are used in a single digestion, the enzyme buffer should be compatible (verified by the supplier and/or tested empirically). In addition, MSRE should have a mechanism for inactivating its activity that is compatible with downstream assay processing. For example, if MSRE digestion is performed before connection, thermal inactivation (>65°C) of MSRE will not be suitable because it will denature dsDNA, making it incompatible with the adapter ligation reaction.

In some embodiments, a methylation-sensitive restriction endonuclease that does not cleave DNA if the specific nucleotide base at the recognition sequence is methylated can be used. Such MSRE can be used only in high partitions to remove unmethylated molecules that are not correctly partitioned in the high partition; thereby improving the detection specificity of methylated nucleic acid molecules. In some embodiments, a methylation-sensitive restriction endonuclease that cleaves DNA if the specific nucleotide base at the recognition sequence is methylated can be used. Such MSRE can be used in low partitions to remove methylated molecules that are not correctly partitioned in the low partition; thereby improving the detection specificity of unmethylated nucleic acid molecules. In some embodiments, both high (and residual) partitions and low partitions are digested with MSRE, so that (i) if there are unmethylated nucleotides at the recognition site, the MSRE that cleaves DNA is used for high (and residual) partitions, and (ii) if there are methylated nucleotides at the recognition site, the MSRE that cleaves DNA is used for low partitions.

In some embodiments, after the adapter is connected, if more than one partition (e.g., a high partition and a residual partition) is to be digested with the same MSRE, each partition can be digested separately, or the partitions can be combined and digested in one reaction. In some embodiments, if the necessary enzyme performance (efficiency, specificity) can only be achieved by using a separate reaction, it can be advantageous to digest each partition separately. In some embodiments, combining the partitions and then performing the MSRE digestion can be beneficial to reduce the assay cost of the goods sold (COGS (SPRI beads, enzymes, PCR plates, pipetting tips, etc.)) and simplify the automated assay of scale (i.e., a single digestion reaction per sample).

In some embodiments, if MSRE digestion is performed before connecting the adapter (wherein MSRE cleaves unmethylated DNA at the recognition site), the cleavage fragments of the molecule can be retained, and the ends of the molecules matching the RE recognition site are used to identify unmethylated molecules in the high partition. In such embodiments, when analyzing cfDNA samples, if there is genomic DNA contamination, the genomic DNA can be cleaved by MSRE (before the adapter is connected), and genomic DNA contamination can be caused. This can be avoided by connecting the adapter before MSRE digestion.

In some embodiments, all or a subset of all partitions may be sequenced.In some embodiments, only one or more partitions to which MSRE digestion was performed may be sequenced to analyze nucleic acid molecules in cancer DMRs.

In some embodiments, polynucleotide sample partition is divided into two partition groups.In some embodiments, polynucleotide sample partition is divided into three partition groups.In some embodiments, nucleic acid molecules in high partition and low partition are carried out MSRE digestion, if wherein recognition site has unmethylated nucleotide, the MSRE cracking DNA used in high partition, and if recognition site has methylated nucleotide, the MSRE cracking DNA used in low partition.This makes it possible to detect high DMR and low DMR sensitively simultaneously.

In some embodiments, the polynucleotide sample is between 1 ng and 500 ng. In some embodiments, the polynucleotide sample is less than 500 ng. In some embodiments, the polynucleotide sample is selected from the group consisting of: a DNA sample, an RNA sample, a cell-free DNA sample, and a cell-free RNA sample. In some embodiments, the polynucleotide sample is a cfDNA sample obtained from the subject's blood. In some embodiments, the polynucleotide sample is a DNA sample obtained from a tumor tissue biopsy.

II. General characteristics of the method

A. Sample

Sample can be any biological sample separated from a subject.Sample can include body tissue, whole blood, platelets, serum, plasma, feces, red blood cells, white blood cells (white blood cell) or white blood cells (leucocyte), endothelial cells, tissue biopsy (e.g., biopsy from known or suspected solid tumors), cerebrospinal fluid, synovial fluid, lymphatic fluid, ascites, interstitial fluid or extracellular fluid (e.g., fluid from intercellular spaces), gingival fluid, gingival sulcus fluid, bone marrow, pleural effusion, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat and urine.Therefore, sample can be body fluid, such as blood and its fractions and urine.Such sample can include nucleic acid falling off from tumor.Nucleic acid can include DNA and RNA, and can be in double-stranded form and single-stranded form.In some embodiments, sample includes cell-free DNA.Sample can be the form initially separated from subject, or can have been further processed to remove or add components, such as cells, enrich a component relative to another component, or convert a form of nucleic acid into another form of nucleic acid, such as converting RNA into DNA or converting single-stranded nucleic acid into double-stranded nucleic acid. Thus, for example, the bodily fluid for analysis can be plasma or serum containing cell-free nucleic acids, such as cell-free DNA (cfDNA).

The sample can be separated or obtained from the subject and transported to the sample analysis site. The sample can be stored and transported at a desired temperature, such as room temperature, 4°C, -20°C and/or -80°C. The sample can be separated or obtained from the subject at the sample analysis site. The subject can be a human, a mammal, an animal, a companion animal, a service animal or a pet. The subject can suffer from cancer. The subject can have no cancer or detectable cancer symptoms. The subject can have been treated with one or more cancer therapies, such as any one or more chemotherapy. The subject can be in remission. The subject may be diagnosed or may not be diagnosed as susceptible to cancer or any cancer-related genetic mutation/disorder.

In some embodiments, the sample volume of the body fluid taken from the subject depends on the read depth of the desired sequencing region. Examples of volumes are about 0.4-40 milliliters (mL), about 5-20 mL, about 10-20 mL. For example, the volume can be about 0.5 mL, about 1 mL, about 5 mL, about 10 mL, about 20 mL, about 30 mL, about 40 mL or more milliliters. The volume of sampled plasma is generally between about 5 mL and about 20 mL.

Samples can contain different amounts of nucleic acid. Typically, the amount of nucleic acid in a particular sample is equivalent to multiple genome equivalents. For example, a sample of about 30 nanograms (ng) of DNA can contain about 10,000 (10 ⁴ ) haploid human genome equivalents, and in the case of cfDNA, can contain about 200 billion (2x 10 ¹¹ ) individual polynucleotide molecules. Similarly, a sample of about 100 ng of DNA can contain about 30,000 haploid human genome equivalents, and in the case of cfDNA, can contain about 600 billion individual molecules.

In some embodiments, the sample comprises nucleic acids from different sources, for example, from cell sources and from cell-free sources (e.g., blood samples, etc.). Typically, the sample includes nucleic acids carrying mutations. For example, the sample optionally includes DNA carrying germline mutations and/or somatic mutations. Typically, the sample includes DNA carrying cancer-associated mutations (e.g., cancer-associated somatic mutations).

Exemplary amounts of cell-free nucleic acid in a sample prior to amplification generally range from about 1 femtogram (fg) to about 1 microgram (μg), for example, about 1 picogram (pg) to about 200 nanograms (ng), about 1 ng to about 100 ng, about 10 ng to about 1000 ng. In some embodiments, the sample includes up to about 600 ng, up to about 500 ng, up to about 400 ng, up to about 300 ng, up to about 200 ng, up to about 100 ng, up to about 50 ng, or up to about 20 ng of cell-free nucleic acid molecules. Optionally, the amount is at least about 1 fg, at least about 10 fg, at least about 100 fg, at least about 1 pg, at least about 10 pg, at least about 100 pg, at least about 1 ng, at least about 10 ng, at least about 100 ng, at least about 150 ng, or at least about 200 ng of cell-free nucleic acid molecules. In some embodiments, the amount is up to about 1 fg, about 10 fg, about 100 fg, about 1 pg, about 10 pg, about 100 pg, about 1 ng, about 10 ng, about 100 ng, about 150 ng, or about 200 ng of cell-free nucleic acid molecules. In some embodiments, the method comprises obtaining between about 1 fg and about 200 ng of cell-free nucleic acid molecules from a sample.

The cell-free nucleic acids typically have a size distribution between about 100 nucleotides in length and about 500 nucleotides in length, with molecules between about 110 nucleotides in length and about 230 nucleotides in length representing about 90% of the molecules in the sample, with a mode of about 168 nucleotides in length (in samples from human subjects), and a second minor peak ranging in length from about 240 nucleotides to about 440 nucleotides. In some embodiments, the cell-free nucleic acids are from about 160 nucleotides to about 180 nucleotides in length, or from about 320 nucleotides to about 360 nucleotides in length, or from about 440 nucleotides to about 480 nucleotides in length.

In some embodiments, cell-free nucleic acid is separated from body fluid by partitioning step, in which the cell-free nucleic acid present in the solution is separated from intact cells and other insoluble components in the body fluid. In some embodiments, partitioning includes techniques such as centrifugation or filtration. Alternatively, cells in body fluid can be lysed, and cell-free nucleic acid and cell nucleic acid can be processed together. Typically, after adding buffer and washing steps, cell-free nucleic acid can be precipitated with, for example, alcohol. In some embodiments, additional cleaning steps such as silica-based columns are used to remove pollutants or salts. For example, non-specific bulk carrier nucleic acid is optionally added throughout the reaction to optimize multiple aspects such as yield of exemplary procedures. After such processing, the sample generally contains various forms of nucleic acid, including double-stranded DNA, single-stranded DNA and/or single-stranded RNA. Optionally, single-stranded DNA and/or single-stranded RNA are converted into double-stranded forms so that they are included in subsequent processing and analysis steps.

Double-stranded DNA molecules in the sample and single-stranded nucleic acid molecules converted into double-stranded DNA molecules can be connected to adapters at one or both ends. Usually, in the presence of all four standard nucleotides, double-stranded molecules are flat-ended by polymerase treatment with 5'-3' polymerase and 3'-5' exonuclease (or proofreading function). Klenow large fragment and T4 polymerase are examples of suitable polymerases. Flat-ended DNA molecules can be connected to at least partially double-stranded adapters (e.g., Y-shaped or bell-shaped adapters). Alternatively, complementary nucleotides can be added to the flat ends of sample nucleic acids and adapters to promote connection. Both flat-ended connections and sticky end connections are contemplated herein. In flat-ended connections, both nucleic acid molecules and adapter tags have flat ends. In sticky end connections, nucleic acid molecules usually have "A" overhangs and adapters have "T" overhangs.

B. Partitioning, adding adapters, and labeling

In another embodiment, the following exemplary program can be used to perform a partitioning scheme. Both ends of the nucleic acid are connected to a Y-shaped adapter comprising a primer binding site and a tag. Amplify the molecule. Then, the amplified molecule is partitioned to produce two partitions by contacting with an antibody that preferentially binds to 5-methylcytosine. One partition contains the original molecule lacking methylation and the amplified copy that loses methylation. The other partition contains the original DNA molecule with methylation. The partition containing the original DNA molecule with methylation is subjected to a program that differently affects the first nucleobase in the DNA of the first partition and the second nucleobase in the DNA, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity. The two partitions are then processed and sequenced separately, and the methylation partition is further amplified. The sequence data of the two partitions can then be compared. In this example, the tag is not used to distinguish between methylated DNA and unmethylated DNA, but to distinguish between different molecules in these partitions, so that people can determine whether the read segments with the same starting point and end point are based on the same or different molecules.

The label can be incorporated into the adapter or otherwise connected to the adapter by methods such as chemical synthesis, connection (e.g., flat end connection or sticky end connection) or overlap extension polymerase chain reaction (PCR). Such an adapter can ultimately be connected to the target nucleic acid molecule. In other embodiments, one or more rounds of amplification cycles (e.g., PCR amplification) are generally applied to introduce the sample index into the nucleic acid molecule using conventional nucleic acid amplification methods. Amplification can be carried out in one or more reaction mixtures (e.g., more than one micropore in the array). Molecular barcodes and/or sample indexes can be introduced simultaneously or in any order. In some embodiments, molecular barcodes and/or sample indexes are introduced before and/or after the sequence capture step is performed. In some embodiments, only molecular barcodes are introduced before the probe capture, and sample indexes are introduced after the sequence capture step is performed. In some embodiments, both molecular barcodes and sample indexes are introduced before the probe-based capture step is performed. In some embodiments, sample indexes are introduced after the sequence capture step is performed. In some embodiments, the molecular barcode is incorporated into a nucleic acid molecule (e.g., cfDNA molecule) in a sample through an adapter by ligation (e.g., blunt end ligation or sticky end ligation). In some embodiments, the sample index is incorporated into a nucleic acid molecule (e.g., cfDNA molecule) in a sample by overlap extension polymerase chain reaction (PCR). Typically, sequence capture schemes include the introduction of a single-stranded nucleic acid molecule complementary to a target nucleic acid sequence, such as a coding sequence of a genomic region, and mutations in such a region are associated with cancer types.

In some embodiments, the label can be located at one end or both ends of the sample nucleic acid molecule. In some embodiments, the label is an oligonucleotide of a predetermined or random or semi-random sequence. In some embodiments, the length of the label can be less than or equal to about 500, 200, 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides. The label can be randomly or non-randomly connected to the sample nucleic acid.

In some embodiments, each sample is uniquely labeled by a sample index or a combination of sample indexes. In some embodiments, each nucleic acid molecule of a sample or subsample is uniquely labeled by a molecular barcode or a combination of molecular barcodes. In other embodiments, more than one barcode may be used so that the molecular barcodes do not have to be unique relative to each other in the more than one barcode (e.g., non-unique molecular barcodes). In these embodiments, molecular barcodes are typically attached to individual molecules (e.g., by connection) so that the combination of molecular barcodes and sequences to which they can be attached produces a unique sequence that can be traced back individually. Detection of a non-unique molecular barcode in combination with endogenous sequence information (e.g., corresponding to the start (start) and/or end (stop) genomic loci/positions of the original nucleic acid molecule sequence in the sample, corresponding to the start and end genomic positions of the original nucleic acid molecule sequence in the sample, the start (start) and/or end (stop) genomic loci/positions of sequence reads mapped to a reference sequence, the start and end genomic positions of sequence reads mapped to a reference sequence, a subsequence of the sequence reads at one or both ends, the length of the sequence reads, and/or the length of the original nucleic acid molecule in the sample) generally allows a unique identity to be assigned to a specific molecule. In some embodiments, the start region includes the first 1, first 2, first 5, first 10, first 15, first 20, first 25, first 30, or at least the first 30 base positions at the 5' end of the sequencing reads aligned to the reference sequence. In some embodiments, the end region includes the last 1, last 2, last 5, last 10, last 15, last 20, last 25, last 30, or at least the last 30 base positions of the 3' end of the sequencing reads aligned to the reference sequence. The length or number of base pairs of individual sequence reads is also optionally used to assign a unique identity to a specific molecule. As described herein, fragments from a single strand of nucleic acid that has been assigned a unique identity can thereby allow subsequent identification of fragments from a parental strand and/or a complementary strand.

In some embodiments, the molecular barcode is introduced with an expected ratio of an identifier group (e.g., a combination of unique or non-unique molecular barcodes) to the molecules in the sample. An exemplary form uses about 2 to about 1,000,000 different molecular barcode sequences, or about 5 to about 150 different molecular barcode sequences, or about 20 to about 50 different molecular barcode sequences connected to both ends of the target molecule. Alternatively, about 25 to about 1,000,000 different molecular barcode sequences can be used. For example, 20-50×20-50 molecular barcode sequences can be used (i.e., one of the 20-50 different molecular barcode sequences can be attached to each end of the target molecule). Such a number of identifiers is generally sufficient to allow different molecules with the same starting and ending points to have a high probability of receiving different identifier combinations (e.g., at least 94%, 99.5%, 99.99% or 99.999%). In some embodiments, about 80%, about 90%, about 95%, or about 99% of the molecules have the same molecular barcode combination.

In some embodiments, the assignment of unique or non-unique molecular barcodes in a reaction is performed using methods and systems described in, for example, U.S. Patent Application Nos. 20010053519, 20030152490, and 20110160078 and U.S. Patent Nos. 6,582,908, 7,537,898, 9,598,731, and 9,902,992, each of which is incorporated herein by reference in its entirety. Alternatively, in some embodiments, only endogenous sequence information (e.g., start and/or end position, subsequences at one or both ends of the sequence, and/or length) can be used to identify different nucleic acid molecules of a sample.

In certain embodiments described herein, it is possible to analyze, for example, before sequencing or labeling and sequencing, to a population of different forms of nucleic acid (for example, hypermethylated DNA and hypomethylated DNA in a sample) physically partitioned. For example, in some embodiments, partitioning includes dividing nucleic acid molecules into partition groups based on different binding affinities of a binding agent that preferentially binds to nucleic acid molecules comprising methylated nucleotides. In some embodiments, the partition group is modified by, for example, digesting at least one subgroup of at least one partition group with MSRE. The method can be used to determine, for example, whether a hypermethylated variable epigenetic target region shows the hypermethylated characteristics of a tumor cell, or whether a hypomethylated variable epigenetic target region shows the hypomethylated characteristics of a tumor cell. In addition, by partitioning a heterogeneous nucleic acid population, rare signals can be increased, for example, by enriching in a rare nucleic acid molecule that is more common in a fraction (or partition) of a population. For example, by partitioning a sample into a hypermethylated nucleic acid molecule and a hypomethylated nucleic acid molecule, it is possible to more easily detect genetic variations that are present in a hypermethylated DNA but are less (or absent) in a hypomethylated DNA. By analyzing more than one fraction of a sample, multidimensional analysis of individual loci of a genome or nucleic acid species can be performed and thus greater sensitivity can be achieved.

In some cases, a heterogeneous nucleic acid sample is partitioned into two or more partitions (e.g., at least 3, 4, 5, 6, or 7 partitions). In some embodiments, each partition is differentially labeled—that is, each partition can have a different set of molecular barcodes. The labeled partitions can then be pooled together for pooled sample preparation and/or sequencing. The partition-labeling-pooling step can occur more than once, with each round of partitioning occurring based on a different feature (examples provided herein) and labeled using a differential label that is distinct from other partitions and partitioning means.

The example of the feature that can be used for partition includes sequence length, methylation level, nucleosome binding, sequence mismatch, immunoprecipitation and/or protein combined with DNA. The partition of gained can include one or more of the following nucleic acid forms: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), shorter DNA fragments and longer DNA fragments. In some embodiments, the partition based on cytosine modification (for example, cytosine methylation) or methylation is usually carried out, and optionally combined with at least one other partitioning step, the step can be based on any aforementioned feature or form of DNA. In some embodiments, heterogeneous nucleic acid population is partitioned into nucleic acids with one or more epigenetic modifications and without the one or more epigenetic modifications. The example of epigenetic modification includes the presence or absence of methylation, methylation level, methylation type (for example, 5-methylcytosine and other types of methylation, such as adenine methylation and/or cytosine hydroxymethylation) and association and association level with one or more proteins (such as histones). Alternatively or additionally, the heterogeneous nucleic acid population can be partitioned into nucleic acid molecules associated with nucleosomes and nucleic acid molecules without nucleosomes. Alternatively or additionally, the heterogeneous nucleic acid population can be partitioned into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Alternatively or additionally, the heterogeneous nucleic acid population can be partitioned based on nucleic acid length (e.g., molecules of maximum 160bp and molecules with a length greater than 160bp).

In some embodiments, the nucleic acid population is partitioned into two or more different partitions. Each partition represents a different nucleic acid form, and the first partition comprises a DNA modified with cytosine having a larger proportion than the second partition. Each partition is labeled differently. The first partition is subjected to a program that differently affects the first nuclear base in the DNA of the first partition and the second nuclear base in the DNA, wherein the first nuclear base is a modified or unmodified nuclear base, the second nuclear base is a modified or unmodified nuclear base different from the first nuclear base, and the first nuclear base and the second nuclear base have the same base pairing specificity. The labeled nucleic acids are brought together and then sequenced. Sequence reads are obtained and analyzed, including distinguishing the first nuclear base and the second nuclear base in the DNA of the first partition by computer (insilico). Labels are used to sort reads from different partitions. Analysis can be performed at the level of each partition and the entire nucleic acid population level to detect genetic variation. For example, analysis can include determining genetic variation with computer analysis, such as CNV, SNV, insertion/deletion, fusion in the nucleic acid of each partition. In some cases, computer analysis can include determining chromatin structure. For example, the coverage of sequence reads can be used to determine the positioning of nucleosomes in chromatin. Higher coverage can be associated with higher nucleosome occupancy in a genomic region, while lower coverage can be associated with lower nucleosome occupancy or nucleosome-depleted regions (NDRs).

The sample may include nucleic acids of various modifications, including post-replicative modifications to the nucleotides and association (usually non-covalently) with one or more proteins.

In embodiments, nucleic acid colony is the nucleic acid colony obtained from serum, plasma or blood sample of the experimenter suspected of suffering from vegetation, tumor or cancer or previously diagnosed as suffering from vegetation, tumor or cancer.Nucleic acid colony includes nucleic acid with different methylation levels.Methylation can be modified after any one or more replication or transcription.Replication and modification include modification of nucleotide cytosine, particularly at the 5-position of nucleobase, such as 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine.

Agents used for partitioning, such as binding agents, can be antibodies with the desired specificity, natural binding partners or variants thereof (Bock et al., Nat Biotech 28:1106-1114 (2010); Song et al., Nat Biotech 29:68-72 (2011)), or artificial peptides with specificity for a given target, for example selected by phage display.

The examples of binding agents contemplated herein include methyl binding domains (MBD) and methyl binding proteins (MBP) as described herein, including proteins such as MeCP2 and antibodies preferentially combined with 5-methylcytosine. When methylated DNA is immunoprecipitated with antibodies, methylated DNA can be recovered in single-stranded form. In such embodiments, the second chain can be synthesized. Then, the partition of high methylation (and optional medium methylation) can be contacted with the MSRE (such as HpaII, BstUI or Hin6I) that does not crack hemimethylated DNA but cracks unmethylated DNA. Alternatively or additionally, the partition of low methylation (and optionally medium methylation) can then be contacted with the MSRE that cracks hemimethylated DNA but does not crack unmethylated DNA.

Likewise, partitioning of different forms of nucleic acids can be performed using histone binding proteins that can separate nucleic acids bound to histones from free or unbound nucleic acids.Examples of histone binding proteins that can be used in the methods disclosed herein include RBBP4, RbAp48, and SANT domain peptides.

For some binding agents and some nucleic acid modifications, although the combination with the agent can depend on whether the nucleic acid is modified and occurs in a substantially all-or-nothing manner, separation can be to a certain degree. In such a case, compared with the underrepresented nucleic acid of the modification, the overrepresented nucleic acid and the agent are combined with the agent to a greater extent. Alternatively, the nucleic acid with the modification can be combined in an all-or-nothing manner. But then, the modification of various levels can be eluted from the binding agent sequence.

For example, in some embodiments, partitioning can be binary or based on the degree/level of modification. For example, methyl binding domain protein (such as MethylMiner methylated DNA enrichment kit (ThermoFisherScientific)) can be used to partition all methylated fragments with unmethylated fragments. Subsequently, other partitioning can include eluting fragments with different methylation levels by adjusting the salt concentration of the solution containing the methyl binding domain and the binding fragment. As the salt concentration increases, the fragment with a larger methylation level is eluted.

In some cases, the final partition represents nucleic acids with varying degrees of modification (overrepresentation or underrepresentation of modification). Overrepresentation and underrepresentation can be defined by the median of the modification of each chain in the population by the number of modifications carried by nucleic acid. For example, if the median of 5-methylcytosine residues in the nucleic acid in the sample is 2, then the modification of the nucleic acid comprising more than two 5-methylcytosine residues is overrepresentation, while the nucleic acid with 1 or 0 5-methylcytosine residues is underrepresentation. The effect of affinity separation is to enrich the nucleic acid molecules that are overrepresented in the binding phase and the nucleic acid molecules that are not fully represented in the non-binding phase (that is, in the solution). The nucleic acid molecules in the binding phase can be eluted before subsequent treatment.

When using MethylMiner methylated DNA enrichment kit (ThermoFisher Scientific), sequential elution can be used to contain the DNA partitions of different methylation levels. For example, low methylation partitions (no methylation) can be separated from methylation partitions by making nucleic acid colonies contact with the MBD attached to magnetic beads from the test kit. Pearl is used to separate methylated nucleic acids from unmethylated nucleic acids. Subsequently, one or more elution steps are sequentially carried out to elute nucleic acids with different methylation levels. For example, the first group of methylated nucleic acids can be eluted at a salt concentration of 160mM or higher, such as at least 150mM, at least 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1000mM or 2000mM. After such methylated nucleic acids are eluted, magnetic separation is used again to separate the nucleic acid of higher methylation level from the nucleic acid with lower methylation level. The elution and magnetic separation steps themselves can be repeated to generate various partitions, such as a hypomethylated partition (representing no methylation), a methylated partition (representing low methylation levels), and a hypermethylated partition (representing high methylation levels).

In some methods, the nucleic acid bound to the agent for partitioning is subjected to a washing step. The washing step washes away nucleic acids weakly bound to the binding agent. Such nucleic acids can be enriched in nucleic acids with a degree of modification close to the mean or median (i.e., the intermediate value between the nucleic acid that remains bound to the solid phase and the nucleic acid that does not bind to the solid phase when the sample and the agent are initially contacted).

Partitioning results in at least two, and sometimes three or more nucleic acid partitions with varying degrees of modification. When the partitions are still separated, the nucleic acids of at least one partition and usually two or three (or more) partitions are connected to nucleic acid tags, which are usually provided as components of adapters, and the nucleic acids in different partitions receive different tags that distinguish members of one partition from members of another partition. The tags connected to the nucleic acid molecules of the same partition can be the same or different from each other. But if they are different from each other, a portion of the tag encoding can be shared, so that the molecules to which they are attached are identified as belonging to a specific partition.

For further details on partitioning nucleic acid samples based on characteristics such as methylation, see WO2018/119452, which is incorporated herein by reference.

In some embodiments, nucleic acid molecules can be partitioned into different partitions based on nucleic acid molecules that bind to a specific protein or fragment thereof and nucleic acid molecules that do not bind to the specific protein or fragment thereof.

Nucleic acid molecules can be partitioned based on DNA-protein binding. Protein-DNA complexes can be partitioned based on specific protein properties. Examples of such properties include various epitopes, modifications (e.g., histone methylation or acetylation) or enzymatic activity. Examples of proteins that can be combined with DNA and used as binding agents for partitioning can include but are not limited to protein A and protein G. Any suitable method can be used for partitioning nucleic acid molecules based on protein binding regions. Examples of methods for partitioning nucleic acid molecules based on protein binding regions include but are not limited to SDS-PAGE, chromatin immunoprecipitation (ChIP), heparin chromatography and asymmetrical field flow fractionation (asymmetrical field flow fractionation, AF4).

Typically, elution varies with the number of methylation sites per nucleic acid molecule, with more methylated molecules eluting at increased salt concentrations. In order to elute DNA into different populations or partitions based on the degree of methylation, one can use a series of elution buffers with increasing NaCl concentrations. Salt concentrations can range from about 100nM to about 2500mM NaCl. In an embodiment, the process results in three (3) partitions. The molecule is contacted with a solution of a first salt concentration, and the solution comprises a molecule containing a methyl binding domain, which can be attached to a capture portion such as streptavidin. At a first salt concentration, a population of molecules will bind to MBD, and a population will remain unbound. Unbound populations can be separated into "hypomethylated" populations. For example, the first partition representing a low-methylated form of DNA is a partition that remains unbound at a low salt concentration (e.g., 100mM or 160mM). The second partition representing a medium methylated DNA is eluted using a medium salt concentration (e.g., a concentration between 100mM and 2000mM). This is also separated from the sample. The third partition, representing the hypermethylated form of DNA, is eluted using a high salt concentration (eg, at least about 2000 mM).

Partitioning program can cause incomplete sorting of DNA molecules in obtained partitions or fractions.For example, a small part of molecules in the low methylation partition may be highly modified (for example, high methylation), and/or a small part of molecules in the high methylation partition may be unmodified or substantially unmodified (for example, unmethylated or substantially unmethylated).Such molecules are considered to be non-specific partitions.Methods described herein include the step of reducing the technical noise of the DNA from non-specific partitions, for example, by making the DNA of non-specific partitions degraded and/or by converting some bases, so that the DNA of non-specific partitions can be identified after sequencing.Therefore, methods described herein can provide the sensitivity of improvement and/or simplified analysis.

In some cases, each partition group (representing a different nucleic acid form) is differentially tagged with a molecular barcode, and the partition groups are pooled together and then sequenced. In other cases, different forms are sequenced separately.

In some embodiments, nucleic acid molecules (from a polynucleotide sample, e.g., after partitioning) can be tagged with sample indices and/or molecular barcodes (commonly referred to as "tags"). Tags can be used to mark nucleic acids in a partition so that a tag (or more than one tag) is associated with a specific partition. Alternatively, tags can be used in embodiments of the present invention that do not employ a partitioning step. A tag or index can be a molecule, such as a nucleic acid, containing information that indicates the characteristics of the molecule associated with the tag. For example, a molecule can carry a sample tag or sample index (which distinguishes molecules in one sample from molecules in different samples), a partition tag (which distinguishes molecules in one partition from molecules in different partitions), or a molecular tag/molecular barcode/barcode (which distinguishes different molecules from each other (in the case of both unique and non-unique tagging)). In certain embodiments, a tag can include a combination of one barcode or more barcodes. In some embodiments, a barcode has, for example, between 10 and 100 nucleotides. As desired for a particular purpose, a collection of barcodes can have a degenerate sequence or can have a sequence with a certain Hamming distance. Thus, for example, a molecular barcode may comprise one barcode or a combination of two barcodes, each barcode attached to a different end of the molecule. Additionally or alternatively, for different partitions and/or samples, different sets of molecular barcodes, molecular tags, or molecular indices may be used, such that the barcodes are used as molecular tags by their individual sequences and also used to identify the partitions and/or samples to which they correspond based on the group of which they are members.

The labeling strategy can be divided into a unique labeling strategy and a non-unique labeling strategy. In unique labeling, all or substantially all molecules in the sample carry different labels, so that the reads can be assigned to the original molecules based on the separate label information. The labels used in such methods are sometimes referred to as "unique labels". In non-unique labeling, different molecules in the same sample can carry the same label, so that other information besides the label information is used to assign sequence reads to the original molecules. Such information may include start and end coordinates, coordinates to which the molecules are mapped, separate start or end coordinates, etc. The labels used in such methods are sometimes referred to as "non-unique labels". Therefore, it is not necessary to uniquely label each molecule in the sample. It is sufficient to uniquely label the molecules in the sample that fall into identifiable categories. Therefore, molecules in different identifiable families can carry the same label without losing information about the identity of the labeled molecules.

In certain embodiments of adding non-unique tags, the number of different tags used can be enough to make all molecules of a particular group with different tags very high (e.g., at least 99%, at least 99.9%, at least 99.99% or at least 99.999%). It should be noted that when barcodes are used as tags, and when barcodes are, for example, randomly attached to the two ends of a molecule, the combination of barcodes together can constitute tags. With regard to this number, it is a function of the number of molecules that fall into the determination. For example, a category can be all molecules mapped to the same start-stop position on a reference genome. A category can be all molecules mapped across a specific genetic locus, for example, a specific base or a specific region (e.g., up to 100 bases or genes or gene exons). In some embodiments, the number of different tags used to uniquely identify the number of molecules in a class (z) can be between any of 2*z, 3*z, 4*z, 5*z, 6*z, 7*z, 8*z, 9*z, 10*z, 11*z, 12*z, 13*z, 14*z, 15*z, 16*z, 17*z, 18*z, 19*z, 20*z, or 100*z (e.g., a lower limit) and any of 100,000*z, 10,000*z, 1000*z, or 100*z (e.g., an upper limit).

For example, in a sample of about 5 ng to 30 ng of cell-free DNA, one would expect about 3000 molecules to map to a particular nucleotide coordinate, and between about 3 and 10 molecules with any starting coordinate to share the same ending coordinate. Thus, about 50 to about 50,000 different tags (e.g., between about 6 and 220 barcode combinations) would be sufficient to uniquely tag all such molecules. To uniquely tag all 3000 molecules mapped across one nucleotide coordinate, about 1 million to about 20 million different tags would be required.

Typically, the assignment of unique or non-unique tag barcodes in a reaction follows the methods and systems described in US Patent Applications 20010053519, 20030152490, 20110160078 and US Patents 6,582,908, 7,537,898 and 9,598,731. Tags can be randomly or non-randomly attached to sample nucleic acids.

In some embodiments, the tagged nucleic acids are sequenced after loading into a microwell plate. The microwell plate can have 96, 384, or 1536 microwells. In some cases, they are introduced with an expected ratio of unique tags to microwells. For example, unique tags can be loaded so that more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000, or 1,000,000,000 unique tags are loaded per genome sample. In some cases, unique tags can be loaded such that less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000, or 1,000,000,000 unique tags are loaded per genomic sample. In some cases, the average number of unique tags loaded per sample genome is less than or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000, or 1,000,000,000 unique tags per genomic sample.

A preferred format uses 20-50 different tags (e.g., barcodes) attached to both ends of the target nucleic acid. For example, 35 different tags (e.g., barcodes) are attached to both ends of the target molecule, resulting in 35×35 arrangements, which is equivalent to 1225 arrangements for 35 tags. The number of such tags is sufficient to make different molecules with the same start and end points have a high probability of receiving different tag combinations (e.g., at least 94%, 99.5%, 99.99%, 99.999%). Other barcode combinations include any number between 10 and 500, for example, about 15x15, about 35x35, about 75x75, about 100x100, about 250x250, about 500x500.

In some cases, the unique tag can have a predetermined sequence or a random sequence or a semi-random sequence. In other cases, more than one barcode can be used so that the barcodes do not have to be unique relative to each other in the more than one barcode. In this example, the barcode can be connected to an individual nucleic acid molecule so that the combination of the barcode and the sequence that can be connected thereto produces a unique sequence that can be traced back individually. As described herein, the detection of non-unique barcodes combined with the sequence data at the beginning (start) and end (termination) parts of the sequence reads can allow a unique identity to be assigned to a specific molecule. The length of the individual sequence reads or the number of base pairs can also be used to assign a unique identity to such a molecule. As described herein, the fragments from the nucleic acid single strands to which a unique identity has been assigned can thereby allow subsequent identification of the fragments from the parental chain.

In some embodiments, adapters (e.g., adapters comprising tags) are added to nucleic acids after nucleic acids are partitioned, and in other embodiments, adapters can be added to nucleic acids before nucleic acids are partitioned. In some such methods, nucleic acid populations with different degrees of modification (e.g., each nucleic acid molecule has 0, 1, 2, 3, 4, 5 or more methyl groups) are contacted with adapters, and then the population is partitioned according to the degree of modification. Adapters are attached to one or both ends of nucleic acid molecules in the population. In some embodiments, adapters include a sufficient number of different tags so that the number of tag combinations results in a high probability of two nucleic acids with the same starting point and end point receiving different tag combinations, such as 95%, 99% or 99.9%. Regardless of whether adapters with the same or different tags can include the same or different primer binding sites, but preferably adapters include the same primer binding sites. In some embodiments, after partitioning, nucleic acids are amplified by primers that bind to primer binding sites in adapters. After amplification, different partitions can then undergo further processing steps in parallel but separately, which steps can include further amplification (e.g., clonal amplification) and sequence analysis. Sequence data from different partitions can then be compared.

In some embodiments, a single label can be used to mark a specific partition. In some embodiments, more than one different label can be used to mark a specific partition group. In the embodiment of using more than one different label to mark a specific partition, the label group for marking a partition can be easily distinguished from the label group for marking other partitions. In some embodiments, the label can be multifunctional-that is, it can be used as a molecule identifier (that is, a molecular barcode), a partition identifier (that is, a partition label) and a sample identifier (that is, a sample index) at the same time. For example, if there are four DNA samples, and each DNA sample is partitioned into three partitions, then the DNA molecules in each partition in twelve partitions (that is, four DNA samples have twelve partitions in total) can be labeled with a separate label group, so that the label sequence attached to the DNA molecule reveals the identity of the DNA molecule, the partition to which the DNA molecule belongs, and the sample from which the DNA molecule is derived. In some embodiments, the label can be used as both a molecular barcode and a partition label. For example, if the DNA sample is partitioned into three partitions, then the DNA molecules in each partition are labeled with a separate label group, so that the label sequence attached to the DNA molecule reveals the identity of the DNA molecule and the partition to which the DNA molecule belongs. In some embodiments, the label can be used as both a molecular barcode and a sample index. For example, if there are four DNA samples, the DNA molecules in each sample will be labeled with a separate set of labels that can distinguish each sample, so that the label sequence attached to the DNA molecule is used as a molecular identifier and a sample identifier.

In some embodiments, the tag can have additional functions, such as the tag can be used to index the sample source or used as a unique molecular identifier (which can be used to improve the quality of sequencing data by distinguishing sequencing errors from mutations, for example, as in Kinde et al., Proc Nat'l Acad Sci USA 108:9530-9535 (2011); Kou et al., PLoS ONE, 11:e0146638 (2016)) or as a non-unique molecular identifier, for example, as described in U.S. Patent No. 9,598,731. Similarly, in some embodiments, the tag can have additional functions, such as the tag can be used to index the sample source or used as a non-unique molecular identifier (which can be used to improve the quality of sequencing data by distinguishing sequencing errors from mutations).

In one embodiment, labeling the partitions includes labeling the molecules in each partition with a partition label. After the partitions are reassembled (e.g., to reduce the number of sequencing runs required and avoid unnecessary costs) and the molecules are sequenced, the partition label identifies the source partition. In another embodiment, different partitions are labeled with different sets of molecular labels (e.g., comprising a pair of barcodes). In this way, each molecular barcode indicates the source partition and can be used to distinguish molecules within the partition. For example, a first set of 35 barcodes can be used to label molecules in a first partition, while a second set of 35 barcodes can be used to label molecules in a second partition.

In some embodiments, after partitioning and labeling with partition labels, the molecules can be pooled for sequencing in a single run. In some embodiments, sample labels are added to the molecules in a step, such as after adding partition labels and pooling. Sample labels can help pool material generated from more than one sample for sequencing in a single sequencing run.

Optionally, in some embodiments, partition labels can be associated with samples and partitions. As a simple example, a first label can indicate a first partition of a first sample; a second label can indicate a second partition of the first sample; a third label can indicate a first partition of a second sample; and a fourth label can indicate a second partition of the second sample.

Although tags can be attached to molecules that have been partitioned based on one or more epigenetic features, the final tagged molecules in the library may no longer have that epigenetic feature. For example, although single-stranded DNA molecules may be partitioned and tagged, the final tagged molecules in the library may be double-stranded. Similarly, although DNA can undergo partitioning based on different methylation levels, the tagged molecules derived from these molecules in the final library may be unmethylated. Therefore, the tags attached to the molecules in the library generally indicate the characteristics of the "parent molecule" from which the final tagged molecules are derived, and not necessarily the characteristics of the tagged molecules themselves.

As an example, barcodes 1, 2, 3, 4, etc. are used to tag and label the molecules in the first partition; barcodes A, B, C, D, etc. are used to tag and label the molecules in the second partition; and barcodes a, b, c, d, etc. are used to tag and label the molecules in the third partition. The differentially tagged partitions can be pooled and then sequenced. The differentially tagged partitions can be sequenced individually or together simultaneously, for example, in the same flow cell of an Illumina sequencer.

After sequencing, the reads can be analyzed at the level of each partition and the entire nucleic acid population level to detect genetic variation. Labels are used to sort the reads from different partitions. Analysis can include using sequence information, genome coordinate length, coverage and/or copy number to determine genetic and epigenetic variation (methylation, chromatin structure, etc., one or more). In some embodiments, higher coverage can be associated with higher nucleosome occupancy in a genomic region, while lower coverage can be associated with lower nucleosome occupancy or nucleosome loss region (NDR).

C. Digestion of nucleic acid molecules with restriction endonucleases

In some embodiments, the partition or partition group is digested by contacting the partition or partition group (e.g., a first, second, or third partition group prepared by partitioning the sample as described herein, such as based on the level of cytosine modification, such as methylation, e.g., 5-methylation) with a methylation-sensitive restriction endonuclease (MSRE). In some embodiments of partitioning based on cytosine modification, the first partition is the partition with a higher modification level; the second partition is the partition with a lower modification level; and, when present, the third partition has a modification level between the first and second partitions.

As discussed above, the partitioning procedure can result in the incomplete sorting of DNA molecules in the partition.MSRE can be selected to degrade the DNA of non-specific partitions.For example, the second partition can contact with the MSRE that selectively digests the methylated nucleic acid molecule.This can make the DNA (for example, methylated DNA) of the non-specific partition in the second partition degraded, to produce the processed second partition.Alternatively or additionally, the first partition can contact with the MSRE that selectively digests the unmethylated nucleic acid molecule, thereby degrade the DNA of the non-specific partition in the first partition, to produce the processed first partition.Degrading the DNA of the non-specific partition in one or both of the first or second partitions is proposed as an improvement in the performance of the method for the accurate partitioning of the DNA modified based on cytosine, for example, the presence of the DNA of abnormal modification in the detection sample, determining the tissue of the DNA source, and/or determining whether the experimenter suffers from cancer.For example, such degradation can provide the sensitivity of improvement and/or simplify downstream analysis.

When making partition and nuclease such as MSRE contact, one or more nucleases can be used.In some embodiments, partition is contacted with more than one nuclease.Partition can be contacted with nuclease sequentially or simultaneously.When nuclease has activity under similar conditions (such as buffer composition), it may be advantageous to use nuclease simultaneously to avoid unnecessary sample operation.Contacting the second partition with more than one MSRE can more completely degrade the high methylated DNA of non-specific partition.Similarly, contacting the first partition with more than one MSRE can more completely degrade the low methylated and/or unmethylated DNA of non-specific partition.

In some embodiments, the MSRE that selectively digests the methylated nucleic acid molecule includes one or more of MspJI, LpnPI, FspEI or McrBC. In some embodiments, at least two MSREs that selectively digest the methylated nucleic acid molecule are used. In some embodiments, at least three MSREs that selectively digest the methylated nucleic acid molecule are used.

In some embodiments, the MSRE that selectively digests unmethylated nucleic acid molecules includes one or more of the following: AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI, Cfr10I, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, MspI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI and SnaBI. In some embodiments, at least two MSREs that selectively digest unmethylated nucleic acid molecules are used. In some embodiments, at least three MSREs that selectively digest unmethylated nucleic acid molecules are used. In some embodiments, MSRE includes BstUI and HpaII. In some embodiments, two kinds of MSREs include HhaI and AccII. In some embodiments, the MSER includes BstUI, HpaII, and Hin6I.

In some embodiments, after the step of tagging or attaching adapters to both ends of the DNA, the partition is contacted with a nuclease as described above. The tag or adapter can be resistant to cleavage by the nuclease using any of the methods described above. In this method, cleavage can prevent the analysis of molecules of non-specific partitions because the cleavage products lack tags or adapters at both ends.

Alternatively, the step of labeling or attaching adapters can be performed after digestion with nucleases as described above. Then, the cleaved molecules can be identified in the sequence reads based on the ends corresponding to the nuclease recognition sites (the points to which the tags or adapters are attached). Treating molecules in this way can also allow information to be obtained from the cleaved molecules, for example, observing somatic mutations. When labeling or attaching adapters after the partitions are contacted with nucleases, and analyzing low molecular weight DNA such as cfDNA, it may be desirable to remove high molecular weight DNA (such as contaminated genomic DNA) from the sample before the contact step. It may also be desirable to use nucleases that can be heat-inactivated at relatively low temperatures (e.g., 65°C or lower, or 60°C or lower) to avoid DNA denaturation, because denaturation can interfere with subsequent connection steps.

In the case where the sample is partitioned into three partitions, including a third partition comprising a medium methylated molecule, in some embodiments, the third partition is contacted with an MSRE (e.g., an MSRE that selectively digests unmethylated nucleic acid molecules). Such a step may have any of the features described elsewhere herein regarding the contact step, and may be performed before or after the step of labeling or attaching an adapter as discussed above. In some embodiments, the first and third partitions are combined before contacting with the MSRE. Such a step may have any of the features described elsewhere herein regarding the contact step, and may be performed before or after the step of labeling or attaching an adapter as discussed above. In some embodiments, the first and third partitions are differentially labeled before combination.

In some embodiments, when the sample is partitioned into three partitions, including the third partition comprising medium methylated molecules, in some embodiments, the third partition is contacted with an MSRE that selectively digests methylated nucleic acid molecules. Such a step can have any of the features described elsewhere herein about the contact step, and can be performed before or after the step of labeling or attaching adapters as discussed above. In some embodiments, before contacting with the MSRE, the second and third partitions are combined. Such a step can have any of the features described elsewhere herein about the contact step, and can be performed before or after the step of labeling or attaching adapters as discussed above. In some embodiments, the second and third partitions are labeled for differences before combination.

In some embodiments, after contacting with the nuclease, the DNA is purified, for example, using SPRI beads. Such purification can occur after heat inactivation of the nuclease. Alternatively, purification can be omitted; thus, for example, the partition containing the heat-inactivated nuclease can be subjected to subsequent steps such as amplification. In another embodiment, the contacting step can be performed in the presence of a purification reagent such as SPRI beads, for example, in order to minimize losses associated with tube transfer. After lysis and heat inactivation, the SPRI beads can be reused for cleanup by adding molecular crowding reagents (e.g., PEG) and salts.

D. Amplification

The sample nucleic acid can be flanked by adapters and amplified by PCR and other amplification methods using nucleic acid primers that bind to primer binding sites in the adapters flanking the DNA molecule to be amplified. In some embodiments, the amplification method includes cycles of extension, denaturation, and annealing generated by thermal cycling, or can be isothermal, for example, in transcription-mediated amplification. Other examples of amplification methods that can optionally be utilized include ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustained sequence-based replication.

Typically, the amplification reaction generates more than one non-uniquely or uniquely tagged nucleic acid amplicon with a molecular barcode and sample index ranging in size from about 150 nucleotides (nt) to about 700 nt, 250 nt to about 350 nt, or about 320 nt to about 550 nt. In some embodiments, the amplicon has a size of about 180 nt. In some embodiments, the amplicon has a size of about 200 nt.

In some embodiments, the method comprises ligating dsDNA with T-tail and C-tail adapters prior to ligation to adapters, which results in amplification of at least 50%, 60%, 70% or 80% of double-stranded nucleic acids. Preferably, the method increases the amount or number of amplified molecules by at least 10%, 15% or 20% relative to a control method performed with T-tail adapters alone.

In some embodiments, nucleic acid molecules digested by MSRE are not amplified.In some such embodiments, substantially all nucleic acid molecules in the sample are amplified except nucleic acid molecules digested by MSRE.

E. Enrichment/Capture

In some embodiments, the methods disclosed herein include capturing or enriching one or more target areas of nucleic acid molecules. Any suitable method known in the art can be used to capture. In some embodiments, capturing includes contacting the DNA to be captured with a target-specific probe group, such as a probe described herein. One or more partitions prepared in the method process disclosed herein can be captured. In some embodiments, DNA is captured from at least the first partition or the second partition, such as at least the first partition and the second partition. Any one, any two or all subgroups of a partition or a partition group can be captured. In some embodiments, the partition differences are labeled (e.g., as described herein), and then collected before capturing.

The capture step can be performed using conditions suitable for specific nucleic acid hybridization, which conditions generally depend to some extent on the characteristics of the probe, such as length, base composition, etc. In view of the general knowledge of nucleic acid hybridization in the art, those skilled in the art will be familiar with appropriate conditions. In some embodiments, a complex of a target-specific probe and DNA is formed.

In some embodiments, the methods described herein include capturing more than one target group of cfDNA obtained from a test subject. The target area includes an epigenetic target area, which can show differences in methylation levels and/or fragmentation patterns, depending on whether they are derived from tumor cells or from healthy cells. The target area also includes a sequence variable target area, which can show sequence differences, depending on whether they are derived from tumor cells or from healthy cells. The capture step produces a capture group of cfDNA molecules. In some embodiments, in the capture group of cfDNA molecules, the cfDNA molecules corresponding to the sequence variable target area group are captured with a greater capture yield than the cfDNA molecules corresponding to the epigenetic target area group. For further discussion of capture steps, capture yields, and related aspects, see WO2020/160414, which is incorporated herein by reference for all purposes.

In some embodiments, the methods described herein include contacting cfDNA obtained from a test subject with a target-specific probe set, wherein the target-specific probe set is configured to capture cfDNA corresponding to a set of sequence variable target regions with a greater capture yield than cfDNA corresponding to a set of epigenetic target regions.

It is beneficial to capture cfDNA corresponding to a set of sequence variable target regions at a greater capture yield than cfDNA corresponding to a set of epigenetic target regions because a greater sequencing depth may be required to analyze sequence variable target regions with sufficient confidence or accuracy than an epigenetic target region. The amount of data required to determine fragmentation patterns (e.g., to test perturbations of transcription start sites or CTCF binding sites) or fragment abundance (e.g., in hypermethylated and hypomethylated partitions) is typically less than the amount of data required to determine the presence or absence of cancer-associated sequence mutations. Capturing target region sets at different yields can facilitate sequencing target regions to different sequencing depths in the same sequencing run (e.g., using pooled mixtures and/or in the same sequencing pool).

In various embodiments, the method further comprises sequencing the captured cfDNA to, for example, varying degrees of sequencing depth for a set of epigenetic target regions and a set of sequence variable target regions, consistent with discussion herein.

In some embodiments, the complex of the target-specific probe and DNA is separated from DNA that is not bound to the target-specific probe. For example, where the target-specific probe is covalently or non-covalently bound to a solid support, a washing or aspiration step can be used to separate the unbound material. Alternatively, where the complex has chromatographic properties different from those of the unbound material (e.g., where the probe comprises a ligand that binds to a chromatographic resin), chromatography can be used.

As discussed in detail elsewhere herein, target-specific probe groups can include more than one group, such as probes for a sequence variable target group and probes for an epigenetic target group. In some such embodiments, a capture step is performed using probes for a sequence variable target and probes for an epigenetic target in the same container, for example, probes for a sequence variable target group and an epigenetic target group are in the same composition. This method provides a relatively more efficient workflow. In some embodiments, the concentration of probes for a sequence variable target group is greater than the concentration of probes for an epigenetic target group.

Optionally, the capture step is performed in the first container with a sequence variable target probe set and in the second container with an epigenetic target probe set, or the contacting step is performed at a first time and in the first container with a sequence variable target probe set and at a second time before or after the first time with an epigenetic target probe set. The method allows the preparation of separate first and second compositions, which contain captured DNA corresponding to the sequence variable target set and captured DNA corresponding to the epigenetic target set. The compositions can be processed separately as desired (e.g., graded based on methylation, as described elsewhere herein), and recombined in appropriate proportions to provide materials for further processing and analysis such as sequencing.

In some embodiments, the DNA is amplified. In some embodiments, the amplification is performed before the capture step. In some embodiments, the amplification is performed after the capture step.

In some embodiments, the DNA includes an adapter. This can be performed simultaneously with the amplification procedure, for example, by providing an adapter in the 5' portion of the primer, for example, as described above. Alternatively, the adapter can be added by other methods such as ligation.

In some embodiments, a label is included in the DNA (e.g., an adapter added to the DNA), which can be a barcode or include a barcode. The label can help identify the source of the nucleic acid. For example, a barcode can be used to allow the source of the DNA to be identified after more than one sample is collected for parallel sequencing, e.g., a subject. This can be performed simultaneously with the amplification procedure, e.g., by providing a barcode in the 5' portion of the primer, e.g., as described above. In some embodiments, the adapter and label/barcode are provided by the same primer or primer set. For example, the barcode can be located at the 3' of the adapter and the 5' of the target hybridization portion of the primer. Alternatively, the barcode can be added by other methods, such as ligation, optionally in the same ligation substrate together with the adapter.

Additional details regarding amplification, labeling, and barcoding are discussed elsewhere herein, which details may be combined, to the extent practicable, with any of the embodiments set forth herein.

In some embodiments, before nucleic acid sequencing, enrichment sequence.Can optionally enrich specific target area, or can non-specifically enrich (" target sequence ").In some embodiments, can use difference tiling (tiling) and capture scheme, with nucleic acid capture probe (" bait ") (such as the target probe group selected for one or more bait set panels) enrichment/capture of interested target region.Different tiling and capture scheme usually use bait set of different relative concentrations to spread in the genomic region related to bait (for example, with different " resolutions "), subject to a group of restrictions (for example, sequencer restrictions, such as sequencing loading, the effectiveness of every kind of bait, etc.), and capture target nucleic acid with the level required for downstream sequencing.These target genomic regions of interest optionally include natural nucleotide sequence or the synthetic nucleotide sequence of nucleic acid construct.In some embodiments, the biotin-labeled pearl with probe for one or more regions of interest can be used to capture the target sequence, and optionally amplify these regions subsequently, to enrich the region of interest. In some embodiments, the nucleic acid capture probe can be a single-stranded RNA or a double-stranded DNA molecule.

Sequence capture generally includes the use of oligonucleotide probes hybridized with the target nucleic acid sequence. In some embodiments, the probe set strategy includes tiling the probe in the region of interest. The length of such probe can be, for example, about 60 to about 120 nucleotides. The group can have a depth (for example, coverage depth) of about 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, 20X, 50X or more than 50X. The effectiveness of sequence capture generally depends in part on the length of the sequence complementary (or almost complementary) to the probe sequence in the target molecule.

In some embodiments, the first target group is captured from the first partition, including at least an epigenetic target area. The epigenetic target area captured from the first partition may include a hypermethylated variable target area. In some embodiments, the hypermethylated variable target area is a CpG-containing region that is unmethylated or has low methylation (e.g., methylation below average relative to a large amount of cfDNA (bulk cfDNA)) in cfDNA from healthy subjects. In some embodiments, the hypermethylated variable target area is a region that shows low methylation in healthy cfDNA than in at least one other tissue type. Without wishing to be bound by any particular theory, cancer cells can shed more DNA into the bloodstream than healthy cells of the same tissue type. Therefore, the distribution of cfDNA-derived tissues can change when cancerous. Therefore, the increase in the level of hypermethylated variable target areas in the first partition can be an indicator of cancer presence (or recurrence, depending on the medical history of the subject).

In some embodiments, the second target group is captured from the second partition, including at least an epigenetic target area. The epigenetic target area may include a low methylation variable target area. In some embodiments, the low methylation variable target area is a CpG-containing area that is methylated in cfDNA from healthy subjects or has high methylation (e.g., methylation higher than average relative to a large amount of cfDNA). In some embodiments, the low methylation variable target area is a region that shows high methylation in healthy cfDNA than in at least one other tissue type. Without wishing to be bound by any particular theory, cancer cells can shed more DNA into the bloodstream than healthy cells of the same tissue type. Therefore, the distribution of cfDNA source tissues can change when cancerous. Therefore, the increase in the level of low methylation variable target areas in the second partition can be an indicator of cancer presence (or recurrence, depending on the medical history of the subject).

In some embodiments, the enriched DNA molecules (or capture groups) may include DNA corresponding to sequence variable target groups and epigenetic target groups. In some embodiments, when normalized for differences in the size (footprint size) of the target area, the amount of captured sequence variable target area DNA is greater than the amount of captured epigenetic target area DNA. In some embodiments, compositions, methods, and systems are described in PCT patent application No. PCT/US2020/016120, which is hereby incorporated by reference in its entirety.

Alternatively, a first capture group and a second capture group may be provided that include DNA corresponding to a set of sequence variable target regions and DNA corresponding to a set of epigenetic target regions, respectively. The first capture group and the second capture group may be combined to provide a combined capture group.

In capture sets that include DNA corresponding to a set of sequence variable target regions and a set of epigenetic target regions (including combined capture sets as discussed above), the DNA corresponding to the set of sequence variable target regions can be present at a greater concentration (e.g., 1.1-fold to 1.2-fold greater concentration, 1.2-fold to 1.4-fold greater concentration, 1.4-fold to 1.6-fold greater concentration, 1.6-fold to 1.8-fold greater concentration, 1.8-fold to 2.0-fold greater concentration, 2.0-fold greater concentration) than the DNA corresponding to the set of epigenetic target regions. .0 times to 2.2 times greater concentration, 2.2 times to 2.4 times greater concentration, 2.4 times to 2.6 times greater concentration, 2.6 times to 2.8 times greater concentration, 2.8 times to 3.0 times greater concentration, 3.0 times to 3.5 times greater concentration, 3.5 times to 4.0, 4.0 times to 4.5 times greater concentration, 4.5 times to 5.0 times greater concentration, 5.0 times to 5.5 times greater concentration, 5.5 times to 6.0 times greater concentration, 6.0 times to 6.5 times greater concentration concentration, 6.5 times to 7.0 times greater, 7.0 times to 7.5 times greater, 7.5 times to 8.0 times greater, 8.0 times to 8.5 times greater, 8.5 times to 9.0 times greater, 9.0 times to 9.5 times greater, 9.5 times to 10.0 times greater, 10 times to 11 times greater, 11 times to 12 times greater, 12 times to 13 times greater, 13 times to 14 times greater, 14 times to 15 times greater, The concentration difference is calculated normalized to the target footprint size, as discussed in the definitions section.

a. Epigenetic target group

The epigenetic target group may include one or more types of target areas that can distinguish DNA from neoplastic (e.g., tumor or cancer) cells from DNA from healthy cells (e.g., non-neoplastic circulating cells). Exemplary types of such regions are discussed in detail herein. In some embodiments, the method according to the present disclosure includes determining whether the cfDNA molecule corresponding to the epigenetic target group includes or indicates cancer-related epigenetic modifications (e.g., hypermethylation in one or more hypermethylated variable target areas; one or more perturbations of CTCF binding; and/or one or more perturbations of transcription start sites) and/or copy number variations (e.g., focused amplification). The epigenetic target group may also include one or more control areas, for example, as described herein.

In some embodiments, the set of epigenetic target regions has a footprint of at least 100 kbp (e.g., at least 200 kbp, at least 300 kbp, or at least 400 kbp). In some embodiments, the set of epigenetic target regions has a footprint in the range of 100 kbp-20 Mbp (e.g., 100-200 kbp, 200-300 kbp, 300-400 kbp, 400-500 kbp, 500-600 kbp, 600-700 kbp, 700-800 kbp, 800-900 kbp, 900-1,000 kbp, 1-1.5 Mbp, 1.5-2 Mbp, 2-3 Mbp, 3-4 Mbp, 4-5 Mbp, 5-6 Mbp, 6-7 Mbp, 7-8 Mbp, 8-9 Mbp, 9-10 Mbp, or 10-20 Mbp). In some embodiments, the set of epigenetic target regions has a footprint of at least 20 Mbp.

i. Hypermethylated variable target regions

In some embodiments, the epigenetic target group includes one or more hypermethylated variable target regions. Generally, a hypermethylated variable target region refers to a region in which the increase in the methylation level observed indicates that the sample (e.g., cfDNA) contains DNA produced by neoplastic cells (such as tumor cells or cancer cells) increases in likelihood. For example, hypermethylation of tumor suppressor gene promoters has been repeatedly observed. See, for example, Kang et al., Genome Biol. 18: 53 (2017) and references cited therein. In another example, as discussed above, a hypermethylated variable target region may include a region in which the methylation of the region in the cancerous tissue is not necessarily different relative to DNA from healthy tissue of the same type, but the methylation of the region is indeed different (e.g., with more methylation) relative to typical cfDNA in healthy subjects.

An extensive discussion of methylated variable targets in colorectal cancer is provided in Lam et al., Biochim Biophys Acta. 1866: 106-20 (2016). These include VIM, SEPT9, ITGA4, OSM4, GATA4, and NDRG4. An exemplary set of hypermethylated variable targets based on colorectal cancer (CRC) studies, including genes or portions thereof, is provided in Table 1. Many of these genes may be associated with cancers other than colorectal cancer; for example, TP53 is widely considered to be a critical tumor suppressor, and hypermethylation-based inactivation of this gene may be a common oncogenic mechanism.

Table 1. Exemplary hypermethylated target regions (genes or parts thereof) based on CRC studies.

In some embodiments, the hypermethylated variable target region includes more than one gene listed in Table 1 or a portion thereof, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the genes listed in Table 1 or a portion thereof. For example, for each locus included as a target region, there can be one or more probes having a hybridization site that binds between the transcription start site and the stop codon (the last stop codon for alternatively spliced genes) of the gene. In some embodiments, one or more probes bind within 300 bp upstream and/or downstream of the genes listed in Table 1 or a portion thereof (e.g., within 200 bp or 100 bp).

Methylation variable targets in various types of lung cancer are discussed in detail in, for example, Ooki et al., Clin. Cancer Res. 23:7141-52 (2017); Belinksy, Annu. Rev. Physiol. 77:453-74 (2015); Hulbert et al., Clin. Cancer Res. 23:1998-2005 (2017); Shi et al., BMC Genomics 18:901 (2017); Schneider et al., BMC Cancer. 11:102 (2011); Lissa et al., Transl Lung Cancer Res 5(5):492-504 (2016); Skvortsova et al., Br. J. Cancer. 94(10):1492–1495 (2006); Kim et al., Cancer Res. 61:3419–3424 (2001); Furonaka et al., Pathology International 55:303-309 (2005); Gomes et al., Rev. Port. Pneumol. 20:20-30 (2014); Kim et al., Oncogene. 20:1765-70 (2001); Hopkins-Donaldson et al., Cell Death Differ. 10:356-64 (2003); Kikuchi et al., Clin. Cancer Res. 11:2954-61 (2005); Heller et al., Oncogene 25:959–968 (2006); Licchesi et al., Carcinogenesis. 29:895–904 (2008); Guo ...11:295 Res.10:7917-24 (2004); Palmisano et al., Cancer Res.63:4620–4625 (2003); and Toyooka et al., Cancer Res.61:4556–4560, (2001). In an example, a hypermethylated variable target region may include a region where the methylation of the region in the cancerous tissue is not necessarily different relative to DNA from healthy tissue of the same type, but the methylation of the region is indeed different (e.g., has more methylation) relative to typical cfDNA in healthy subjects. For example, when the presence of cancer leads to increased cell death (such as apoptosis of the tissue type corresponding to the cancer), such a hypermethylated variable target region can be used, at least in part, to detect such cancer. In some embodiments, the hypermethylated variable target regions include one or more genomic regions, wherein the methylation status of cfDNA molecules in these regions is not different in cancer subjects relative to cfDNA from healthy subjects, but the presence/increased amount of hypermethylated cfDNA in these regions is indicative of a specific tissue type (e.g., cancer origin) and is present in cfDNA that enters the circulation with increased apoptosis (e.g., tumor shedding).

An exemplary set of hypermethylated variable target regions based on lung cancer research is provided in Table 2, including genes or portions thereof. Many of these genes may be associated with cancers other than lung cancer; for example, Casp8 (caspase 8) is a key enzyme for programmed cell death, and hypermethylation-based inactivation of this gene may be a common carcinogenic mechanism, not limited to lung cancer. In addition, some genes appear in both Table 1 and Table 2, indicating generality.

Table 2. Exemplary hypermethylated target regions (genes or portions thereof) based on lung cancer research.

Any of the foregoing embodiments involving target regions identified in Table 2 can be combined with any of the foregoing embodiments involving target regions identified in Table 1. In some embodiments, the hypermethylated variable target regions include more than one gene listed in Table 1 or Table 2, or portions thereof, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the genes listed in Table 1 or Table 2, or portions thereof.

Additional hypermethylated target areas can be obtained, for example, from the Cancer Genome Atlas. Kang et al., Genome Biology 18: 53 (2017) describes the use of hypermethylated target areas from breast, colon, kidney, liver and lung to construct a probabilistic method called Cancer Locator. In some embodiments, the hypermethylated target area can be specific to one or more types of cancer. Therefore, in some embodiments, the hypermethylated target area includes one, two, three, four or five hypermethylated target area subgroups, which collectively show hypermethylation in one, two, three, four or five types of breast cancer, colon cancer, kidney cancer, liver cancer and lung cancer.

In some embodiments, where different epigenetic target regions are captured from the first and second partitions, the epigenetic target regions captured from the first partition include hypermethylated variable target regions.

ii. Hypomethylated variable target regions

Comprehensive hypomethylation is a phenomenon commonly observed in a variety of cancers. See, for example, Hon et al., Genome Res. 22: 246-258 (2012) (breast cancer); Ehrlich, Epigenomics 1: 239-259 (2009) (review of observations of hypomethylation in colon cancer, ovarian cancer, prostate cancer, leukemia, hepatocellular carcinoma, and cervical cancer). For example, regions that are normally methylated in healthy cells (such as repeat elements (e.g., LINE1 elements, Alu elements, centromere tandem repeats, centromere peri-tandem repeats, and satellite DNA) and intergenic regions) may show reduced methylation in tumor cells. Therefore, in some embodiments, the epigenetic target region group includes a hypomethylated variable target region, wherein the observed reduction in methylation levels indicates that the sample (e.g., cfDNA) contains DNA produced by neoplastic cells (such as tumor cells or cancer cells) increases in likelihood. In an example, a hypomethylated variable target region may include a region where the methylation state of the region in the cancerous tissue is not necessarily different relative to DNA from healthy tissue of the same type, but the methylation of the region is indeed different (e.g., less methylated) relative to typical cfDNA in healthy subjects. For example, when the presence of cancer leads to increased cell death (such as apoptosis of cells corresponding to the tissue type of the cancer), such a hypomethylated variable target region may be used at least in part to detect such cancer. In some embodiments, the hypomethylated variable target region includes one or more genomic regions, wherein the methylation state of cfDNA molecules in these regions in cancer subjects is not different relative to cfDNA from healthy subjects, but the presence/increased amount of hypomethylated cfDNA in these regions indicates a specific tissue type (e.g., cancer origin) and is presented as cfDNA entering the circulation with increased apoptosis (e.g., tumor shedding).

In some embodiments, the hypomethylated variable target region comprises a repetitive element and/or an intergenic region. In some embodiments, the repetitive element comprises one, two, three, four or five of a LINE1 element, an Alu element, a centromeric tandem repeat sequence, a pericentromeric tandem repeat sequence and/or a satellite DNA.

Exemplary specific genomic regions showing cancer-associated hypomethylation include nucleotides 8403565-8953708 and 151104701-151106035 of human chromosome 1, for example according to hg19 or hg38 human genome constructs. In some embodiments, the hypomethylated variable target region overlaps with or includes one or both of these regions.

In some embodiments, wherein different epigenetic target regions are captured from the first and second partitions, the epigenetic target regions captured from the second partition include hypomethylated variable target regions.

iii. CTCF binding region

CTCF is a DNA binding protein that contributes to chromatin organization and often colocalizes with cohesin. Perturbations of CTCF binding sites have been reported in many different cancers. See, for example, Katainen et al., Nature Genetics, doi: 10.1038/ng.3335, published online on June 8, 2015; Guo et al., Nat. Commun. 9: 1520 (2018). CTCF binding results in recognizable patterns in cfDNA, which can be detected by sequencing, such as by fragment length analysis. For example, details about fragment length analysis based on sequencing are provided in Snyder et al., Cell 164: 57-68 (2016); WO 2018/009723 and US20170211143A1, each of which is incorporated herein by reference.

Thus, perturbations in CTCF binding lead to variations in cfDNA fragmentation patterns. CTCF binding sites therefore represent types of variable targets for fragmentation.

There are many known CTCF binding sites. See, for example, CTCFBSDB (CTCF binding site database), available on the Internet at insulatordb.uthsc.edu/; Cuddapah et al., Genome Res.19:24-32 (2009); Martin et al., Nat.Struct.Mol.Biol.18:708-14 (2011); Rhee et al., Cell.147:1408-19 (2011), each of which is incorporated by reference. Exemplary CTCF binding sites are located at nucleotides 56014955-56016161 on chromosome 8 and nucleotides 95359169-95360473 on chromosome 13, for example according to hg19 or hg38 human genome constructs.

Thus, in some embodiments, the set of epigenetic target regions includes CTCF binding regions. In some embodiments, the CTCF binding regions include at least 10, 20, 50, 100, 200, or 500 CTCF binding regions, or 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 CTCF binding regions, for example, such as the CTCF binding regions described above, or the CTCFBSDB or the articles cited above, Cuddapah et al., Martin et al., or Rhee et al. One or more of the CTCF binding regions.

In some embodiments, at least some CTCF sites can be methylated or unmethylated, wherein the methylation state is associated with whether the cell is a cancer cell. In some embodiments, the epigenetic target region group includes at least 100bp, at least 200bp, at least 300bp, at least 400bp, at least 500bp, at least 750bp, at least 1000bp upstream and/or downstream of the CTCF binding site.

iv. Transcription start site

Transcription start sites in neoplastic cells can also show disturbances.For example, the nucleosome organization at each transcription start site in healthy cells of hematopoietic lineages (which has a substantial contribution to cfDNA in healthy individuals) can be different from the nucleosome organization at these transcription start sites in neoplastic cells.This leads to different cfDNA patterns, which can be detected by sequencing, for example, as in Snyder et al., Cell 164:57-68 (2016); generally discussed in WO 2018/009723 and US20170211143A1.In another example, the transcription start site in cancerous tissue is not necessarily different in epigenetics relative to DNA from healthy tissues of the same type, but is indeed different in epigenetics (for example, for nucleosome organization) relative to typical cfDNA in healthy subjects.For example, when the presence of cancer causes cell death (such as apoptosis corresponding to the tissue type of cancer) to increase, such a transcription start site can be used at least in part to detect such cancer.

Therefore, perturbations in the transcription start site also lead to variations in the fragmentation pattern of cfDNA. Therefore, the transcription start site also represents a type of variable target region for fragmentation.

Human transcription start sites are available from DBTSS (Database of Human Transcription Start Sites), available on the internet at dbtss.hgc.jp, and described in Yamashita et al., Nucleic Acids Res. 34(Database Issue):D86-D89 (2006), which is incorporated herein by reference.

Thus, in some embodiments, the epigenetic target region group includes a transcription start site. In some embodiments, the transcription start site includes at least 10, 20, 50, 100, 200, or 500 transcription start sites, or 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 transcription start sites, for example, such as the transcription start sites listed in DBTSS. In some embodiments, at least some of the transcription start sites may be methylated or unmethylated, wherein the methylation state is associated with whether the cell is a cancer cell. In some embodiments, the epigenetic target region group includes at least 100bp, at least 200bp, at least 300bp, at least 400bp, at least 500bp, at least 750bp, at least 1000bp upstream and/or downstream of the transcription start site.

v. Copy number variation; focused amplification

Although copy number variations such as focused amplification are somatic mutations, they can be detected by sequencing based on read frequency in a manner similar to methods for detecting certain epigenetic changes such as methylation changes. Therefore, regions that can show copy number variations such as focused amplification in cancer can be included in the epigenetic target region group and can include one or more of AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, and RAF1. For example, in some embodiments, the epigenetic target region group includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the aforementioned targets.

iv. Methylation control region

Inclusion of control regions to aid in data validation may be useful. In some embodiments, the epigenetic target region set includes control regions that are expected to be methylated or unmethylated in substantially all samples, regardless of whether the DNA is derived from cancer cells or normal cells. In some embodiments, the epigenetic target region set includes control hypomethylated regions that are expected to be hypomethylated in substantially all samples. In some embodiments, the epigenetic target region set includes control hypermethylated regions that are expected to be hypermethylated in substantially all samples.

b. Sequence variable target group

In some embodiments, the sequence variable target region group includes more than one region known to undergo somatic mutations in cancer (referred to herein as cancer-associated mutations). Thus, the method can include determining whether a cfDNA molecule corresponding to the sequence variable target region group comprises a cancer-associated mutation.

In some embodiments, the set of sequence variable target regions targets more than one different gene or genomic region selected ("panel") so that a determined proportion of subjects with cancer exhibit genetic variation or tumor markers in one or more different genes or genomic regions in the panel. The panel can be selected to limit the region for sequencing to a fixed number of base pairs. The panel can be selected to sequence a desired amount of DNA, for example, by adjusting the affinity and/or amount of the probe, as described elsewhere herein. The panel can also be selected to achieve a desired sequence read depth. The panel can be selected to achieve a desired sequence read depth or sequence read coverage for a certain amount of sequenced base pairs. The panel can be selected to achieve theoretical sensitivity, theoretical specificity, and/or theoretical accuracy for detecting one or more genetic variations in a sample.

The probes for detecting the set of regions can include probes for detecting genomic regions of interest (hotspot regions) and nucleosome-aware probes (e.g., KRAS codons 12 and 13), and can be designed to optimize capture based on analyzing cfDNA coverage and fragment size variation and GC sequence composition affected by nucleosome binding patterns. The regions used herein can also include non-hotspot regions optimized based on nucleosome positions and GC patterns.

Examples of lists of genomic locations of interest can be found in Tables 3 and 4. In some embodiments, the set of sequence variable target regions used in the methods of the present disclosure comprises at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 genes in Table 3. In some embodiments, the set of sequence variable target regions used in the methods of the present disclosure comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 SNVs in Table 3. In some embodiments, the set of sequence variable target regions used in the methods of the present disclosure comprises at least 1, at least 2, at least 3, at least 4, at least 5, or 6 fusions in Table 3. In some embodiments, the set of sequence variable target regions used in the methods of the present disclosure comprises at least a portion of at least 1, at least 2, or 3 indels in Table 3. In some embodiments, the set of sequence variable target regions used in the methods of the present disclosure comprises at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 genes in Table 4. In some embodiments, the set of sequence variable target regions used in the methods of the present disclosure comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 SNVs in Table 4. In some embodiments, the sequence variable target region group used in the methods of the present disclosure comprises at least 1, at least 2, at least 3, at least 4, at least 5, or 6 fusions in Table 4. In some embodiments, the sequence variable target region group used in the methods of the present disclosure comprises at least a portion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or 18 insertions/deletions in Table 4. Each of these genomic positions of interest can be identified as a backbone region or hotspot region of a particular group. An example of a list of hotspot genomic positions of interest can be found in Table 5. The coordinates in Table 5 are based on the hg19 human genome assembly, but those skilled in the art will be familiar with other assemblies and can identify coordinate groups corresponding to exons, introns, codons, etc. indicated in their selected assembly. In some embodiments, the set of sequence variable target regions used in the methods of the present disclosure comprises at least a portion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 genes in Table 5. Several features are listed for each hotspot genomic region, including the associated gene, the chromosome on which it is located, the start and end positions of the locus of the representative gene in the genome, the length of the locus in base pairs, the exons covered by the gene, and key features (e.g., mutation types) that the specific genomic region of interest can seek to capture.

Table 3

Table 4

Table 5

Additionally or alternatively, suitable target groups can be obtained from the literature. For example, Gale et al., PLoS One 13: e0194630 (2018), which is incorporated herein by reference, describes a group of 35 cancer-related gene targets that can be used as part or all of a sequence variable target group. These 35 targets are AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, FGFR3, FOXL2, GATA3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MED12, MET, MYC, NFE2L2, NRAS, PDGFRA, PIK3CA, PPP2R1A, PTEN, RET, STK11, TP53, and U2AF1.

In some embodiments, the set of sequence variable target regions comprises target regions from at least 10, 20, 30, or 35 cancer-associated genes, such as those listed above.

In some embodiments, the set of sequence variable target regions has a footprint of at least 50 kbp (e.g., at least 100 kbp, at least 200 kbp, at least 300 kbp, or at least 400 kbp). In some embodiments, the set of sequence variable target regions has a footprint in the range of 100-2000 kbp (e.g., 100-200 kbp, 200-300 kbp, 300-400 kbp, 400-500 kbp, 500-600 kbp, 600-700 kbp, 700-800 kbp, 800-900 kbp, 900-1,000 kbp, 1-1.5 Mbp, or 1.5-2 Mbp). In some embodiments, the set of sequence variable target regions has a footprint of at least 2 Mbp.

c. Collection of target-specific probes

In some embodiments, a collection of target-specific probes is used in the methods described herein. In some embodiments, the collection of target-specific probes includes target binding probes specific to a sequence variable target region group and target binding probes specific to an epigenetic target region group. In some embodiments, the capture yield of the target binding probes specific to the sequence variable target region group is higher than the capture yield of the target binding probes specific to the epigenetic target region group (e.g., at least 2 times higher). In some embodiments, the collection of target-specific probes is configured to have a capture yield specific to the sequence variable target region that is higher than its capture yield specific to the epigenetic target region (e.g., at least 2 times higher).

In some embodiments, the capture yield of a target binding probe specific for a set of sequence variable target regions is at least 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold greater than the capture yield of a target binding probe specific for a set of epigenetic target regions. In some embodiments, the capture yield of a target binding probe specific for a set of sequence variable target regions is 1.25-fold to 1.5-fold, 1.5-fold to 1.75-fold, 1.75-fold to 2-fold, 2-fold to 2.25-fold, 2.25-fold to 2.5-fold, 2.5-fold to 2.75-fold, 2.75-fold to 3-fold, 3-fold to 3.5-fold, 3.5-fold to 4-fold, 4-fold to 4.5-fold, 4.5-fold to 5-fold, 5-fold to 5.5-fold, 5.5-fold to 6-fold, 6-fold to 7-fold, 7-fold to 8-fold, 8-fold to 9-fold, 9-fold to 10-fold, 10-fold to 11-fold, 11-fold to 12-fold, 13-fold to 14-fold, or 14-fold to 15-fold higher than the capture yield of a target binding probe specific for a set of epigenetic target regions.

In some embodiments, the collection of target-specific probes is configured to have a capture yield specific for a set of sequence variable target regions that is at least 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold higher than its capture yield for a set of epigenetic target regions. In some embodiments, a collection of target-specific probes is configured to have a capture yield specific for a set of sequence variable target regions that is 1.25-fold to 1.5-fold, 1.5-fold to 1.75-fold, 1.75-fold to 2-fold, 2-fold to 2.25-fold, 2.25-fold to 2.5-fold, 2.5-fold to 2.75-fold, 2.75-fold to 3-fold, 3-fold to 3.5-fold, 3.5-fold to 4-fold, 4-fold to 4.5-fold, 4.5-fold to 5-fold, 5-fold to 5.5-fold, 5.5-fold to 6-fold, 6-fold to 7-fold, 7-fold to 8-fold, 8-fold to 9-fold, 9-fold to 10-fold, 10-fold to 11-fold, 11-fold to 12-fold, 13-fold to 14-fold, or 14-fold to 15-fold higher than its capture yield specific for a set of epigenetic target regions.

The collection of probes can be configured to provide higher capture yields for sets of sequence-variable targets in a variety of ways, including concentration, different lengths, and/or chemistries (e.g., chemistries that affect affinity), and combinations thereof. Affinity can be adjusted by adjusting probe length and/or including nucleotide modifications, as discussed below.

In some embodiments, target-specific probes specific for the set of sequence variable target regions are present at a higher concentration than target-specific probes specific for the set of epigenetic target regions. In some embodiments, the concentration of target binding probes specific for the set of sequence variable target regions is at least 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold higher than the concentration of target binding probes specific for the set of epigenetic target regions. In some embodiments, the concentration of target binding probes specific for the set of sequence variable target regions is 1.25 to 1.5 times, 1.5 to 1.75 times, 1.75 to 2 times, 2 to 2.25 times, 2.25 to 2.5 times, 2.5 to 2.75 times, 2.75 to 3 times, 3 to 3.5 times, 3.5 to 4 times, 4 to 4.5 times, 4.5 to 5 times, 5 to 5.5 times, 5.5 to 6 times, 6 to 7 times, 7 to 8 times, 8 to 9 times, 9 to 10 times, 10 to 11 times, 11 to 12 times, 13 to 14 times, or 14 to 15 times higher than the concentration of target binding probes specific for the set of epigenetic target regions. In such embodiments, the concentration can refer to the average mass/volume concentration of the individual probes in each set.

In some embodiments, a target-specific probe specific for a sequence variable target region group has a higher affinity for its target than a target-specific probe specific for an epigenetic target region group. Affinity can be adjusted in any manner known to those skilled in the art, including by using different probe chemistries. For example, certain nucleotide modifications, such as cytosine 5-methylation (in the case of certain sequences), modifications providing heteroatoms at the 2' sugar position, and LNA nucleotides, can increase the stability of double-stranded nucleic acids, which means that oligonucleotides with such modifications have a relatively higher affinity for their complementary sequences. See, for example, Severin et al., Nucleic Acids Res. 39: 8740–8751 (2011); Freier et al., Nucleic Acids Res. 25: 4429–4443 (1997); U.S. Patent No. 9,738,894. In addition, longer sequence lengths will generally provide increased affinity. Other nucleotide modifications, such as substitution of the nucleobase hypoxanthine for guanine, reduce affinity by reducing the amount of hydrogen bonding between the oligonucleotide and its complementary sequence. In some embodiments, a target-specific probe specific for a sequence variable target region group has a modification that increases its affinity for its target. In some embodiments, alternatively or additionally, a target-specific probe specific for an epigenetic target region group has a modification that reduces its affinity for its target. In some embodiments, a target-specific probe specific for a sequence variable target region group has a longer average length and/or a higher average melting temperature than a target-specific probe specific for an epigenetic target region group. These embodiments can be combined with each other and/or have concentration differences as discussed above to achieve a desired multiple difference in capture yield, such as any multiple difference described above or a range thereof.

In some embodiments, the target-specific probe comprises a capture moiety. The capture moiety can be any capture moiety described herein, such as biotin. In some embodiments, the target-specific probe is connected to a solid support, for example, covalently or non-covalently, such as by the interaction of a binding pair of the capture moiety. In some embodiments, the solid support is a bead, such as a magnetic bead.

In some embodiments, target-specific probes specific for a set of sequence-variable target regions and/or target-specific probes specific for a set of epigenetic target regions are bait sets as discussed above, e.g., probes comprising capture moieties and sequences selected to tile across a set of regions, such as genes.

In some embodiments, the target-specific probes are provided in a single composition. The single composition can be a solution (liquid or frozen). Alternatively, the single composition can be a lyophilisate.

Alternatively, target-specific probes can be provided as more than one composition, for example, including a first composition comprising probes specific to a set of epigenetic target regions and a second composition comprising probes specific to a set of sequence variable target regions. These probes can be mixed in appropriate proportions to provide probe compositions having combinations of any of the aforementioned fold differences in concentration and/or capture yield. Alternatively, they can be used in separate capture procedures (e.g., for aliquots of a sample or sequentially for the same sample) to provide a first composition and a second composition comprising a captured epigenetic target region and a captured sequence variable target region, respectively.

ii. Probes specific for epigenetic target regions

The probes for the epigenetic target region group can include probes specific to one or more types of target regions that can distinguish DNA from neoplastic (e.g., tumor or cancer) cells from healthy cells (e.g., non-neoplastic circulating cells). Exemplary types of such regions are discussed in detail herein (e.g., in the above section on capture groups). The probes for the epigenetic target region group can also include probes for one or more control regions, for example, as described herein.

In some embodiments, the probes of the epigenetic target region probe set have a footprint of at least 100 kbp (e.g., at least 200 kbp, at least 300 kbp, or at least 400 kbp). In some embodiments, the epigenetic target region set has a footprint in the range of 100 kbp-20 Mbp (e.g., 100-200 kbp, 200-300 kbp, 300-400 kbp, 400-500 kbp, 500-600 kbp, 600-700 kbp, 700-800 kbp, 800-900 kbp, 900-1,000 kbp, 1-1.5 Mbp, 1.5-2 Mbp, 2-3 Mbp, 3-4 Mbp, 4-5 Mbp, 5-6 Mbp, 6-7 Mbp, 7-8 Mbp, 8-9 Mbp, 9-10 Mbp, or 10-20 Mbp). In some embodiments, the set of epigenetic target regions has a footprint of at least 20 Mbp.

a. Hypermethylated variable target regions

In some embodiments, probes for the epigenetic target region group include probes specific to one or more hypermethylated variable target regions. Hypermethylated variable target regions may also be referred to herein as hypermethylated DMRs (differentially methylated regions). Hypermethylated variable target regions may be any hypermethylated variable target regions listed above. For example, in some embodiments, probes specific to hypermethylated variable target regions include probes specific to more than one locus listed in Table 1 (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the loci listed in Table 1). In some embodiments, probes specific to hypermethylated variable target regions include probes specific to more than one locus listed in Table 2 (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the loci listed in Table 2). In some embodiments, probes specific for hypermethylated variable target regions include probes specific for more than one locus listed in Table 1 or Table 2 (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 1 or Table 2). In some embodiments, for each locus included as a target region, there can be one or more probes having a hybridization site that binds between the transcription start site and the stop codon (the last stop codon for alternatively spliced genes) of the gene. In some embodiments, one or more probes bind within 300 bp (e.g., within 200 bp or 100 bp) of the listed positions. In some embodiments, the probes have hybridization sites that overlap with the positions listed above. In some embodiments, probes specific to hypermethylated target regions include probes specific to one, two, three, four, or five subsets of hypermethylated target regions, which collectively show hypermethylation in one, two, three, four, or five of breast cancer, colon cancer, kidney cancer, liver cancer, and lung cancer.

b. Hypomethylated variable target regions

In some embodiments, probes for the epigenetic target region group include probes specific to one or more hypomethylated variable target regions. Hypomethylated variable target regions may also be referred to herein as hypomethylated DMRs (regions of differential methylation). Hypomethylated variable target regions may be any of the hypomethylated variable target regions listed above. For example, probes specific to one or more hypomethylated variable target regions may include probes for the following regions: such as repetitive elements (e.g., LINE1 elements, Alu elements, centromere tandem repeats, centromere peri-tandem repeats, and satellite DNA) and intergenic regions, which are typically methylated in healthy cells and may show reduced methylation in tumor cells.

In some embodiments, probes specific for hypomethylated variable target regions include probes specific for repetitive elements and/or intergenic regions. In some embodiments, probes specific for repetitive elements include probes specific for one, two, three, four, or five of LINE1 elements, Alu elements, centromeric tandem repeats, pericentromeric tandem repeats, and/or satellite DNA.

Exemplary probes specific for genomic regions showing cancer-associated hypomethylation include probes specific for nucleotides 8403565-8953708 and/or 151104701-151106035 of human chromosome 1. In some embodiments, probes specific for hypomethylated variable target regions include probes specific for regions overlapping or containing nucleotides 8403565-8953708 and/or 151104701-151106035 of human chromosome 1.

c.CTCF binding region

In some embodiments, probes for the epigenetic target group include probes specific to CTCF binding regions. In some embodiments, probes specific to CTCF binding regions include probes specific to at least 10, 20, 50, 100, 200, or 500 CTCF binding regions, or 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 CTCF binding regions, for example, such as the CTCF binding regions described above, or CTCFBSDB or the articles cited above Cuddapah et al., Martin et al., or Rhee et al. One or more of the CTCF binding regions. In some embodiments, probes for the epigenetic target group include at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, or at least 1000 bp upstream and downstream of the CTCF binding site.

d. Transcription start site

In some embodiments, probes for the epigenetic target region group include probes specific for transcription start sites. In some embodiments, probes specific for transcription start sites include probes for at least 10, 20, 50, 100, 200, or 500 transcription start sites, or 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 transcription start sites, for example, probes specific for transcription start sites such as those listed in DBTSS. In some embodiments, probes for the epigenetic target region group include probes for sequences at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, or at least 1000 bp upstream and downstream of the transcription start site.

e. Focused Amplification

As mentioned above, although focal amplifications are somatic mutations, they can be detected by sequencing based on read frequency in a manner similar to methods for detecting certain epigenetic changes such as methylation changes. Therefore, regions in cancer that may show focal amplification can be included in the epigenetic target group, as discussed above. In some embodiments, probes specific to the epigenetic target group include probes specific for focal amplification. In some embodiments, probes specific for focal amplification include probes specific for one or more of AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, and RAF1. For example, in some embodiments, probes specific for focused amplification include probes specific for one or more of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the aforementioned targets.

f. Control area

It is useful to include control regions to aid in data validation. In some embodiments, probes specific to a panel of epigenetic target regions include probes specific to control methylated regions that are expected to be methylated in substantially all samples. In some embodiments, probes specific to a panel of epigenetic target regions include probes specific to control hypomethylated regions that are expected to be hypomethylated in substantially all samples.

ii. Probes specific for sequence variable target regions

Probes for sequence variable target groups can include probes specific to more than one region known to undergo somatic mutations in cancer. Probes can be specific to any sequence variable target group described herein. Exemplary sequence variable target groups are discussed in detail herein (e.g., in the section above on capture groups).

In some embodiments, the sequence variable target probe group has a footprint of at least 0.5 kb (e.g., at least 1 kb, at least 2 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 30 kb, or at least 40 kb). In some embodiments, the epigenetic target probe group has a footprint in the range of 0.5-100 kb (e.g., 0.5-2 kb, 2-10 kb, 10-20 kb, 20-30 kb, 30-40 kb, 40-50 kb, 50-60 kb, 60-70 kb, 70-80 kb, 80-90 kb, and 90-100 kb). In some embodiments, the sequence variable target probe group has a footprint of at least 50 kbp (e.g., at least 100 kbp, at least 200 kbp, at least 300 kbp, or at least 400 kbp). In some embodiments, the sequence variable target probe set has a footprint in the range of 100-2000 kbp (e.g., 100-200 kbp, 200-300 kbp, 300-400 kbp, 400-500 kbp, 500-600 kbp, 600-700 kbp, 700-800 kbp, 800-900 kbp, 900-1,000 kbp, 1-1.5 Mbp, or 1.5-2 Mbp). In some embodiments, the sequence variable target probe set has a footprint of at least 2 Mbp.

In some embodiments, probes specific for the set of sequence variable target regions include probes specific for at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 genes in Table 3. In some embodiments, probes specific for the set of sequence variable target regions include probes specific for at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 SNVs in Table 3. In some embodiments, probes specific for the set of sequence variable target regions include probes specific for at least 1, at least 2, at least 3, at least 4, at least 5, or 6 fusions in Table 3. In some embodiments, probes specific for the set of sequence variable target regions include probes specific for at least a portion of at least 1, at least 2, or 3 indels in Table 3. In some embodiments, probes specific for the set of sequence variable target regions include probes specific for at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 genes in Table 4. In some embodiments, probes specific for the set of sequence variable target regions include probes specific for at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 SNVs in Table 4. In some embodiments, probes specific for the set of sequence variable target regions include probes specific for at least 1, at least 2, at least 3, at least 4, at least 5, or 6 fusions in Table 4. In some embodiments, probes specific for the set of sequence variable target regions include probes specific for at least a portion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or 18 indels in Table 4. In some embodiments, probes specific for a set of sequence variable target regions include probes specific for at least a portion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 of the genes in Table 5.

In some embodiments, probes specific for a set of sequence variable target regions include probes specific for targets from at least 10, 20, 30, or 35 cancer-related genes, such as AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, FGFR3, FOXL2, GATA3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MED12, MET, MYC, NFE2L2, NRAS, PDGFRA, PIK3CA, PPP2R1A, PTEN, RET, STK11, TP53, and U2AF1.

F. Sequencing

Sample nucleic acid (optionally flanked by adapters) is usually sequenced in advance or without pre-amplification. The sequencing method or commercially available form optionally utilized includes, for example, Sanger sequencing, high-throughput sequencing, pyrophosphate sequencing, synthetic sequencing, single-molecule sequencing, sequencing based on nanopores, semiconductor sequencing, connection sequencing, hybridization sequencing, RNA-Seq (Illumina), digital gene expression (Helicos), next generation sequencing (NGS), single-molecule synthetic sequencing (SMSS) (Helicos), large-scale parallel sequencing, cloned single-molecule array (Solexa), shotgun sequencing, Ion Torrent, Oxford nanopore, Roche Genia, Maxim-Gilbert sequencing, primer walking, sequencing using PacBio, SOLiD, Ion Torrent or nanopore platform. Sequencing reaction can be carried out in various sample processing units, and the sample processing unit can include multiple channels, multichannels, multiporous or other devices that substantially process more than one sample group at the same time. The sample processing unit can also include multiple sample chambers so that multiple operations can be processed simultaneously.

In some embodiments, a library comprising a captured target region set is subjected to a sequencing step, and the target region set may comprise any target region set described herein. In some embodiments, a library comprising a partition (e.g., a whole genome sample) that has not undergone capture/enrichment is subjected to a sequencing step. For example, a target region may be captured from a first partition and a second sample and then sequenced; or a target region may be captured from a first partition and combined with a second partition after treatment such as a contact and labeling step; or a target region may be captured from a second partition and combined with a first partition after treatment such as a contact and labeling step; or both the first and second partitions may be processed and combined without capture/enrichment.

Sequencing reaction can be carried out to one or more nucleic acid fragment types or regions comprising the marker of cancer or other diseases. Sequencing reaction can also be carried out to any nucleic acid fragment present in the sample. Sequencing reaction can be carried out to at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or 100% of a genome. In other cases, sequencing reaction can be carried out to less than about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or 100% of a genome. Sequence coverage can be performed on at least 5%, 10%, 20%, 70%, 100% of the genome, at least 200 or 500 different genes, or up to 5000, 2500, 1000, 500 or 100 different genes.

Multiple sequencing techniques can be used to perform simultaneous sequencing reactions. In some embodiments, the cell-free polynucleotides are sequenced with at least about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000 or 100,000 sequencing reactions. In other embodiments, the cell-free polynucleotides are sequenced with less than about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000 or 100,000 sequencing reactions. Sequencing reactions are typically performed sequentially or simultaneously. Subsequent data analysis is typically performed on all or part of the sequencing reactions. In some embodiments, data analysis is performed on at least about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000 or 100,000 sequencing reactions. In other embodiments, data analysis is performed on less than about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000 or 100,000 sequencing reactions. An example of read depth is about 1000 to about 50000 reads per locus (e.g., base position). Another example of read depth has at least 50000 reads per locus (e.g., base position).

1. Differential sequencing depth

In some embodiments, nucleic acids corresponding to the set of sequence variable target regions are sequenced to a greater sequencing depth than nucleic acids corresponding to the set of epigenetic target regions. For example, the sequencing depth of nucleic acids corresponding to the set of sequence variable target regions can be at least 1.25 times, 1.5 times, 1.75 times, 2 times, 2.25 times, 2.5 times, 2.75 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, or 15 times greater, or 1.25 times to 1.5 times, 1.5 times to 1.7 ... In some embodiments, the sequencing depth is at least 2 times greater. In some embodiments, the sequencing depth is at least 5 times greater. In some embodiments, the sequencing depth is at least 10 times greater. In some embodiments, the sequencing depth is 4 times to 10 times greater. In some embodiments, the sequencing depth is 4 times to 10 times greater. In some embodiments, the sequencing depth is 4 times to 10 times greater. Each of these embodiments involves sequencing nucleic acids corresponding to a set of sequence variable target regions to a greater degree of sequencing depth than nucleic acids corresponding to a set of epigenetic target regions.

In some embodiments, captured DNA corresponding to a set of sequence variable target regions and captured DNA corresponding to a set of epigenetic target regions are sequenced simultaneously, for example, in the same sequencing pool (such as a flow cell of an Illumina sequencer) and/or in the same composition; the same composition can be a pooled composition generated by recombining individually captured groups, or a composition obtained by capturing cfDNA corresponding to a set of sequence variable target regions and captured DNA corresponding to a set of epigenetic target regions in the same container.

G. Additional Features of Certain Methods

a. Subjecting a sample or partition to a procedure that differentially affects a first nucleobase in the DNA and a second nucleobase in the DNA

The methods disclosed herein may include subjecting a sample or a first partition to a procedure that differentially affects a first nucleobase in the DNA and a second nucleobase in the DNA of the first partition, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity (e.g., when the second partition is contacted with an MSRE according to any embodiment described elsewhere herein). In some embodiments, if the first nucleobase is a modified or unmodified adenine, the second nucleobase is a modified or unmodified adenine; if the first nucleobase is a modified or unmodified cytosine, the second nucleobase is a modified or unmodified cytosine; if the first nucleobase is a modified or unmodified guanine, the second nucleobase is a modified or unmodified guanine; if the first nucleobase is a modified or unmodified thymine, the second nucleobase is a modified or unmodified thymine (wherein for the purpose of this step, modified and unmodified uracil are included in modified thymine). Such a procedure can be used to identify nucleotides with or lacking certain modifications (such as methylation) in a partition.

In some embodiments, the first core base is a modified or unmodified cytosine, and then the second core base is a modified or unmodified cytosine. For example, the first core base may include unmodified cytosine (C), and the second core base may include one or more of 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC). Alternatively, the second core base may include C, and the first core base may include one or more of mC and hmC. Other combinations are also possible, as indicated in the above overview and the following discussion, such as wherein one of the first core base and the second core base includes mC and another includes hmC.

In some embodiments, the procedure of the first core base in the DNA of the first partition and the second core base in the DNA includes bisulfite conversion. Unmodified cytosine and certain modified cytosine nucleotides (e.g., 5-formylcytosine (fC) or 5-carboxylcytosine (caC)) are converted into uracil by bisulfite treatment, while other modified cytosines (e.g., 5-methylcytosine and 5-hydroxymethylcytosine) are not converted. Therefore, in the case of using bisulfite conversion, the first core base includes one or more of unmodified cytosine, 5-formylcytosine, 5-carboxylcytosine or other cytosine forms affected by bisulfite, and the second core base can include one or more of mC and hmC, such as mC and optionally hmC. The sequencing of the DNA treated with bisulfite identifies the position read as cytosine as an mC position or an hmC position. At the same time, positions that read as T are identified as T or bisulfite susceptible forms of C, such as unmodified cytosine, 5-formylcytosine, or 5-carboxylcytosine. Thus, bisulfite conversion of the first partition as described herein facilitates identification of positions containing mC or hmC using sequence reads obtained from the first partition. For an exemplary description of bisulfite conversion, see, e.g., Moss et al., Nat Commun. 2018; 9: 5068.

In some embodiments, the program that differently affects the first nucleobase in the DNA of the first partition and the second nucleobase in the DNA includes oxidative bisulfite (Ox-BS) conversion. The program first converts hmC into fC susceptible to bisulfite, and then performs bisulfite conversion. Therefore, when using oxidative bisulfite conversion, the first nucleobase includes one or more of unmodified cytosine, fC, caC, hmC or other cytosine forms affected by bisulfite, and the second nucleobase includes mC. The sequencing of the DNA converted by Ox-BS identifies the position read as cytosine as the mC position. At the same time, the position read as T is identified as T, hmC or bisulfite susceptible form of C, such as unmodified cytosine, fC or hmC. Therefore, as described herein, the first partition is converted by Ox-BS to help identify the position containing mC using the sequence reads obtained from the first partition. For an exemplary description of oxidative bisulfite conversion, see, eg, Booth et al., Science 2012; 336:934-937.

In some embodiments, the procedure that differently affects the first nucleobase in the DNA of the first partition and the second nucleobase in the DNA includes TET-assisted bisulfite (TAB) conversion. In TAB conversion, hmC is protected from conversion, and mC is oxidized before bisulfite treatment, so the position initially occupied by mC is converted to U, while the position initially occupied by hmC remains as a protected form of cytosine. For example, as described in Yu et al., Cell 2012; 149: 1368-80, β-glucosyltransferase can be used to protect hmC (forming 5-glucosylhydroxymethylcytosine (ghmC)), then TET proteins such as mTet1 can be used to convert mC to caC, and then bisulfite treatment can be used to convert C and caC to U, while ghmC remains unaffected. Thus, when TAB conversion is used, the first nucleobase includes one or more of unmodified cytosine, fC, caC, mC, or other bisulfite-affected cytosine forms, and the second nucleobase includes hmC. Sequencing of the TAB-converted DNA identifies positions that read as cytosine as hmC positions. At the same time, positions that read as T are identified as T, mC, or a bisulfite-susceptible form of C, such as unmodified cytosine, fC, or caC. Thus, TAB conversion of the first partition as described herein facilitates identification of positions containing hmC using sequence reads obtained from the first partition.

In some embodiments, the procedure that differently affects the first nucleobase in the DNA of the first partition and the second nucleobase in the DNA includes a Tet-assisted substituted borane reducing agent conversion, optionally wherein the substituted borane reducing agent is 2-methylpyridine borane, pyridine borane, tert-butylamine borane or ammonia borane. In the Tet-assisted substituted borane reducing agent conversion, mC and hmC are converted to caC using TET protein without affecting unmodified C. Then, caC and fC (if present) are converted to dihydrouracil (DHU) by treatment with 2-methylpyridine borane (pic-borane) or other substituted borane reducing agents (such as pyridine borane, tert-butylamine borane or ammonia borane), without affecting unmodified C. See, e.g., Liu et al., Nature Biotechnology 2019; 37: 424–429 (e.g., in Supplementary Figure 1 and Supplementary Note 7). DHU is read as T in sequencing. Therefore, when this type of conversion is used, the first nucleobase includes one or more of mC, fC, caC or hmC, and the second nucleobase includes unmodified cytosine. The sequencing of the converted DNA identifies the position read as cytosine as an unmodified C position. At the same time, the position read as T is identified as T, mC, fC, caC or hmC. Therefore, TAP conversion of the first partition as described herein helps to identify the position containing unmodified C using the sequence reads obtained from the first partition. The program includes Tet-assisted pyridine borane sequencing (TAPS), which is described in further detail below: Liu et al. 2019, supra.

Alternatively, protection of hmC (e.g., using βGT) can be combined with Tet-assisted substituted borane reducing agent conversion. hmC can be protected by glucosylation using βGT to form ghmC as mentioned above. Treated with a TET protein such as mTet1, mC is then converted to caC but not C or ghmC. CaC is then converted to DHU by treatment with pic-borane or other substituted borane reducing agents (such as pyridine borane, tert-butylamine borane or ammonia borane), without affecting unmodified C or ghmC. Therefore, when converted using a Tet-assisted substituted borane reducing agent, the first nucleobase includes mC, and the second nucleobase includes one or more of unmodified cytosine or hmC, such as unmodified cytosine and optionally hmC, fC and/or caC. Sequencing of the converted DNA identifies the position read as cytosine as a hmC position or an unmodified C position. At the same time, the position read as T is identified as T, fC, caC or mC. Thus, performing TAPSβ conversion on the first partition as described herein facilitates using sequence reads obtained from the first partition to distinguish positions containing unmodified C or hmC from positions containing mC on the one hand. For an exemplary description of this type of conversion, see, e.g., Liu et al., Nature Biotechnology 2019; 37: 424–429.

In some embodiments, the procedure that differentially affects the first nucleobase in the DNA of the first partition and the second nucleobase in the DNA comprises a chemically assisted substituted borane reducing agent conversion, optionally wherein the substituted borane reducing agent is 2-methylpyridine borane, pyridine borane, tert-butylamine borane or ammonia borane. In a chemically assisted substituted borane reducing agent conversion, an oxidant such as potassium perruthenate (KRuO4) (also suitable for ox-BS conversion) is used to specifically oxidize hmC to fC. Treatment with pic-borane or other substituted borane reducing agents (such as pyridine borane, tert-butylamine borane or ammonia borane) converts fC and caC to DHU, but does not affect mC or unmodified C. Thus, when this type of conversion is used, the first nucleobase comprises one or more of hmC, fC and caC, and the second nucleobase comprises one or more of unmodified cytosine or mC, such as unmodified cytosine and optionally mC. Sequencing of the converted DNA identifies positions that read as cytosine as mC positions or unmodified C positions. At the same time, positions that read as T are identified as T, fC, caC, or hmC. Thus, performing this type of conversion on the first partition as described herein facilitates using the sequence reads obtained from the first partition to distinguish positions containing unmodified C or mC from positions containing hmC on the one hand. For an exemplary description of this type of conversion, see, e.g., Liu et al., Nature Biotechnology 2019; 37: 424–429.

In some embodiments, the program of the first core base in the DNA of the first partition and the second core base in the DNA differently includes the epigenetic (ACE) conversion of APOBEC coupling. In ACE conversion, AID/APOBEC family DNA deaminase such as APOBEC3A (A3A) is used to deaminize unmodified cytosine and mC, without deaminating hmC, fC or caC. Therefore, when using ACE conversion, the first core base includes unmodified C and/or mC (for example, unmodified C and optionally mC), and the second core base includes hmC. The sequencing of the DNA converted by ACE will be identified as the position of cytosine as hmC position, fC position or caC position. At the same time, the position of T is identified as T, unmodified C or mC. Therefore, as described herein, ACE conversion of the first partition helps to distinguish the position containing hmC from the position containing mC or unmodified C using the sequence reads obtained from the first subsample. For an exemplary description of ACE conversion, see, e.g., Schutsky et al., Nature Biotechnology 2018; 36: 1083–1090.

In some embodiments, the procedure that differently affects the first nucleobase in the DNA of the first partition and the second nucleobase in the DNA comprises an enzymatic conversion of the first nucleobase, e.g., as in EM-SEQ. See, e.g., Vaisvila R, et al. (2019) EM-seq: Detection of DNA methylation at single base resolution from picograms of DNA. bioRxiv; DOI: 10.1101/2019.12.20.884692, available at www.biorxiv.org/content/10.1101/2019.12.20.884692v1. For example, TET2 and T4-βGT can be used to convert 5mC and 5hmC into substrates that cannot be deaminated by a deaminase (e.g., APOBEC3A), and then a deaminase (e.g., APOBEC3A) can be used to deaminate unmodified cytosine, converting it to uracil.

In some embodiments, the method of the first nucleobase in the DNA of the first partition and the second nucleobase in the DNA differently affects includes separating the DNA initially comprising the first nucleobase from the DNA initially not comprising the first nucleobase. In some such embodiments, the first nucleobase is hmC. The DNA initially comprising the first nucleobase can be separated from other DNAs using a labeling procedure including biotinylation of the position initially comprising the first nucleobase. In some embodiments, the first nucleobase is first derivatized with a part containing an azido group (such as a part containing a glucosyl-azido group). Then, the part containing an azido group can be used as a reagent for attaching biotin, for example, by Huisgen cycloaddition chemistry. Then, a biotin binding agent can be used, such as avidin, neutravidin (neutravidin) (deglycosylated avidin with an isoelectric point of about 6.3) or streptavidin, the DNA initially comprising the first nucleobase now being biotinylated is separated from the DNA initially not comprising the first nucleobase. An example of a method for separating DNA that originally contained a first nucleobase from DNA that originally did not contain the first nucleobase is hmC-seal, which labels hmC to form β-6-azido-glucosyl-5-hydroxymethylcytosine, and then attaches a biotin moiety by Huisgen cycloaddition, followed by the use of a biotin binder to separate the biotinylated DNA from the other DNA. For an exemplary description of hmC-seal, see, e.g., Han et al., Mol. Cell 2016; 63: 711-719. This method can be used to identify fragments containing one or more hmC nucleobases.

In some embodiments, after such separation, the method further comprises differentially tagging each of the DNA that initially comprises the first nucleobase, the DNA that initially does not comprise the first nucleobase, and the DNA of the second partition. The method may also comprise pooling the DNA that initially comprises the first nucleobase, the DNA that initially does not comprise the first nucleobase, and the DNA of the second partition after differential tagging. The DNA that initially comprises the first nucleobase, the DNA that initially does not comprise the first nucleobase, and the DNA of the second partition may then be sequenced in the same sequencing pool while maintaining the ability to use the differential tags to distinguish whether a particular read is from a molecule of the DNA that initially comprises the first nucleobase, the DNA that initially does not comprise the first nucleobase, or the DNA of the second partition.

In some embodiments, the first core base is a modified or unmodified adenine, and the second core base is a modified or unmodified adenine. In some embodiments, the modified adenine is N6-methyladenine (mA). In some embodiments, the modified adenine is one or more of N6-methyladenine (mA), N6-hydroxymethyladenine (hmA) or N6-formyladenine (fA).

Techniques including methylated DNA immunoprecipitation (MeDIP) can be used to separate DNA containing modified bases (such as mA) from other DNA. See, for example, Kumar et al., Frontiers Genet.2018; 9: 640; Greer et al., Cell 2015; 161: 868-878. Antibodies specific to mA are described in Sun et al., Bioessays 2015; 37: 1155-62. Antibodies against various modified nucleobases (such as forms of thymine/uracil, including halogenated forms, such as 5-bromouracil) are commercially available. Various modified bases can also be detected based on changes in their base pairing specificity. For example, hypoxanthine is a modified form of adenine, which can be produced by deamination and is read as G in sequencing. See, e.g., U.S. Patent No. 8,486,630; Brown, Genomes, 2nd ed., John Wiley & Sons, Inc., New York, N.Y., 2002, Chapter 14, “Mutation, Repair, and Recombination.”

b. Subjects

In some embodiments, nucleic acid molecules such as DNA (e.g., cfDNA) are obtained from subjects with cancer. In some embodiments, DNA (e.g., cfDNA) is obtained from subjects suspected of having cancer. In some embodiments, DNA (e.g., cfDNA) is obtained from subjects with tumors. In some embodiments, DNA (e.g., cfDNA) is obtained from subjects suspected of having tumors. In some embodiments, DNA (e.g., cfDNA) is obtained from subjects with vegetation. In some embodiments, DNA (e.g., cfDNA) is obtained from subjects suspected of having vegetation. In some embodiments, DNA (e.g., cfDNA) is obtained from subjects in remission (e.g., after chemotherapy, surgical resection, radiation, or a combination thereof) from tumors, cancers, or vegetation. In any of the foregoing embodiments, cancer, tumors, or vegetations, or suspected cancer, tumors, or vegetations, can be lungs, colons, rectums, kidneys, breasts, prostates, or livers. In some embodiments, cancer, tumors, or vegetations, or suspected cancer, tumors, or vegetations are lungs. In some embodiments, cancer, tumors, or vegetations, or suspected cancer, tumors, or vegetations are colonic or rectal. In some embodiments, the cancer, tumor or neoplasm or suspected cancer, tumor or neoplasm is of the breast. In some embodiments, the cancer, tumor or neoplasm or suspected cancer, tumor or neoplasm is of the prostate. In any of the foregoing embodiments, the subject can be a human subject.

c. Quantification

In some embodiments, the epigenetic target regions captured from one or more of the first partition, the processed first partition, or the processed second partition are quantified. For example, the low methylation variable target regions can be quantified in the processed second partition, and/or the high methylation variable target regions can be quantified in the first partition or the processed first partition. Quantification can be performed by any appropriate technique, such as quantitative amplification, such as quantitative PCR. In some embodiments, quantification is performed based on sequencing data (e.g., the number of sequencing reads or the number of unique molecules sequenced).

As described above, quantification of epigenetic target regions can be used to determine the presence, absence, or likelihood of cancer in a subject. For example, the presence or absence of cancer can be determined based at least in part on whether the amount of hypermethylated variable target regions in the first partition or the treated first partition and/or the amount of hypomethylated variable target regions in the treated second partition exceeds a predetermined threshold. In some embodiments, such amounts can be used together with other data collected from the sample, such as the presence of mutations and/or other epigenetic features described elsewhere herein, such as perturbations of transcription start sites and/or CTCF binding sites.

d. Pooling DNA from the first and second partitions or portions thereof

In some embodiments, the method includes preparing a pool comprising at least a portion of the DNA of the second partition (e.g., a hypomethylated partition) and at least a portion of the DNA of the first partition (e.g., a hypermethylated partition). Target regions can be captured from the pool, for example, including epigenetic target regions and/or sequence variable target regions. The steps of capturing the target region group from at least a portion of the partition described elsewhere herein include performing a capture step on a pool comprising DNA from the first and second partitions. Prior to capturing the target region from the pool, a step of amplifying the DNA in the pool can be performed. The capture step can have any of the features described elsewhere herein.

Epigenetic target regions may show differences in methylation levels and/or fragmentation patterns depending on whether they are derived from tumor or healthy cells, or what type of tissue they are derived from, as discussed elsewhere in this article. Sequence variable target regions may show differences in sequence depending on whether they are derived from tumor or healthy cells.

In some applications, analyzing the epigenetic target area from the hypomethylation partition may be less than analyzing the sequence variable target area from the hypermethylation and hypomethylation partitions and the epigenetic target area information from the hypermethylation partition. Therefore, in the method for capturing the sequence variable target area and the epigenetic target area, the latter can be captured with a degree less than one or more sequence variable target areas from the hypermethylation and hypomethylation partitions and the epigenetic target area from the hypermethylation partition. For example, the sequence variable target area can be captured from the part of the hypomethylation partition that is never collected with the hypermethylation partition, and a pool can be prepared with some (for example, most of, substantially all or all) DNA from the hypermethylation partition and zero or some (for example, a small part) DNA from the hypomethylation partition. Such a method can reduce or eliminate the sequencing of the epigenetic target area from the hypomethylation partition, thereby reducing the sequencing data amount that is enough for further analysis.

In some embodiments, comprising a small portion of DNA from a hypomethylated partition in a pool facilitates quantification of one or more epigenetic features (eg, methylation or other epigenetic features discussed in detail elsewhere herein), for example, on a relative basis.

In some embodiments, the pool comprises a small portion of the DNA of the hypomethylated partition, for example, less than about 50% of the DNA of the hypomethylated partition, such as less than or equal to about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the DNA of the hypomethylated partition. In some embodiments, the pool comprises about 5%-25% of the DNA of the hypomethylated partition. In some embodiments, the pool comprises about 10%-20% of the DNA of the hypomethylated partition. In some embodiments, the pool comprises about 10% of the DNA of the hypomethylated partition. In some embodiments, the pool comprises about 15% of the DNA of the hypomethylated partition. In some embodiments, the pool comprises about 20% of the DNA of the hypomethylated partition.

In some embodiments, the pool comprises a portion of a hypermethylated partition, which may be at least about 50% of the DNA of the hypermethylated partition. For example, the pool may comprise at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the DNA of the hypermethylated partition. In some embodiments, the pool comprises 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95% or 95%-100% of the DNA of the hypermethylated partition. In some embodiments, the second pool comprises all or substantially all of the hypermethylated partition.

In some embodiments, the method includes preparing a first pool comprising at least a portion of the DNA of the hypomethylated partitions. In some embodiments, the method includes preparing a second pool comprising at least a portion of the DNA of the hypermethylated partitions. In some embodiments, the first pool also comprises a portion of the DNA of the hypermethylated partitions. In some embodiments, the second pool also comprises a portion of the DNA of the hypomethylated partitions. In some embodiments, the first pool comprises a majority of the DNA of the hypomethylated partitions, and optionally a minority of the DNA of the hypermethylated partitions. In some embodiments, the second pool comprises a majority of the DNA of the hypermethylated partitions and a minority of the DNA of the hypomethylated partitions. In some embodiments comprising a medium methylated partition, the second pool comprises at least a portion of the DNA of the medium methylated partitions, for example, a majority of the DNA of the medium methylated partitions. In some embodiments, the first pool comprises a majority of the DNA of the hypomethylated partitions, and the second pool comprises a majority of the DNA of the hypermethylated partitions and a majority of the DNA of the medium methylated partitions.

In some embodiments, the method includes capturing at least a first target region group from a first pool, for example, wherein the first pool is as described in any of the above embodiments. In some embodiments, the first group includes sequence variable target regions. In some embodiments, the first group includes hypomethylated variable target regions and/or fragmented variable target regions. In some embodiments, the first group includes sequence variable target regions and fragmented variable target regions. In some embodiments, the first group includes sequence variable target regions, hypomethylated variable target regions, and fragmented variable target regions. Prior to this capture step, a step of amplifying the DNA in the first pool may be performed. In some embodiments, capturing the first target region group from the first pool includes contacting the DNA of the first pool with a first target-specific probe group. In some embodiments, the first target-specific probe group includes a target binding probe specific for a sequence variable target region. In some embodiments, the first target-specific probe group includes a target binding probe specific for a sequence variable target region, a hypomethylated variable target region, and/or a fragmented variable target region.

In some embodiments, the method includes capturing a second target area group or more than one target area group from a second pool, for example, wherein the second pool is as described in any of the above embodiments. In some embodiments, the second more than one target area group includes epigenetic target areas, such as hypermethylated variable target areas and/or fragmented variable target areas. In some embodiments, the second more than one target area group includes sequence variable target areas and epigenetic target areas, such as hypermethylated variable target areas and/or fragmented variable target areas. Prior to this capture step, a step of amplifying the DNA in the second pool can be performed. In some embodiments, capturing the second more than one target area group from the second pool includes contacting the DNA of the second pool with a second target-specific probe group, wherein the second target-specific probe group includes a target binding probe specific to the sequence variable target area and a target binding probe specific to the epigenetic target area. In some embodiments, the first target area group and the second target area group are not the same. For example, the first target area group may include one or more target areas that are not present in the second target area group. Alternatively or in addition, the second target area group may include one or more target areas that are not present in the first target area group. In some embodiments, at least one hypermethylated variable target region is captured from the second pool but not from the first pool. In some embodiments, more than one hypermethylated variable target region is captured from the second pool but not from the first pool. In some embodiments, the first target region group includes sequence variable target regions and/or the second target region group includes epigenetic target regions. In some embodiments, the first target region group includes sequence variable target regions and fragmented variable target regions; and the second target region group includes epigenetic target regions, such as hypermethylated variable target regions and fragmented variable target regions. In some embodiments, the first target region group includes sequence variable target regions, fragmented variable target regions, and includes hypomethylated variable target regions; and the second target region group includes epigenetic target regions, such as hypermethylated variable target regions and fragmented variable target regions.

In some embodiments, the first pool comprises a majority of the DNA of the hypomethylated partitions and a portion (e.g., about half) of the DNA of the hypermethylated partitions, and the second pool comprises a portion (e.g., about half) of the DNA of the hypermethylated partitions. In some such embodiments, the first target region set comprises sequence variable target regions and/or the second target region set comprises epigenetic target regions. The sequence variable target regions and/or epigenetic target regions may be as described in any of the embodiments described elsewhere herein.

f. Capture part, bait set

As described above, the nucleic acid in the sample can be subjected to a capture step, wherein molecules with target sequences are captured for subsequent analysis. Target capture can include the use of a bait set comprising an oligonucleotide bait, such as a target-specific probe labeled with a capture moiety (such as biotin or other examples mentioned below). The probe can have a sequence selected to tile across a group of regions (such as a gene). In some embodiments, the bait set can have a higher and lower capture yield for a target area group (such as a sequence variable target area group and a target area group of an epigenetic target area), respectively, as discussed elsewhere herein. Under conditions that allow target molecules to hybridize with baits, these bait sets are combined with samples. Then, capture moieties are used to separate captured molecules. For example, a biotin capture moiety based on the streptavidin of a bead. For example, such a method is further described in U.S. Patent No. 9,850,523, issued on December 26, 2017, which is incorporated herein by reference.

Capture moieties include but are not limited to biotin, avidin, streptavidin, nucleic acids comprising specific nucleotide sequences, haptens recognized by antibodies, and magnetically attractable particles. Extraction moieties may be members of binding pairs, such as biotin/streptavidin or haptens/antibodies. In some embodiments, the capture moiety attached to the analyte is captured by its binding pair, which is attached to a separable portion, such as magnetically attractable particles or large particles that can be precipitated by centrifugation. Capture moieties may be molecules of any type that allow affinity separation of nucleic acids with capture moieties from nucleic acids lacking capture moieties. Exemplary capture moieties are biotin or oligonucleotides, the biotin allowing affinity separation by binding to streptavidin that is connected or can be connected to a solid phase, the oligonucleotides allowing affinity separation by binding to complementary oligonucleotides that are connected or can be connected to a solid phase.

H. Analysis

In some embodiments, the methods described herein include identifying the presence of DNA produced by a tumor (or neoplastic cell or cancer cell).

In some embodiments, the method herein includes analyzing nucleic acid molecules, wherein at least some nucleic acids include one or more modified cytosine residues, such as 5-methylcytosine and any other modification described previously. In some such methods, after partitioning, the nucleic acid partition is contacted with an adapter comprising one or more cytosine residues modified at the 5C position (such as 5-methylcytosine). In some embodiments, all cytosine residues in such adapters are also modified, or all such cytosines in the primer binding region of the adapter are modified. The adapter is attached to the two ends of the nucleic acid molecule in the population. In some embodiments, the adapter includes a sufficient number of different tags so that the number of tag combinations causes the probability of two nucleic acids with the same starting point and end point receiving different tag combinations to be high, such as 95%, 99% or 99.9%. The primer binding sites in such adapters can be the same or different, but preferably the same. After the adapter is attached, the nucleic acid is amplified by a primer bound to the primer binding site of the adapter. The amplified nucleic acid is divided into a first aliquot and a second aliquot. In the case of carrying out or not carrying out further processing, the first aliquot is subjected to sequence data determination. Thus the sequence data of the molecule in the first aliquot is determined regardless of the initial methylation state of the nucleic acid molecule. The nucleic acid molecule experience in the second aliquot affects the program of the first core base in DNA and the second core base in DNA differently, wherein the first core base includes the cytosine modified at position 5, and the second core base includes unmodified cytosine. The program can be another program of bisulfite treatment or unmodified cytosine converted into uracil. Then the nucleic acid undergoing the program is amplified with the primer for the original primer binding site of the adapter connected to the nucleic acid. Now only the nucleic acid molecules (different from its amplification product) initially connected to the adapter are amplified, because these nucleic acids retain cytosine in the primer binding site of the adapter, and the amplification product has lost the methylation of these cytosine residues, and these cytosine residues that lose methylation have been transformed into uracil in the bisulfite treatment. Therefore, only the original nucleic acid molecules in the population (at least some of which are methylated) are subjected to amplification. After amplification, these nucleic acids are subjected to sequence analysis. Comparison of the sequences determined from the first aliquot and the second aliquot can in particular indicate which cytosines in the nucleic acid population have undergone methylation.

Such analysis can be carried out using the following exemplary program.After partitioning, the two ends of the methylated DNA are connected to a Y-shaped adapter containing a primer binding site and a label.The cytosine in the adapter is modified (e.g., 5-methylated) at position 5. The modification of the adapter is used to protect the primer binding site in subsequent conversion steps (e.g., bisulfite treatment, TAP conversion, or any other conversion that does not affect modified cytosine but affects unmodified cytosine).After the adapter is attached, the DNA molecule is amplified.The amplified product is divided into two aliquots for sequencing with and without conversion.The aliquots that have not undergone conversion can be subjected to sequence analysis in the case of carrying out or not carrying out further processing.Another aliquot experience differently affects the first core base in DNA and the second core base in DNA The program, wherein the first core base includes the cytosine modified at position 5, and the second core base includes unmodified cytosine.The program can be another program of bisulfite treatment or conversion of unmodified cytosine into uracil. When contacted with primers specific to the original primer binding site, only primer binding sites protected by cytosine modification can support amplification. Therefore, only the original molecule, rather than the copy from the first amplification, undergoes further amplification. The further amplified molecules are then subjected to sequence analysis. The sequences from the two aliquots can then be compared. As in the separation scheme discussed above, the nucleic acid tags in the adapter are not used to distinguish between methylated DNA and unmethylated DNA, but are used to distinguish nucleic acid molecules within the same partition.

Sequencing can generate more than one sequence read or reads. Sequence reads or reads can include data of nucleotide sequences having a length of less than about 150 bases or a length of less than about 90 bases. In some embodiments, the length of the read is between about 80 bases and about 90 bases, for example, about 85 bases. In some embodiments, the method of the present disclosure is applied to very short reads, for example, having a length of less than about 50 bases or about 30 bases. Sequence read data can include sequence data and meta information. Sequence read data can be stored in any suitable file format, including, for example, VCF files, FASTA files, or FASTQ files.

FASTA may refer to a computer program for searching sequence databases, and the name FASTA may also refer to a standard file format. FASTA is described by, for example, Pearson & Lipman, 1988, Improved tools for biological sequence comparison, PNAS 85: 2444-2448, which is incorporated herein by reference in its entirety. A sequence in FASTA format begins with a single-line description, followed by a sequence data row. The description row is distinguished from the sequence data by a greater than (">") symbol in the first column. The word after the ">" symbol is an identifier for the sequence, and the rest of the row is a description (all optional). There may be no space between the ">" and the first letter of the identifier. It is recommended that all lines of text be less than 80 characters. If another line beginning with ">" appears, the sequence ends; this indicates the beginning of another sequence.

The FASTQ format is a text-based format for storing biological sequences (usually nucleotide sequences) and their corresponding quality scores. It is similar to the FASTA format, but has a quality score after the sequence data. For simplicity, both the sequence letters and the quality scores are encoded using a single ASCII character. The FASTQ format is a conventional standard for storing the output results of high-throughput sequencing instruments such as Illumina Genome Analyzer, such as Cock et al. ("The SangerFASTQ file format for sequences with quality scores, and the Solexa/IlluminaFASTQ variants," Nucleic Acids Res 38 (6): 1767-1771, 2009) described, which is incorporated herein by reference in its entirety.

For FASTA and FASTQ files, the meta information includes the description line but does not include the sequence data line. In some embodiments, for FASTQ files, the meta information includes a quality score. For FASTA and FASTQ files, the sequence data begins after the description line and is typically presented using a subset of IUPAC fuzzy codes that are optionally preceded by "-". In one embodiment, the sequence data may use A, T, C, G, and N characters, optionally including "-" or including U (e.g., to represent a vacancy or uracil) as desired.

In some embodiments, at least one of the master sequence read file and the output file is stored as a plain text file (e.g., using an encoding such as ASCII, ISO/IEC 646, EBCDIC, UTF-8, or UTF-16). The computer system provided by the present disclosure may include a text editor program capable of opening a plain text file. A text editor program may refer to a computer program capable of presenting the contents of a text file (such as a plain text file) on a computer screen, allowing a person to edit the text (e.g., using a display, keyboard, and mouse). Examples of text editors include, but are not limited to, Microsoft Word, emacs, pico, vi, BBEdit, and TextWrangler. A text editor program may be capable of displaying a plain text file on a computer screen in a human-readable format, displaying meta-information and sequence reads (e.g., not binary encoding but using alphanumeric characters as they may be used for printing or human writing).

Although methods have been discussed with reference to FASTA or FASTQ files, the methods and systems of the present disclosure can be used to compress any suitable sequence file format, including, for example, files in Variant Call Format (VCF) format. A typical VCF file can include a header portion and a data portion. The header contains any number of meta information rows, each starting with the characters '##', and TAB-delimited field definition rows starting with a single '#' character. The field definition row names eight required columns, and the body portion contains data rows that fill in the columns defined by these field definition rows. The VCF format is described by, for example, Danecek et al. ("The variant call format and VCFtools," Bioinformatics 27(15):2156-2158, 2011), which is incorporated herein by reference in its entirety. The header portion can be viewed as meta information to be written to the compressed file, and the data portion can be viewed as rows, each of which can be stored in the master file only if it is unique.

Some embodiments provide assembly of sequence reads. For example, in assembly by alignment, sequence reads are aligned to each other or to a reference sequence. By aligning each read, and then to a reference genome, all reads are positioned in relation to each other to create an assembly. In addition, aligning or mapping sequence reads to a reference sequence can also be used to identify variant sequences in sequence reads. Identifying variant sequences can be used in combination with the methods and systems described herein to further aid in the diagnosis or prognosis of a disease or condition or to guide treatment decisions.

In some embodiments, any or all steps are automated.Alternatively, the method of the present disclosure can be realized in whole or in part in one or more special programs, for example, each is optionally written in a compiled language such as C++, then compiled and distributed in binary.The method of the present disclosure can be realized in whole or in part as a module in an existing sequence analysis platform or by calling the function in an existing sequence analysis platform.In some embodiments, the method of the present disclosure includes a plurality of steps that are all automatically called in response to a single startup queue (for example, an event or combination of events in a triggering event derived from human activity, another computer program or machine).Therefore, the present disclosure provides a method in which any step or any combination of steps can occur automatically in response to a queue. "Automatically" generally means not intervening in human input, influence or interaction (for example, only in response to original or pre-queued human activity).

The method of the present disclosure may also include various forms of output, including accurate and sensitive interpretation of the nucleic acid sample of the subject. The output of the retrieval may be provided in the format of a computer file. In some embodiments, the output is a FASTA file, a FASTQ file or a VCF file. The output may be processed to generate a text file or XML file containing sequence data such as a nucleic acid sequence aligned with a reference genome. In other embodiments, the processing generates an output containing a coordinate or a string describing one or more mutations in the subject's nucleic acid relative to a reference genome. The alignment string may include Simple UnGapped Alignment Report (SUGAR), Verbose Useful Labeled Gapped Alignment Report (VALGAR) and Compact Idiosyncratic Gapped Alignment Report (CIGAR) (e.g., Ning et al., Genome Research 11 (10): 1725-9, 2001 described, which are incorporated herein by reference in their entirety). These words can be implemented, for example, in the Exonerate sequence alignment software from the European Bioinformatics Institute (Hinxton, UK).

In some embodiments, a sequence alignment containing CIGAR strings is generated, such as, for example, a sequence alignment map (SAM) or a binary alignment map (BAM) file (the SAM format is described in, for example, Li et al., "The Sequence Alignment/Map format and SAMtools," Bioinformatics, 25(16): 2078-9, 2009, which is incorporated herein by reference in its entirety). In some embodiments, CIGAR displays or includes alignments with one gap per line. CIGAR is a compressed, pairwise alignment format reported as CIGAR strings. CIGAR strings can be used to present long (e.g., genomic) pairwise alignments. CIGAR strings can be used in the SAM format to represent alignments of reads to a reference genomic sequence.

CIGAR strings can follow established motifs. Each character is preceded by a number giving the base count of the event. The characters used can include M, I, D, N, and S (M = match; I = insertion; D = deletion; N = gap; S = substitution). CIGAR strings define sequences of matches and/or mismatches and deletions (or gaps). For example, the CIGAR string 2MD3M2D2M can indicate that the alignment contains 2 matches, 1 deletion (the number 1 is omitted to save some space), 3 matches, 2 deletions, and 2 matches.

In some embodiments, a nucleic acid population for sequencing is prepared by enzymatically forming a flat end on a double-stranded nucleic acid with a single-stranded overhang at one or both ends. In these embodiments, the population is usually treated with an enzyme with 5'-3' DNA polymerase activity and 3'-5' exonuclease activity in the presence of nucleotides (e.g., A, C, G and T or U). The example of an enzyme or its catalytic fragment that can be optionally used includes Klenow large fragment and T4 polymerase. At 5' overhang, the enzyme usually extends the 3' end of the depression on the relative chain until it is flush with the 5' end to produce a flat end. At 3' overhang, the enzyme usually digests from the 3' end, reaches the 5' end of the relative chain and sometimes exceeds the 5' end of the relative chain. If the digestion advances beyond the 5' end of the relative chain, the gap can be filled by an enzyme with the same polymerase activity as used for the 5' overhang. The formation of a flat end on a double-stranded nucleic acid is conducive to the attachment of, for example, an adaptor and subsequent amplification.

In some embodiments, the nucleic acid population undergoes additional processing, such as converting single-stranded nucleic acids into double-stranded nucleic acids and/or converting RNA into DNA (e.g., complementary DNA or cDNA). These forms of nucleic acids are also optionally ligated to adapters and amplified.

The nucleic acid subjected to the above-described flat-end treatment and optionally other nucleic acids in the sample, with or without prior amplification, can be sequenced to produce sequenced nucleic acid. Sequenced nucleic acid can refer to the sequence (e.g., sequence information) of a nucleic acid or a nucleic acid whose sequence has been determined. Sequencing can be performed so as to provide sequence data of individual nucleic acid molecules in the sample directly or indirectly from the consensus sequence of the amplification products of the individual nucleic acid molecules in the sample.

In some embodiments, after the double-stranded nucleic acid with single-stranded overhangs in the sample is formed with a barcode adapter at both ends, and sequencing determines the nucleic acid sequence and the in-line barcode introduced by the adapter. The flat-ended DNA molecule is optionally connected to the flat end of an adapter (e.g., a Y-shaped or bell-shaped adapter) that is at least partially double-stranded. Optionally, the flat ends of the sample nucleic acid and the adapter can be tailed with complementary nucleotides to facilitate connection (e.g., sticky end connection).

The nucleic acid sample is typically contacted with a sufficient number of adapters so that the probability that any two copies of the same nucleic acid receive the same adapter barcode combination from the adapters connected at both ends is low (e.g., less than about 1% or 0.1%). Using adapters in this manner can allow identification of a family of nucleic acid sequences that have the same start and end points on a reference nucleic acid and are connected to the same barcode combination. Such a family can represent the sequence of the amplified product of the nucleic acid in the sample before amplification. The sequences of the family members can be compiled to obtain the common nucleotides or complete common sequences of the nucleic acid molecules in the original sample, which are modified by blunt end formation and adapter attachment. In other words, the nucleotides occupying a specific position of the nucleic acid in the sample can be determined as the common nucleotides of the nucleotides occupying the corresponding position in the family member sequence. The family can include the sequence of one or two chains of a double-stranded nucleic acid. If the members of the family include sequences from two chains of a double-stranded nucleic acid, the sequence of one chain can be converted into their complementary sequences for the purpose of assembling the sequence to obtain the common nucleotides or sequences. Some families include only a single member sequence. In this case, the sequence can be obtained as the sequence of the nucleic acid in the sample before amplification. Alternatively, families with only a single member sequence can be eliminated from subsequent analysis.

By comparing the sequenced nucleic acid with the reference sequence, the nucleotide variation (e.g., SNV or insertion/deletion) in the sequenced nucleic acid can be determined. The reference sequence is generally a known sequence, for example, a known full genome or partial genome sequence from a subject (e.g., a full genome sequence of a human subject). The reference sequence can be an external reference sequence, such as hG19 or hG38. As described above, the sequenced nucleic acid can represent the sequence of the nucleic acid in the sample directly determined or the consensus sequence of the amplification product of such nucleic acid. Comparison can be performed at one or more designated positions on the reference sequence. When the corresponding sequence is aligned to the greatest extent, a subgroup of sequenced nucleic acid can be identified, which includes a position corresponding to the designated position of the reference sequence. In such a subgroup, it can be determined which (if any) sequenced nucleic acids contain nucleotide variations at designated positions, and optionally which (if any) contain reference nucleotides (e.g., the same as in the reference sequence). If the number of sequenced nucleic acids including nucleotide variants in the subgroup exceeds a selected threshold, variant nucleotides can be called at designated positions. The threshold value can be a number, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 sequenced nucleic acids in the subset comprising nucleotide variations, or the threshold value can be a ratio of sequenced nucleic acids in the subset comprising nucleotide variations, such as at least about 0.5, 1, 2, 3, 4, 5, 10, 15 or 20 and other possibilities. Any specified position of interest in the reference sequence can be compared repeatedly. Sometimes, a specified position occupying at least about 20, 100, 200 or 300 consecutive positions on the reference sequence, for example, about 20-500 or about 50-300 consecutive positions can be compared.

Additional details about nucleic acid sequencing, including the formats and applications described herein, are also provided in the following references: for example, Levy et al., Annual Review of Genomics and Human Genetics, 17:95-115 (2016); Liu et al., J. of Biomedicine and Biotechnology, Volume 2012, Article ID 251364:1-11 (2012); Voelkerding et al., Clinical Chem., 55:641-658 (2009); MacLean et al., Nature Rev. Microbiol., 7:287-296 (2009), Astier et al., J Am Chem. Soc., 128(5):1705-10(2006); U.S. Patent No. 6,210,891, U.S. Patent No. 6,258,568, U.S. Patent No. 6,833,246, U.S. Patent No. 7,115,400, U.S. Patent No. 6,969,488, U.S. Patent No. 5,912,148, U.S. Patent No. 6,130,073, U.S. Patent No. 7,169,560, U.S. Patent No. 7,282,337, U.S. Patent No. No. 7,482,120, U.S. Pat. No. 7,501,245, U.S. Pat. No. 6,818,395, U.S. Pat. No. 6,911,345, U.S. Pat. No. 7,501,245, U.S. Pat. No. 7,329,492, U.S. Pat. No. 7,170,050, U.S. Pat. No. 7,302,146, U.S. Pat. No. 7,313,308, and U.S. Pat. No. 7,476,503, each of which is hereby incorporated by reference in its entirety.

I. Exemplary Workflow

Exemplary workflows are provided herein. In some embodiments, some or all features of the partitioning and library preparation workflows can be used in combination with each other or with other features of the methods described herein.

a. Partition

In some embodiments, sample nucleic acid molecules, such as DNA (e.g., between 5ng and 200ng), are mixed with a methyl binding domain (MBD) buffer and magnetic beads conjugated to the MBD protein, and incubated overnight. During this incubation, methylated DNA (highly methylated DNA) binds to the MBD protein on the magnetic beads. Unmethylated (lowly methylated DNA) or less methylated (medium methylated) DNA is washed off the beads with a buffer containing increasing salt concentrations. For example, one, two or more fractions containing unmethylated DNA, lowly methylated DNA and/or medium methylated DNA can be obtained from such washings. Finally, the heavily methylated DNA (highly methylated DNA) is eluted from the MBD protein using a high salt buffer. In some embodiments, these washes produce three partitions (lowly methylated partitions, medium methylated fractions, and high methylated partitions) of DNA with increasing methylation levels.

In some embodiments, three partitions of DNA are desalted and concentrated in preparation for the enzymatic steps of library preparation.

b. Library Preparation

In some embodiments (e.g., after the DNA in the partition is concentrated), the DNA of the partition is made ligatable, for example, by extending the terminal overhangs of the DNA molecules, and adding adenosine residues to the 3' ends of the fragments, and phosphorylating the 5' ends of each DNA fragment. DNA ligase and adapters are added to connect adapters at each end of the DNA molecules of each partition. These adapters contain partition tags (e.g., non-random, non-unique barcodes) that are distinguishable from the partition tags in the adapters for other partitions. Before or after making the DNA of the partition ligatable and connecting, digest at least one partition (e.g., a high methylation partition, or, if applicable, a high methylation partition and a medium methylation partition) with an MSRE (e.g., an MSRE that preferentially cleaves unmethylated DNA, such as one or more or each of HpaII, BstUI, and Hin6I). Optionally, the low methylation partition can be digested with an MSRE such as FspEI that preferentially cleaves methylated DNA. Optionally, the high methylation partition can undergo a program that differently affects the first nucleobase in the DNA and the second nucleobase in the DNA, such as any of the programs described herein. In the case where a procedure that differentially affects a first nucleobase in DNA and a second nucleobase in DNA further partitions a hypermethylated partition, the procedure should be followed by ligation of adapters so that sub-partitions of the hypermethylated partition can be differentially tagged. The three (or more) partitions are then pooled together and amplified (e.g., by PCR, such as using primers specific for the adapters).

After PCR, the amplified DNA can be cleaned and concentrated before enrichment. The amplified DNA is contacted with a probe set (which can be, for example, a biotinylated RNA probe) targeting a specific region of interest as described herein. The mixture is incubated, for example, overnight, for example, in a salt buffer. The probe is captured (for example, using streptavidin magnetic beads) and separated from the uncaptured amplified DNA, such as by a series of salt washes, thereby enriching the sample. After enrichment, the enriched sample is amplified by PCR. In some embodiments, the PCR primer contains a sample tag, thereby incorporating the sample tag into the DNA molecule. In some embodiments, DNA from different samples is pooled together, and then multiple sequencing is performed, for example, using an Illumina NovaSeq sequencer.

J. Compositions Comprising Captured Nucleic Acid Molecules

Provided herein is a combination comprising a first and a second DNA population, wherein the second population is included in a DNA fragment having an end or attached label or adapter at the recognition site of at least one MSRE, and the MSRE can be any one or any combination of the MSRE described herein. In some embodiments, the first and second population differences are labeled. The first population can include or be derived from a DNA modified with a larger proportion of cytosine than the second population. The first population can include a form of the first nuclear base originally present in DNA with a base pairing specificity that has been changed and a second nuclear base of the base pairing specificity that has not been changed, wherein the form of the first nuclear base originally present in DNA before the base pairing specificity changes is a modified or unmodified nuclear base, the second nuclear base is a modified or unmodified nuclear base different from the first nuclear base, and the form of the first nuclear base originally present in DNA before the base pairing specificity changes and the second nuclear base have the same base pairing specificity. In some embodiments, cytosine modification is cytosine methylation. In some embodiments, the first nuclear base is a modified or unmodified cytosine, and the second nuclear base is a modified or unmodified cytosine. The first core base and the second core base can be any core base discussed herein or with respect to subjecting the first partition to a procedure that differently affects the first core base in the DNA of the first partition and the second core base in the DNA. In some embodiments, the first population comprises DNA fragments having ends, or attached tags or adapters at the recognition site of at least one MSRE, which can be any one or any combination of the MSREs described herein.

In some embodiments, the first population comprises sequence tags selected from a first set of one or more sequence tags, and the second population comprises sequence tags selected from a second set of one or more sequence tags, and the second set of sequence tags is different from the first set of sequence tags.The sequence tags may include barcodes.

In some embodiments, the first population comprises protected hmC, such as glucosylated hmC.

In some embodiments, the first population undergoes any conversion procedure discussed herein, such as bisulfite conversion, Ox-BS conversion, TAB conversion, ACE conversion, TAP conversion, TAPSβ conversion, or CAP conversion. In some embodiments, the first population undergoes protection of hmC and then undergoes deamination of mC and/or C.

In some embodiments of the combination, the first population comprises or is derived from a DNA modified with a greater proportion of cytosine than the second population, and the first population includes a first subgroup and a second subgroup, and the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity. In some embodiments, the second population does not comprise the first nucleobase. In some embodiments, the first nucleobase is a modified or unmodified cytosine, and the second nucleobase is a modified or unmodified cytosine, optionally wherein the modified cytosine is mC or hmC. In some embodiments, the first nucleobase is a modified or unmodified adenine, and the second nucleobase is a modified or unmodified adenine, optionally wherein the modified adenine is mA.

In some embodiments, the first nucleobase (e.g., modified cytosine) is biotinylated. In some embodiments, the first nucleobase (e.g., modified cytosine) is the product of Huisgen cycloaddition of β-6-azido-glucosyl-5-hydroxymethylcytosine, which comprises an affinity tag (e.g., biotin).

In any of the combinations described herein, the captured DNA may include cfDNA.

The captured DNA can have any of the features described herein about the capture group, including, for example, a greater concentration of DNA corresponding to a sequence variable target group than a concentration of DNA corresponding to an epigenetic target group (normalized for footprint size as discussed above). In some embodiments, the DNA of the capture group comprises a sequence tag, which can be added to the DNA as described herein. Typically, the inclusion of sequence tags causes DNA molecules to be different from their naturally occurring, untagged forms. The combination can also include probe groups or sequencing primers described herein, each of which can be different from naturally occurring nucleic acid molecules. For example, the probe groups described herein can include a capture portion, and a sequencing primer can include a non-naturally occurring label.

III. Computer System

The methods of the present disclosure can be implemented using or with the aid of a computer system. For example, such methods may include: (a) providing a biological sample of nucleic acid molecules, wherein the nucleic acid molecules include methylated nucleic acid molecules and unmethylated nucleic acid molecules; (b) partitioning at least one subset of nucleic acid molecules in the biological sample into more than one partition group based on the methylation state of the nucleic acid molecules; (c) digesting at least one subset of one or more of the partition groups with at least one methylation-sensitive restriction endonuclease; (d) enriching at least one subset of nucleic acid molecules in more than one partition group for a genomic region of interest, wherein at least one subset of nucleic acid molecules comprises digested nucleic acid molecules in one or more partition groups; and (e) determining the methylation state of one or more genetic loci of nucleic acid molecules in at least one of the partition groups, which is then used to determine the presence or absence of cancer in the subject.

Fig. 5 shows a computer system 501 programmed or otherwise configured to implement the method of the present disclosure. Computer system 501 can control various aspects of sample preparation, sequencing and/or analysis. In some instances, computer system 501 is configured to perform sample preparation and sample analysis, including nucleic acid sequencing.

In some embodiments, the method further includes obtaining more than one sequence read generated by a nucleic acid sequencer from sequencing; mapping the more than one sequence read to one or more reference sequences to generate mapped sequence reads; and processing the mapped sequence reads to determine the likelihood that the subject has cancer.

The computer system 501 includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor") 505, which may be a single-core or multi-core processor or more than one processor for parallel processing. The computer system 501 also includes a memory or memory location 510 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 515 (e.g., a hard disk), a communication interface 520 (e.g., a network adapter) for communicating with one or more other systems, and peripherals 525, such as cache, other memory, data storage, and/or an electronic display adapter. The memory 510, storage unit 515, interface 520, and peripherals 525 communicate with the CPU 505 via a communication network or bus (real line) such as a motherboard. The storage unit 515 may be a data storage unit (or data repository) for storing data. The computer system 501 may be operably coupled to a computer network 530 by means of the communication interface 520. Computer network 530 can be the Internet, an intranet and/or an extranet, or an intranet and/or an extranet that communicates with the Internet. In some cases, computer network 530 is a telecommunications and/or data network. Computer network 530 can include one or more computer servers, which can enable distributed computing, such as cloud computing. In some cases, with the aid of computer system 501, computer network 530 can implement a peer-to-peer network that can enable devices coupled to computer system 501 to operate as clients or servers.

The CPU 505 may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 510. Examples of operations performed by the CPU 405 may include reading, decoding, executing, and writing back.

Storage unit 515 can store files, such as drivers, libraries, and saved programs. Storage unit 515 can store user-generated programs and recorded sessions and outputs related to programs. Storage unit 515 can store user data, such as user preferences and user programs. In some cases, computer system 501 may include one or more additional data storage units, which are external to computer system 501, such as located on a remote server that communicates with computer system 501 via an intranet or the Internet. Data can be transferred from one location to another using, for example, a communication network or a physical data transfer (e.g., using a hard drive, a thumb drive, or other data storage mechanisms).

Computer system 501 can communicate with one or more remote computer systems via network 530. For embodiments, computer system 501 can communicate with a remote computer system of a user (e.g., an operator). Examples of remote computer systems include personal computers (e.g., portable PCs), slate or tablet PCs (e.g., iPad, Galaxy Tab), phones, smartphones (e.g. iPhone, Android supported devices, ) or a personal digital assistant. A user can access the computer system 501 through a network 530.

The methods as described herein may be implemented by means of machine (e.g., computer processor) executable code stored in an electronic storage location of the computer system 501, such as, for example, the memory 510 or the electronic storage unit 515. The machine executable code or machine readable code may be provided in the form of software. During use, the code may be executed by the processor 505. In some cases, the code may be retrieved from the storage unit 515 and stored on the memory 510 for immediate access by the processor 505. In some cases, the electronic storage unit 515 may be excluded and the machine executable instructions may be stored on the memory 510.

In one aspect, the present disclosure provides a non-transitory computer-readable medium comprising computer-executable instructions that, when executed by at least one electronic processor, perform at least a portion of a method comprising: (a) providing a biological sample of nucleic acid molecules, wherein the nucleic acid molecules include methylated nucleic acid molecules and unmethylated nucleic acid molecules; (b) partitioning at least a subset of the nucleic acid molecules in the biological sample into more than one partitioning group based on the methylation status of the nucleic acid molecules; (c) digesting at least one subset of one or more of the more than one partitioning groups with at least one methylation-sensitive restriction endonuclease; (d) enriching at least one subset of the nucleic acid molecules in the more than one partitioning group for a genomic region of interest, wherein at least one subset of the nucleic acid molecules comprises digested nucleic acid molecules in one or more partitioning groups; and (e) determining the methylation status at one or more genetic loci of the nucleic acid molecules in at least one of the partitioning groups, which is then used to detect the presence or absence of cancer in a subject.

The code may be precompiled and configured for use with a machine having a processor suitable for executing the code or may be compiled during runtime. The code may be provided in a programming language that may be selected so that the code can be executed in a precompiled or as-compiled manner.

Aspects of the systems and methods provided herein, such as computer system 501, can be embodied in programming. Various aspects of the technology can be considered to be "products" or "articles of manufacture," which are generally in the form of machine (or processor) executable code and/or related data carried on or embodied in some type of machine-readable medium. The machine executable code can be stored in an electronic storage unit such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. "Storage" type media can include any or all tangible memories of a computer, processor, etc., or their related modules, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-temporary storage for software programming at any time.

All or part of the software can sometimes communicate via the Internet or various other telecommunication networks. For example, such communication can enable software to be loaded from one computer or processor to another, for example, from a management server or mainframe to a computer platform of an application server. Therefore, another type of medium that can carry software elements includes optical waves, electric waves, and electromagnetic waves such as those used across physical interfaces between local devices, through wired and optical fiber landline networks, and on various air links (air-link). Physical elements carrying such waves, such as wired or wireless links, optical links, etc., can also be considered as media carrying software. As used herein, unless limited to non-temporary, tangible "storage" media, terms such as computer or machine "readable media" refer to any medium that participates in providing instructions to a processor for execution.

Thus, machine-readable media, such as computer executable code, may take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, such as any storage device in any one or more computers, etc., such as the storage device shown in the accompanying drawings that can be used to implement a database, etc. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and optical fiber, including the wires that make up the bus within a computer system. Carrier transmission media may take the form of electrical or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer-readable media include, for example: a floppy disk, a flexible disk, a hard disk, magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, punch cards, paper tape, any other physical storage medium with a pattern of holes, a RAM, a ROM, a PROM and an EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave that transports data or instructions, a cable or link that transports such a carrier wave, or any other medium from which a computer can read programming code and/or data. Many of these forms of computer-readable media may participate in transmitting one or more sequences of one or more instructions to a processor for execution.

Computer system 501 may include or communicate with an electronic display 535 that includes a user interface (UI) 540 to provide, for example, one or more results of a sample analysis. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

Additional details relating to computer systems and networks, databases, and computer program products are also provided in, for example, Peterson, Computer Networks: A Systems Approach, Morgan Kaufmann, 5th Edition (2011); Kurose, Computer Networking: A Top-Down Approach, Pearson, 7th Edition (2016); Elmasri, Fundamentals of Database Systems, Addison Wesley, 6th Edition (2010); Coronel, Database Systems: Design, Implementation, & Management, Cengage Learning, 11th Edition (2014); Tucker, Programming Languages, McGraw-Hill Science/Engineering/Math, 2nd Edition (2006); and Rhoton, Cloud Computing Architected: Solution Design Handbook, Recursive Press (2011), each of which is hereby incorporated by reference in its entirety.

IV. Application

A. Cancer and other diseases

The present method can be used to diagnose the presence or absence of a condition, particularly cancer, in a subject, to characterize the condition (e.g., to stage cancer or determine the heterogeneity of cancer), monitor the response of the condition to treatment, and achieve a prognosis of the risk of the development of the condition or the subsequent course of the condition. The present disclosure can also be used to determine the efficacy of a particular treatment option. If the treatment is successful, a successful treatment option may increase the amount of copy number variation or rare mutation detected in the subject's blood as more cancers may die and cause DNA to fall off. In other instances, this may not happen. In another instance, perhaps certain treatment options may be associated with the genetic spectrum of cancer over time. This correlation can be used to select therapy. In some embodiments, hypermethylated variable epigenetic target regions are analyzed to determine whether they show the high methylation characteristics of tumor cells or cells that do not usually contribute significantly to cfDNA, and/or low methylated variable epigenetic target regions are analyzed to determine whether they show the low methylation characteristics of tumor cells or cells that do not usually contribute significantly to cfDNA.

Additionally, if a cancer is observed to be in remission following treatment, the present method can be used to monitor for residual disease or recurrence of disease.

In some embodiments, the methods and systems disclosed herein can be used to identify tailored or targeted therapies to treat a patient's specific disease or condition based on classifying nucleic acid variations as somatic or germline in origin. Typically, the disease under consideration is a type of cancer. Non-limiting examples of such cancers include biliary tract cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, glioma, astrocytoma, breast cancer, metaplastic carcinoma, cervical cancer, cervical squamous cell carcinoma, rectal cancer, colorectal cancer, colon cancer, hereditary nonpolyposis colorectal cancer, colon adenocarcinoma, gastrointestinal stromal tumor (GIST), endometrial cancer, endometrial stromal sarcoma, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, ocular melanoma, uveal melanoma, gallbladder cancer, gallbladder adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, transitional cell carcinoma, urothelial carcinoma, Wilms tumor, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), liver cancer, liver cancer, carcinoma), hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, B-cell lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma, precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma, multiple myeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharyngeal cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary tumor, follicular cell carcinoma. Prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, stomach cancer, gastric carcinoma, gastrointestinal stromal tumor (GIST), uterine cancer or uterine sarcoma. The type and/or stage of cancer can be detected based on genetic variations, including mutations, rare mutations, insertions/deletions, copy number variations, transversions, translocations, inversions, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structural changes, gene fusions, chromosome fusions, gene truncations, gene amplifications, gene duplications, chromosomal damage, DNA damage, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, and abnormal changes in nucleic acid 5-methylcytosine.

Genetic data can also be used to characterize specific forms of cancer. Cancer is often heterogeneous in both composition and staging. Genetic profile data can allow for characterization of a specific subtype of cancer, which may be important in the diagnosis or treatment of that specific subtype. This information can also provide clues to the prognosis of a specific type of cancer to a subject or practitioner, and allow the subject or practitioner to adjust treatment options based on the progression of the disease. Some cancers can progress and become more aggressive and genetically unstable. Other cancers can remain benign, inactive, or dormant. The systems and methods of the present disclosure can be used to determine disease progression.

In addition, the method of the present disclosure can be used to characterize the heterogeneity of the abnormal condition of the subject. Such a method may include, for example, generating a genetic spectrum of extracellular polynucleotides derived from the subject, wherein the genetic spectrum includes more than one data obtained by copy number variation and rare mutation analysis. In some embodiments, the abnormal condition is cancer. In some embodiments, the abnormal condition can be a condition that leads to a heterogeneous genome population. In the example of cancer, it is known that some tumors contain tumor cells in different stages of cancer. In other examples, heterogeneity can include multiple lesions of the disease. Again, in the example of cancer, multiple tumor lesions may be present, perhaps one or more of which are the result of a metastasis that has spread from the primary site.

The method can be used to generate or analyze a fingerprint or data set that is the sum of genetic information from different cells in a heterogeneous disease. The data set can include copy number variation, epigenetic variation, and mutation analysis alone or in combination.

The present method can be used for diagnosis, prognosis, monitoring or observation of cancer or other diseases. In some embodiments, the methods herein do not involve diagnosis, prognosis or monitoring of a fetus, and therefore do not involve non-invasive prenatal testing. In other embodiments, these methods can be used in pregnant subjects to diagnose, prognose, monitor or observe cancer or other diseases in unborn subjects, whose DNA and other polynucleotides can co-circulate with maternal molecules.

Non-limiting examples of other genetically based diseases, disorders, or conditions that are optionally assessed using the methods and systems disclosed herein include achondroplasia, alpha-1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Charcot-Marie-Tooth disease (CMT), Cry-a-Cat syndrome, Crohn's disease, cystic fibrosis, Dercum's disease, Down syndrome, Duane syndrome, Duchenne muscular dystrophy, Factor V Leiden's thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, Klinefelter syndrome, Marfan syndrome, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, retinitis pigmentosa, severe combined immunodeficiency (scid), sickle cell disease, spinal muscular atrophy, Tay-Sachs disease, thalassemia, trimethylaminuria, Turner syndrome, velocardiofacial syndrome, WAGR syndrome, Wilson's disease, etc.

In some embodiments, the methods described herein include detecting the presence or absence of DNA derived from or originating from tumor cells using a sequence information set obtained as described herein at a preselected time point after a previous cancer treatment in a subject previously diagnosed with cancer. The method may also include determining a cancer recurrence score indicating the presence or absence of DNA derived from or originating from tumor cells of the test subject.

In the case where a cancer recurrence score is determined, it can be further used to determine a cancer recurrence state. For example, when the cancer recurrence score is above a predetermined threshold, the cancer recurrence state may be at risk of cancer recurrence. For example, when the cancer recurrence score is below a predetermined threshold, the cancer recurrence state may be at a low or lower risk of cancer recurrence. In a specific embodiment, a cancer recurrence score equal to a predetermined threshold can result in a cancer recurrence state that is at risk of cancer recurrence or at a low or lower risk of cancer recurrence.

In some embodiments, the cancer recurrence score is compared to a predetermined cancer recurrence threshold, and when the cancer recurrence score is above the cancer recurrence threshold, the test subject is classified as a candidate for subsequent cancer treatment, or when the cancer recurrence score is below the cancer recurrence threshold, the test subject is classified as a non-candidate for treatment. In specific embodiments, a cancer recurrence score equal to the cancer recurrence threshold can result in classification as a candidate for subsequent cancer treatment or a non-candidate for treatment.

The methods discussed above may also include any compatible features or features set forth elsewhere herein, including in the sections regarding methods of determining a test subject's risk of cancer recurrence and/or classifying a test subject as a candidate for subsequent cancer treatment.

B. Methods for Determining a Test Subject's Risk of Cancer Recurrence and/or Classifying a Test Subject as a Candidate for Subsequent Cancer Treatment

In some embodiments, the methods provided herein are methods of determining a test subject's risk of cancer recurrence. In some embodiments, the methods provided herein are methods of classifying a test subject as a candidate for subsequent cancer treatment.

Any of such methods may include collecting DNA (e.g., derived from or derived from tumor cells) from a test subject diagnosed with cancer at one or more preselected time points after one or more previous cancer treatments have been performed on the test subject. The subject may be any subject described herein. The DNA may be cfDNA. The DNA may be obtained from a tissue sample.

Any of such methods may include capturing more than one target region set from DNA from a subject, wherein the more than one target region set includes a sequence variable target region set and an epigenetic target region set, thereby generating a captured set of DNA molecules. The capture step may be performed according to any embodiment described elsewhere herein.

In any of such methods, prior cancer treatment may include surgery, administration of a therapeutic composition, and/or chemotherapy.

Any of such methods may include sequencing the captured DNA molecules, thereby generating a sequence information set.The captured DNA molecules of the sequence variable target region set may be sequenced to a greater sequencing depth than the captured DNA molecules of the epigenetic target region set.

Any of such methods may include using the sequence information set to detect the presence or absence of DNA derived from or derived from tumor cells at a preselected time point. The detection of the presence or absence of DNA derived from or derived from tumor cells may be carried out according to any of its embodiments described elsewhere herein.

The method for determining the risk of cancer recurrence of a test subject may include determining a cancer recurrence score, the cancer recurrence score indicating the presence or absence or amount of DNA derived from or derived from a tumor cell of the test subject. The cancer recurrence score may be further used to determine a cancer recurrence state. For example, when the cancer recurrence score is higher than a predetermined threshold, the cancer recurrence state may be at a risk of cancer recurrence. For example, when the cancer recurrence score is lower than a predetermined threshold, the cancer recurrence state may be at a low or lower risk of cancer recurrence. In a specific embodiment, a cancer recurrence score equal to a predetermined threshold can be obtained to be at a risk of cancer recurrence or at a low or lower risk of cancer recurrence.

The method of classifying a test subject as a candidate for a subsequent cancer treatment may include comparing the test subject's cancer recurrence score to a predetermined cancer recurrence threshold, thereby classifying the test subject as a candidate for a subsequent cancer treatment when the cancer recurrence score is above the cancer recurrence threshold, or classifying the test subject as a non-candidate for treatment when the cancer recurrence score is below the cancer recurrence threshold. In certain embodiments, a cancer recurrence score equal to the cancer recurrence threshold can result in a classification as a candidate for a subsequent cancer treatment or a non-candidate for treatment. In some embodiments, the subsequent cancer treatment includes chemotherapy or administration of a therapeutic composition.

Any of such methods may include determining a disease-free survival (DFS) period for the test subject based on the cancer recurrence score; for example, the DFS period may be 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years.

In some embodiments, the sequence information set includes sequence variable target region sequences, and determining the cancer recurrence score can include determining at least a first sub-score that indicates the amount of SNVs, indels, CNVs, and/or fusions present in the sequence variable target region sequences.

In some embodiments, the number of mutations selected from 1, 2, 3, 4, or 5 in the sequence variable target region is sufficient for the first subscore to result in the cancer recurrence score being classified as cancer recurrence positive. In some embodiments, the number of mutations is selected from 1, 2, or 3.

In some embodiments, the sequence information set includes an epigenetic target region sequence, and determining the cancer recurrence score includes determining a second sub-score indicating a change in an epigenetic feature in the epigenetic target region sequence (e.g., methylation of a hypermethylated variable target region and/or perturbed fragmentation of a fragmented variable target region, where "perturbed" means different from DNA found in a corresponding sample from a healthy subject). In some such embodiments, determining the cancer recurrence score includes determining a second sub-score indicating the amount of a molecule (obtained from the epigenetic target region sequence) that represents an epigenetic state different from DNA found in a corresponding sample from a healthy subject (e.g., cfDNA found in a blood sample from a healthy subject, or DNA found in a tissue sample from a healthy subject, where the tissue sample is the same type of tissue as the tissue obtained from the test subject). These abnormal molecules (i.e., molecules having an epigenetic state different from DNA found in a corresponding sample from a healthy subject) can be consistent with epigenetic changes associated with cancer (e.g., methylation of a hypermethylated variable target region and/or perturbed fragmentation of a fragmented variable target region).

In some embodiments, the proportion of molecules corresponding to the hypermethylated variable target region group and/or the fragmented variable target region group that is greater than or equal to a value in the range of 0.001%-10% indicating hypermethylation in the hypermethylated variable target region group and/or aberrant fragmentation in the fragmented variable target region group is sufficient for the second subscore to be classified as positive for cancer recurrence. The range may be 0.001%-1%, 0.005%-1%, 0.01%-5%, 0.01%-2%, or 0.01%-1%.

In some embodiments, any of such methods may include determining a tumor DNA score based on the fraction of molecules in a sequence information group indicating one or more features indicating a source from a tumor cell. This can be used for molecules corresponding to some or all of the epigenetic target regions, for example, including one or both of a hypermethylated variable target region and a fragmented variable target region (hypermethylation of a hypermethylated variable target region and/or abnormal fragmentation of a fragmented variable target region can be considered to indicate a source from a tumor cell). This can be used for molecules corresponding to sequence variable target regions, for example, molecules containing changes consistent with cancer (such as SNVs, insertions/deletions, CNVs, and/or fusions). The tumor DNA score can be determined based on a combination of molecules corresponding to epigenetic target regions and molecules corresponding to sequence variable target regions.

The determination of the cancer recurrence score can be based at least in part on the tumor DNA score, wherein a tumor DNA score greater than a threshold value in the range of ^10-11 to 1 or ^10-10 to 1 is sufficient for the cancer recurrence score to be classified as positive for cancer recurrence. In some embodiments, a tumor DNA score greater than or equal to a threshold value in the range of ^10-10 to ^10-9 , ^10-9 to ^10-8 , ^10-8 to ^10-7 , ^10-7 to ^10-6 , ^10-6 to ^10-5 , ^10-5 to ^10-4 , ^10-4 to ^10-3 , ^10-3 to ^10-2 , or ^10-2 to ^10-1 is sufficient for the cancer recurrence score to be classified as positive for cancer recurrence. In some embodiments, a tumor DNA score greater than a threshold value of at least ^10-7 is sufficient for the cancer recurrence score to be classified as positive for cancer recurrence. The tumor DNA score being greater than a threshold value, such as a threshold value corresponding to any of the foregoing embodiments, can be determined based on a cumulative probability. For example, if the cumulative probability that the tumor score is greater than a threshold in any of the aforementioned ranges exceeds a probability threshold of at least 0.5, 0.75, 0.9, 0.95, 0.98, 0.99, 0.995, or 0.999, the sample is considered positive. In some embodiments, the probability threshold is at least 0.95, such as 0.99.

In some embodiments, the sequence information group includes sequence variable target region sequences and epigenetic target region sequences, and determining the cancer recurrence score includes determining a first sub-score indicating the amount of SNVs, insertions/deletions, CNVs, and/or fusions present in the sequence variable target region sequences and a second sub-score indicating the amount of abnormal molecules in the epigenetic target region sequences, and combining the first sub-score and the second sub-score to provide a cancer recurrence score. In the case of combining the first sub-score and the second sub-score, they can be combined by applying a threshold to each sub-score independently (e.g., greater than a predetermined number of mutations (e.g., >1) in the sequence variable target region, and greater than a predetermined number of abnormal molecules (i.e., molecules with an epigenetic state different from that of DNA found in a corresponding sample from a healthy subject; e.g., a tumor) score), or training a machine learning classifier to determine a state based on more than one positive training sample and a negative training sample.

In some embodiments, a value for the combined score in the range of -4 to 2 or -3 to 1 is sufficient for the cancer recurrence score to be classified as positive for cancer recurrence.

In any embodiment in which the cancer recurrence score is classified as cancer recurrence positive, the cancer recurrence status of the subject may be at risk for cancer recurrence and/or the subject may be classified as a candidate for subsequent cancer treatment.

In some embodiments, the cancer is any of the cancer types described elsewhere herein, e.g., colorectal cancer.

C. Treatment and related management

In certain embodiments, methods disclosed herein relate to identifying customized therapy and administering customized therapy to patients in view of the state that nucleic acid variation is somatic cell source or germline source. In some embodiments, substantially any cancer therapy (e.g., surgical therapy, radiotherapy, chemotherapy and/or similar therapy) can be included as part of these methods. Typically, customized therapy includes at least one immunotherapy (or immunotherapeutic agent). Immunotherapy generally refers to a method for enhancing the immune response for a specific cancer type. In certain embodiments, immunotherapy refers to a method for enhancing the T cell response for a tumor or cancer.

In certain embodiments, the state of the nucleic acid variation of the sample from the subject as a somatic cell source or a germline source can be compared with a database of comparator results (comparator results) from a reference population to identify a customized or targeted therapy for the subject. Typically, the reference population includes patients with the same cancer or disease type as the test subject and/or patients who are receiving or have received the same treatment as the test subject. When the nucleic acid variation and the comparator result meet certain classification criteria (e.g., a substantial or approximate match), customized or targeted therapy (or more than one treatment) can be identified.

In certain embodiments, the customized therapy described herein is generally administered parenterally (e.g., intravenously or subcutaneously). Pharmaceutical compositions containing immunotherapeutics are generally administered intravenously. Certain therapeutic agents are administered orally. However, customized therapy (e.g., immunotherapeutics, etc.) can also be administered by the following methods, such as, for example, buccal, sublingual, rectal, vaginal, intraurethral, topical, intraocular, intranasal and/or intraauricular, and the administration may include tablets, capsules, granules, aqueous suspensions, gels, sprays, suppositories, salve, ointment, etc.

D. Test kit

Also provided is a test kit comprising a composition as described herein.Test kit can be used for carrying out a method as described herein.Test kit comprises at least one MSRE.In some embodiments, test kit also comprises the first reagent for partitioning a sample into more than one partition as described herein, such as any partition reagent described elsewhere herein.In some embodiments, test kit comprises a second reagent (for example, for converting a core base such as cytosine or methylated cytosine into any reagent described elsewhere herein of different core bases), and the second reagent is used to make the first partition experience differently affect the first core base in the DNA of the first partition and the program of the second core base in the DNA, wherein the first core base is a modified or unmodified core base, the second core base is a modified or unmodified core base different from the first core base, and the first core base and the second core base have identical base pairing specificity.Test kit can comprise the first reagent and the second reagent and the other elements discussed below and/or elsewhere herein.

The kit may also contain more than one oligonucleotide probe that selectively hybridizes to at least 5, 6, 7, 8, 9, 10, 20, 30, 40 or all genes selected from the group consisting of: ALK, APC, BRAF, CDKN2A, EGFR, ERBB2, FBXW7, KRAS, MYC, NOTCH1, NRAS, PIK3CA, PTEN, RBI, TP53, MET, AR, ABL1, AKT1, ATM, CDH1, CSFIR, CTNNB1, ERBB4, EZ , FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, MLH1, MPL, NPM1, PDGFRA, PROC, PTPN11, RET, SMAD4, SMARCB1, SMO, SRC, STK11, VHL, TERT, CCND1, CDK4, CDKN2B, RAF1, BRCA1, CCND2, CDK6, NF1, TP53, ARID1A, BRCA2, CCNE1, ESR1, RIT1, GATA3, MAP2K1, RHEB, ROS1, ARAF, MAP2K2, NFE2L2, RHOA, and NTRK1. The number of genes to which the oligonucleotide probe can selectively hybridize can vary. For example, the number of genes can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54. The kit can include a container comprising more than one oligonucleotide probe and instructions for performing any of the methods described herein.

Oligonucleotide probes can selectively hybridize with the exon region of a gene (e.g., at least 5 genes). In some cases, oligonucleotide probes can selectively hybridize with at least 30 exons of a gene (e.g., at least 5 genes). In some cases, more than one probe can selectively hybridize with each of at least 30 exons. The probe hybridized with each exon can have a sequence overlapping with at least 1 other probe. In some embodiments, oligoprobes can selectively hybridize with the non-coding region (e.g., the intron region of a gene) of a gene disclosed herein. Oligoprobes can also selectively hybridize with the region of a gene comprising both the exon region and the intron region of a gene disclosed herein.

The oligonucleotide probes can target any number of exons. For example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 250, 251, 252, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265 70, 80 ...

The kit may include at least 4, 5, 6, 7 or 8 different library adapters with different molecular barcodes and the same sample barcode. The library adapter may not be a sequencing adapter. For example, the library adapter does not include a flow cell sequence or a sequence that allows the formation of a hairpin loop for sequencing. Different variations and combinations of molecular barcodes and sample barcodes are described in the full text and are applicable to the kit. In addition, in some cases, the adapter is not a sequencing adapter. In addition, the adapter provided with the kit may also include a sequencing adapter. The sequencing adapter may include a sequence that hybridizes with one or more sequencing primers. The sequencing adapter may also include a sequence that hybridizes with a solid support, such as a flow cell sequence. For example, the sequencing adapter may be a flow cell adapter. The sequencing adapter may be attached to one or both ends of a polynucleotide fragment. In some cases, the kit may include at least 8 different library adapters with different molecular barcodes and the same sample barcode. The library adapter may not be a sequencing adapter. The kit can also include a sequencing adapter, the sequencing adapter having a first sequence selectively hybridized with a library adapter and a second sequence selectively hybridized with a flow cell sequence. In another example, the sequencing adapter can be hairpin-shaped. For example, a hairpin-shaped adapter can include a complementary double-stranded portion and a loop portion, wherein the double-stranded portion can be attached (e.g., connected) to a double-stranded polynucleotide. A hairpin-shaped sequencing adapter can be attached to the two ends of a polynucleotide fragment to produce a circular molecule, which can be sequenced multiple times. 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, The sequencing adapter may comprise 20-30, 20-40, 30-50, 30-60, 40-60, 40-70, 50-60, 50-70 bases from end to end. In a particular example, the sequencing adapter may comprise 20-30 bases from end to end. In another example, the sequencing adapter may comprise 50-60 bases from end to end. The sequencing adapter may comprise one or more barcodes. For example, the sequencing adapter may comprise a sample barcode. The sample barcode may comprise a predetermined sequence. The sample barcode may be used to identify the source of the polynucleotide. The sample barcode can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more (or any length as described throughout) nucleic acid bases, such as at least 8 bases. The barcode can be a continuous or non-continuous sequence, as described above.

The library adapter can be blunt-ended and Y-shaped, and can be less than or equal to 40 nucleic acid bases in length. Other variations of library adapters can be found throughout the text and are suitable for use in this kit.

Example

The following examples are provided to illustrate certain aspects of the disclosed methods. The examples do not limit the present disclosure.

Example 1: Reduction of technical noise by digestion of nonspecifically partitioned DNA

Combine the cfDNA pools from two healthy normal samples, of which 18.6ng is used as the input of the MBD partition assay described herein. The cfDNA from the colorectal cancer sample (CRC) with 0.5% MAF (somatic allele fraction) is added to the subgroup of the sample to obtain the diluted CRC sample with 0.16% MAF. Three groups of normal samples and diluted CRC samples are used for determination. Then the three groups of samples are partitioned into three partitions (high methylation (high) partition, medium (residual) partition and low methylation (low) partition) using MBD protein. After cleaning, the cfDNA molecules in each partition are connected to the partition-specific adapters comprising molecular barcodes. The molecular barcodes of high partitions and residual partitions are selected so that they do not have MSRE recognition sites, so they are not digested in downstream processing (regardless of cfDNA methylation status). After connection, connection cleaning is performed. After connection cleaning, high partitions and residual partitions are subjected to MSRE digestion reaction. The first sample group (normal and diluted CRC samples) was treated with BstUI and HpaII, and another sample group was treated with BstUI, HpaII and Hin6I enzymes. The third sample group was simulated digested (without MSRE) as a control in the MBD partitioning assay. After MSRE digestion, the enzyme was heat inactivated (65°C, 20 minutes) and cleaned up using SPRI beads. After digestion and cleaning, the high partition, residual partition and (undigested) low partition (cfDNA connected to the adapter) were merged and processed by an NGS assay workflow including the following: PCR amplification; Enrichment of molecules in the genomic region of interest; Pooling samples to allow multiple sequencing, and sequencing the pooled samples using NovaSeq. In an optional method, the low partition can be additionally contacted with one or more MSREs with methylation recognition sites to cleave DNA from nonspecific partitions in the low partition.

Figure 6 clearly shows the increase in cancer methylation signals of DMRs relative to technical noise from unmethylated molecules in normal samples when MSRE digestion is applied. In the negative control area shown in Figure 6 (where DNA molecules are unmethylated almost all the time, regardless of disease state), "a" clearly indicates that MSRE digestion significantly removes unmethylated molecules that were incorrectly partitioned into high partitions - in the mock digestion, 90 molecules were partitioned into high partitions, while in BstUI, HpaII, and Hin6I digestions, the molecule count was reduced to 10. In the classified DMRs shown in Figure 6, after digestion with MSRE, cfDNA molecules were removed at a much higher ratio in normal samples (b; 350→100) than in diluted CRC samples (c; 1500→1100).

Example 2: Analysis of cfDNA to detect the presence or absence of tumor

A group of patient samples were analyzed by blood-based NGS assays at Guardant Health (Redwood City, CA, USA) to detect the presence or absence of cancer. cfDNA was extracted from the plasma of these patients. The cfDNA of the patient sample was then combined with a methyl binding domain (MBD) buffer and magnetic beads conjugated to the MBD protein and incubated overnight. During this incubation period, methylated cfDNA (if present in the cfDNA sample) binds to the MBD protein. Unmethylated or less methylated DNA was washed off the beads with a buffer containing increasing salt concentrations. Finally, heavily methylated DNA was washed off the MBD protein using a high salt buffer. These washes produce three partitions of cfDNA with increasing methylation (low methylation partitions, residual methylation partitions, and high methylation partitions).

Optionally, cfDNA molecules in the hypermethylated partition undergo enzymatic modification (EM) whereby unmodified cytosine, but not mC and hmC, undergo deamination, thereby marking the hypomethylated molecules of the non-specific partition in the first partition by converting unmodified cytosine to uracil.

After the cfDNA in the partition is concentrated, the terminal overhangs of the partitioned cfDNA are extended, and adenosine residues are added to the 3' ends of the cfDNA fragments by a polymerase during the extension. The 5' end of each fragment is phosphorylated. These modifications make the partitioned cfDNA connectable. DNA ligase and adapters are added to connect each end of the cfDNA molecule of each partition to the adapter. These adapters contain non-unique molecular barcodes, and each partition is connected to an adapter with a non-unique molecular barcode that is distinguishable from the barcodes in the adapters used for other partitions.

The cfDNA in the low methylation partition is contacted with one or more MSREs having methylation recognition sites. These enzymes cleave the DNA of at least a portion of non-specific partitions in the low methylation partition. Alternatively or additionally, the cfDNA in the high methylation partition is contacted with one or more MSREs having unmethylated recognition sites. These enzymes cleave the DNA of at least a portion of non-specific partitions in the high methylation partition.

After ligation, the four partitions are pooled together and amplified by PCR. Molecules cleaved by one or more MSREs do not undergo exponential amplification because they do not have an adaptor at each end.

After PCR, the amplified DNA is washed and concentrated before enrichment. After concentration, the amplified DNA is combined with a salt buffer and a biotinylated RNA probe (including a probe for a sequence variable target group and a probe for an epigenetic target group), and the mixture is incubated overnight. The probe for the sequence variable group has a footprint of about 50kb, and the probe for the epigenetic target group has a footprint of about 500kb. The probe for the sequence variable target group includes an oligonucleotide targeting at least one gene subset identified in Tables 3-5, and the probe for the epigenetic target group includes an oligonucleotide targeting the following selections: a high methylation variable target region, a low methylation variable target region, a CTCF binding target region, a transcription start site target region, a focused amplification target region, and a methylation control region.

Biotinylated RNA probes (hybridized with DNA) are captured by streptavidin magnetic beads and separated from uncaptured amplified DNA by a series of salt-based washings to enrich the sample. After enrichment, aliquots of the enriched sample are sequenced using the Illumina NovaSeq sequencer. The sequence reads generated by the sequencer are then analyzed using bioinformatics tools/algorithms. Molecular barcodes are used to identify unique molecules and to deconvolute samples into molecules of differential MBD partitions. The method described in this embodiment, in addition to providing information about the overall methylation level (i.e., methylated cytosine residues) of the molecule based on the partition of the molecule (including increased accuracy and/or confidence due to the cleavage of cfDNA of non-specific partitions in low methylation partitions), can also provide information about the position of methylated cytosine based on the conversion of unmethylated cytosine in high methylation partitions. Sequence variable target region sequences are analyzed by detecting genomic changes (such as SNV, insertion, deletion, and fusion) that can be determined, and the determination has sufficient support for distinguishing true tumor mutations from technical errors (e.g., PCR errors, sequencing errors). The epigenetic target sequence is analyzed independently to detect the methylation status of cfDNA molecules in regions that have shown differential methylation (e.g., in potentially cancerous tissue compared to healthy cfDNA). Finally, the results of the two analyses are combined to produce a final tumor presence/absence decision.

Example 3: Analysis of methylation at single nucleotide resolution in cfDNA samples from healthy subjects and subjects with early-stage colorectal cancer

The cfDNA samples from healthy subjects and subjects with early colorectal cancer were analyzed as follows. cfDNA was partitioned using MBD to provide high methylation partitions, medium methylation partitions, and low methylation partitions. The DNA of the partitions in each partition was connected to an adapter and the EM-seq conversion procedure was performed, whereby unmodified cytosines, rather than mC and hmC, were deaminized, although in an optional procedure, the DNA of the partitions in the high methylation partitions could be contacted with the MSRE with unmethylated recognition sites as described herein. After such deamination, the partitions were prepared for sequencing and whole genome sequencing was performed. Sequencing was performed separately for each partition, although in an optional procedure, the partition differences could be labeled (e.g., after the partitions and before EM-seq conversion, or after the partitions and EM-seq conversions and before further sequencing preparations), pooled, processed, and sequenced in parallel.

Sequence data from the hypermethylated variable target regions were bioinformatically separated, although in an optional procedure the target regions can be enriched in vitro prior to sequencing. Per-base methylation of the hypermethylated variable target regions was quantified, as shown in FIG7 , which shows the number of methylated CpGs per molecule in the hypermethylated variable target regions from the hypermethylated partitions. The x-axis indicates the total number of CpGs per molecule, so the points along the diagonal represent molecules with methylation at each CpG. Thus, methylation can be analyzed at single-base resolution and per-base methylation and fractional molecule methylation of MBD partitioned material quantified. Samples from subjects with colorectal cancer exhibited much higher overall methylation in these regions compared to samples from healthy subjects.

Example 4: Analysis of MDRE-digested cfDNA

Multiple aliquots of cfDNA from two healthy donors are separated, and methylated cfDNA is subjected to MBD-based partitioning. Then the low-methylated cfDNA partition is subjected to the connection of NGS adapters to cfDNA molecules. Then the cfDNA from each donor connected is subjected to the digestion of MSRE that preferentially cleaves methylated DNA, also known as methylation-dependent restriction endonuclease (MDRE) digestion. The MDRE used is FspEI, LpnPI, MspJI or SgeI, or "simulated" digestion (no enzyme is added to the digestion), or the undigested condition of skipping MDRE reaction as a control reaction. After the MDRE step, low-methylated cfDNA partitions are amplified in universal PCR, where the DNA cleaved by MDRE is not exponentially amplified because adapters are not present at each end. Then the PCR product is enriched in the targeted genomic region using a hybrid capture group, amplified in the second PCR, and sequenced by NGS. The hybrid capture group target includes a "positive control (ctrl)" area and a "negative control (ctrl)" area for the enriched genome. The positive control region is a CpG-dense region in the genome that is found to be generally highly methylated (>85% methylated, by bisulfite-seq) in all human tissues including blood and cancerous tissues. In contrast, the negative control region is generally unmethylated (<15% methylated) in all human tissues. According to NGS analysis, the number of positive control molecules (i.e., molecules in the positive control region) and negative control molecules (i.e., molecules in the negative control region) sequenced under all conditions is compared to estimate the MDRE sensitivity and specificity, respectively. Figures 8A-8B show that compared with the "simulation" conditions, FspEI enzyme treatment reduces the number of positive control molecules by >100 times, demonstrating a ~99% sensitivity for digestion of methylated molecules. Figures 8C-8D show that FspEI treatment does not significantly reduce negative control molecules, indicating the high specificity of FspEI digestion (unmethylated molecules are not digested). It is noted that MspJI shows a certain sensitivity, but poor specificity compared to FspEI, while LpnI and SgeI show little/no sensitivity.

MDRE digestion efficiency is calculated using molecules with different recognition sites and the number of sites for each molecule. Digestion efficiency is calculated as 1-[number of positive control molecules under MDRE conditions]/[number of positive control molecules under simulation conditions]. The general recognition sequence of FspEI including ^5m CpG is C ^5m CGH (H=A, C or T), wherein cracking occurs in the downstream 12-16 bases. The FspEI palindromic site C ^5m CGG comprises two FspEI recognition sites-on the upper and lower chains in opposite directions. The consensus sequence generally comprising ^5m CpG is ^5m CpGNR, which can overlap with the FspEI consensus sequence. Figure 9 A-Figure 9D shows that digestion efficiency increases with the minimum number of C ^5m CGH or C ^5m CGG sites for each molecule, and is more effective at palindromic sites (C ^5m CGG). Positive control molecules with at least one C ^5m CGG or at least two C ^5m CGH sites are cracked with an efficiency of 95%.

In addition, to being tested with FspEI and MspJI digestion simultaneously or sequentially.Sequential digestion with two kinds of MDRE (FspEI then MspJI) has the highest efficiency.It may be that in digestion simultaneously (FspEI and MspJI), MspJI sometimes binds to DNA but does not crack (lower individual efficiency), thereby spatially blocking FspEI activity.Although FspEI then MspJI has a higher overall efficiency than independent FspEI here, independent FspEI has better cracking specificity.Therefore, in different situations, it may be preferred to digest with FspEI alone or to digest with FspEI then MspJI.Notice that the number of minimum sites is more, the positive control molecules observed are less (Fig. 9 C-Fig. 9 D) and therefore digestion efficiency estimation becomes more noisy.

Example 5: Detection of tumor DNA after MDRE treatment

cfDNA isolated from four healthy donors was used to create "normal" and simulated "cancer" cfDNA samples. Pure donor samples were used as "normal" samples, and "cancer" samples were created by incorporating cfDNA from patients with colorectal cancer (CRC). The circulating tumor DNA fraction of CRC cfDNA samples has been previously measured, and the calculated amount of CRC cfDNA was incorporated into the normal donor cfDNA using this fraction, so that the resulting "cancer" sample contained 0.5% of circulating tumor DNA ("0.5% CRC" in Figure 10A-Figure 10J). MBD-based partitioning was performed on all samples, and cfDNA was divided into high-methylation and low-methylation cfDNA partitions. The low-methylation cfDNA partition was then connected to the NGS adapter. Then FspEI, MspJI, or FspEI+MspJI were used to perform MDRE digestion on the cfDNA from each donor connected. "Simulated digestion" (no enzyme added to the digestion reaction) and "no digestion" conditions (completely skipping the MDRE reaction) were used as control reactions. After the MDRE step, undigested low-methylation partitioned cfDNA is amplified in a universal PCR, and then a hybrid capture group is used for enrichment of the targeted genomic region, and then amplified in a second PCR and sequenced by NGS. The hybrid capture group target includes a low-methylation variable target region and a "negative control (ctrl)" region for the enriched genome. The negative control region is a CpG-dense region in the genome that is found to be generally low-methylated (<15% methylated, bisulfite-seq) in all human tissues including blood and cancerous tissues. The low-methylation variable target region is a genomic region annotated in the literature as having a reduced percentage of methylation in CRC tissues compared to healthy colon tissues and blood. According to NGS analysis, the number of low-methylation variable target region molecules (which should be digested with high efficiency by MDRE) with 2 or more CCGG sites between "normal" and "cancer" samples is compared under all digestion conditions (Figures 10A-10E). The ratio of the low methylation variable target molecule count to the negative control molecule count was also compared, and the ratio was normalized for the variable cfDNA input amount that could affect the low methylation variable target molecule count (Figure 10F-Figure 10J). No distinguishable detection of low methylation variable target cancer signals was observed under MDRE-free digestion conditions ("no digestion" and "simulated digestion"). That is, the low methylation variable target molecule and normalized ratio levels were indistinguishable (no significant difference) between "cancer" and "normal" samples (this is marked by horizontal arrows in Figure 10C, Figure 10E, Figure 10H, and Figure 10J). In contrast, when there was MDRE treatment, the movement (increase) of the low methylation variable target count and normalized ratio was detected in the "cancer" sample compared with the "normal" sample (marked by the upper right arrows in Figure 10A, Figure 10B, Figure 10D, Figure 10F, Figure 10G, and Figure 10I). Thus, MDRE treatment enabled the detection of a cancer hypomethylated variable target signal in a “cancer” sample of 0.5% CRC ctDNA, which was undetectable by the MBD partitioning assay alone.

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Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only by way of example. It is not intended that the present invention be limited to the specific examples provided in the specification. Although the present invention has been described with reference to the foregoing description, the description and illustration of the embodiments herein should not be interpreted in a restrictive sense. Without departing from the present invention, those skilled in the art will now think of many variations, changes and substitutions. In addition, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations or relative proportions set forth herein, which depend on various conditions and variables. It should be understood that various alternatives of the embodiments of the present disclosure described herein may be adopted when practicing the present invention. It is therefore contemplated that the present disclosure should also encompass any such alternatives, modifications, variations or equivalents. It is intended that the appended claims define the scope of the present invention, and thus encompass methods and structures and their equivalents within the scope of these claims.

Although the foregoing disclosure has been described in some detail by way of illustration and example for the purpose of clarity and understanding, it will be clear to those skilled in the art upon reading this disclosure that various changes in form and detail may be made without departing from the true scope of the disclosure and may be implemented within the scope of the appended claims. For example, all methods, systems, computer-readable media and/or component features, steps, elements or other aspects may be used in various combinations.

All patents, patent applications, websites, other publications or documents, accession numbers, etc. cited herein are incorporated by reference in their entirety for all purposes, to the extent that each individual item is specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, it is intended to refer to the version associated with that accession number at the effective filing date of the present application. If applicable, the effective filing date means the earlier of the actual filing date or the filing date of the priority application that mentions the registration number. Similarly, if different versions of a publication, website, etc. are published at different times, it is intended to refer to the version that was published most recently on the actual filing date of the present application, unless otherwise indicated.

Claims (44)

1. A method for analyzing nucleic acid molecules in a biological sample, said method comprising: a) partitioning at least a subgroup of said nucleic acid molecules in said biological sample into more than one partition group based on the methylation status of said nucleic acid molecules, wherein said biological sample comprises methylated nucleic acid molecules and Unmethylated nucleic acid molecules; b) digesting at least a subset of one or more of the more than one compartments with at least one methylation-sensitive restriction enzyme; and c) determining the methylation status at one or more genetic loci of said nucleic acid molecules in at least one of said partition groups. 2. A method for determining the methylation status of a nucleic acid molecule, said method comprising: a) providing a biological sample of nucleic acid molecules, wherein said nucleic acid molecules include methylated nucleic acid molecules and unmethylated nucleic acid molecules; b) partitioning at least a subgroup of said nucleic acid molecules in said biological sample into more than one partition group based on the methylation status of said nucleic acid molecules; c) digesting at least a subset of one or more of the more than one partitions with at least one methylation-sensitive restriction enzyme; d) enriching at least a subset of said nucleic acid molecules in said group of more than one partitions for a genomic region of interest, wherein said at least one subset of nucleic acid molecules comprises said one or more partitions digested nucleic acid molecules in the set; and e) determining the methylation status at one or more genetic loci of said nucleic acid molecules in at least one of said partition groups. 3. A method for analyzing nucleic acid molecules in a biological sample, said method comprising: a) partitioning at least a subgroup of said nucleic acid molecules in said biological sample into more than one partition group based on the methylation status of said nucleic acid molecules, wherein said biological sample comprises methylated nucleic acid molecules and unmethylated nucleic acid molecules, and the more than one partition group comprises a first partition group and a second partition group, wherein methylated nucleic acid molecules are in the first partition group relative to the second partition group overrepresented in b) digesting at least a subset of said first subgroup of said more than one subgroup with at least one methylation sensitive restriction enzyme; and c) capturing a first target segment comprising the epigenetic target region from at least a portion of the first compartment and capturing a second target segment comprising the epigenetic target region from at least a portion of the second segment. 4. The method of claim 3, wherein capturing the first target region comprises contacting DNA in the first region with a first target-specific probe set, and capturing the second target region Grouping includes contacting DNA in said second subgroup with a second set of target-specific probes. 5. The method of claim 3 or 4, further comprising determining the methyl group at one or more genetic loci of the nucleic acid molecule in at least one of the partition or target block. status. 6. The method according to any one of the preceding claims, wherein the genomic region of interest, the first set of target regions and/or the second set of target regions comprise sequence variable target regions. 7. The method according to any one of the preceding claims, further comprising, prior to the digesting step, attaching one or more adapters to at least a portion of the more than one partitioned groups. at least one end of the nucleic acid molecule. 8. A method for determining the methylation status of a nucleic acid molecule, said method comprising: a) providing a biological sample of nucleic acid molecules, wherein said nucleic acid molecules include methylated nucleic acid molecules and unmethylated nucleic acid molecules; b) partitioning at least a subgroup of said nucleic acid molecules in said biological sample into more than one partition group based on the methylation status of said nucleic acid molecules; c) attaching one or more adapters to at least one end of said nucleic acid molecules in said more than one partition; d) digesting at least a subset of one or more of the more than one partitions with at least one methylation-sensitive restriction enzyme; e) enriching at least a subset of said nucleic acid molecules in said group of more than one partitions for a genomic region of interest; wherein said at least one subset of nucleic acid molecules comprises said one or more partitions digested nucleic acid molecules in the set; and f) determining the methylation status at one or more genetic loci of said nucleic acid molecules in at least one of said partition groups. 9. The method of claim 7 or 8, wherein adapters are attached to both ends of at least a portion of the nucleic acid molecules in the more than one partitioned group. 10. The method of claim 1, further comprising, prior to c), enriching at least a subset of the nucleic acid molecules in the more than one partition group for a genomic region of interest , wherein at least a subset of said nucleic acid molecules comprises digested nucleic acid molecules in said one or more partitions. 11. The method according to any one of the preceding claims, further comprising detecting the presence or absence of cancer in the biological sample. 12. The method according to any one of the preceding claims, further comprising determining the level of cancer in the biological sample. 13. The method of any preceding claim, wherein determining the methylation status comprises sequencing at least a subset of the digested nucleic acid molecules. 14. The method of any one of claims 7-13, wherein the one or more adapters comprise at least one tag. 15. The method according to any one of the preceding claims, wherein the methylation sensitive restriction enzyme selectively digests at the recognition site of the methylation sensitive restriction endonuclease Unmethylated nucleic acid molecules. 16. The method according to any one of the preceding claims, wherein at least a portion of the nucleic acid molecule is amplified and/or sequenced after said digestion step and digested by said methylation sensitive restriction endonuclease The nucleic acid molecules are not amplified and/or not sequenced. 17. The method according to any one of the preceding claims, comprising digesting one or more of the more than one compartmental groups with at least two methylation-sensitive restriction endonucleases at least one subgroup of . 18. The method of claim 17, wherein the at least two methylation-sensitive restriction enzymes consist of two methylation-sensitive restriction enzymes. 19. The method according to claim 17 or 18, wherein the methylation sensitive restriction enzyme comprises or consists of BstUI and HpaII. 20. The method according to claim 17 or 18, wherein the methylation sensitive restriction enzyme comprises or consists of HhaI and AccII. 21. The method of claim 17 or 18, wherein the at least two methylation-sensitive restriction enzymes comprise three methylation-sensitive restriction enzymes or consist of three methylation-sensitive restriction enzymes. constituting restriction endonucleases. 22. The method according to claim 17 or 21, wherein the methylation sensitive restriction enzyme comprises or consists of BstUI, HpaII and Hin6I. 23. The method according to any one of the preceding claims, wherein the methylation-sensitive restriction enzyme is selected from the group consisting of AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI , Cfr10I, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, MspI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, and SnaBI. 24. The method of any one of claims 7-23, wherein the one or more adapters are resistant to digestion by the methylation-sensitive restriction enzyme. 25. The method of claim 24, wherein the one or more tolerant adapters comprise one or more methylated nucleotides, optionally wherein the methylated nucleotides comprise 5-methylcytosine and/or 5-hydroxymethylcytosine. 26. The method of claim 24, wherein the one or more tolerant adapters comprise one or more nucleotide analogs that are tolerant to methylation-sensitive restriction enzymes. 27. The method of claim 24, wherein the one or more tolerant adapters comprise a nucleotide sequence that is not recognized by a methylation-sensitive restriction enzyme. 28. The method of any one of claims 14-27, wherein the tag comprises a molecular barcode. 29. The method of claim 28, wherein the molecular barcode attached to the nucleic acid molecule in the first partition group of the more than one partition group is different from the second partition in the more than one partition group. Molecular barcodes attached to the nucleic acid molecules in the set. 30. The method of claims 1-29, wherein a first of the more than one partition groups and a second of the more than one partition groups differences are tagged. 31. The method of claim 30, wherein a first partition label is attached to the nucleic acid molecules in the first partition group, and a second partition label is attached to the nucleic acid molecules in the second partition group . 32. The method according to any one of the preceding claims, wherein the methylated nucleic acid molecule comprises 5-methylcytosine and/or 5-hydroxymethylcytosine. 33. The method of any one of claims 13-32, wherein the sequencing is performed by a next generation sequencer. 34. The method of any preceding claim, wherein the biological sample is selected from the group consisting of a DNA sample, an RNA sample, a polynucleotide sample, a cell-free DNA sample, and a cell-free RNA sample. 35. The method of any one of the preceding claims, wherein the biological sample is a cell-free DNA sample. 36. The method of claim 35, wherein the cell-free DNA is between 1 ng and 500 ng. 37. The method according to any one of the preceding claims, wherein the partitioning comprises classifying the nucleic acid molecule based on the different binding affinities of the nucleic acid molecule to binding agents that preferentially bind nucleic acid molecules comprising methylated nucleotides. Partitioned. 38. The method of claim 37, wherein the binding agent is a methyl binding domain (MBD) protein. 39. The method of claim 37, wherein the binding agent is an antibody specific for one or more methylated nucleotide bases. 40. The method of any one of claims 2-39, wherein the genomic region or epigenetic target region of interest comprises a differentially methylated region for cancer detection. 41. The method of any one of claims 13-40, further comprising amplifying at least a portion of the nucleic acid molecule prior to sequencing. 42. The method of claim 41, wherein primers used in said amplification comprise at least one sample index. 43. The method of any one of the preceding claims, wherein the one or more genetic loci comprise more than one genetic locus. 44. The method of claim 43, wherein the more than one genetic locus comprises one or more genomic regions.