WO2023060270A1

WO2023060270A1 - Single cell analysis for epigenomic profiling

Info

Publication number: WO2023060270A1
Application number: PCT/US2022/077817
Authority: WO
Inventors: David Ruff; Dalia Dhingra
Original assignee: Mission Bio Inc
Current assignee: Mission Bio Inc
Priority date: 2021-10-08
Filing date: 2022-10-07
Publication date: 2023-04-13
Anticipated expiration: 2024-04-08

Abstract

This disclosure relates to methods of analyzing a target nucleic acid (e.g., a DNA) in a single cell, and, more particularly, to regions of said nucleic acid bound by a protein. Such methods are useful for determining epigenomic profiles of individual cells.

Description

SINGLE CELL ANALYSIS FOR EPIGENOMIC PROFILING

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and U.S. priority to Provisional Patent Application Number 63/253,590, filed on October 8, 2021, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

FIELD OF THE DISCLOSURE

[0002] The present disclosure relates generally to methods of analyzing a target nucleic acid (e.g., a DNA) in a single cell, and, more particularly, to regions of said nucleic acid bound by a protein.

SUMMARY OF THE DISCLOSURE

[0003] The present disclosure relates to methods of analyzing a target nucleic acid (e.g., a DNA) in a single cell, and, more particularly, to regions of said nucleic acid bound by a protein. Such methods are useful for determining epigenomic profiles of individual cells. As used herein, the epigenomic profile refers to chromatin characteristics of genomic DNA. Examples of chromatin characteristics include open chromatin regions, histone characteristics, nucleosome characteristics, presence of transcription factors, presence of polymerases (e.g., RNA polymerase II). The chromatin characteristics enable profiling of genomic DNA and reveals regulatory information of genomes of individual cells. Disclosed methods described herein are able to determine epigenomic profiles of single cells more effectively than conventional methods, such as conventional ATAC-seq methods or CUT&Tag methodologies performed in bulk. CUT&Tag is described in further detail in Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. etal. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 10, 1930 (2019), which is incorporated by reference in its entirety.

[0004] Accordingly, in one aspect, provided herein is a method of analyzing a target nucleic acid in a single cell, the method comprising: a) providing a sample comprising a plurality of individual cells or cellular nuclei, a primary detection reagent, a secondary detection reagent, and a first reaction mixture comprising a transposase; b) forming a first microdroplet comprising a single cell or cellular nucleus isolated within the first microdroplet from the plurality of individual cells or cellular nuclei, the first reaction mixture, and a second reaction mixture comprising magnesium or manganese and a protease; c) forming a second microdroplet comprising components of the first microdroplet and reagents for nucleic acid amplification; d) incubating the microdroplet under conditions allowing for nucleic acid amplification to produce amplification products; and e) sequencing the amplification products.

[0005] In some embodiments, the target nucleic acid is a DNA or an RNA.

[0006] In some embodiments, the target nucleic acid is bound to a protein. In some embodiments, the protein is selected from the group consisting of a histone, a chromatinmodifying enzyme, or a transcription factor.

[0007] In some embodiments, the primary detection reagent is an antibody. In some embodiments, the primary detection reagent binds the target nucleic acid.

[0008] In some embodiments, the secondary detection reagent is an antibody. In some embodiments, the secondary detection reagent binds the primary detection reagent.

[0009] In some embodiments, the transposase is a prokaryotic transposase pAG-Tn5. In some embodiments, the transposase binds the secondary detection reagent.

[0010] In some embodiments, the first reaction mixture does not comprise magnesium or manganese. In some embodiments, the first reaction mixture comprises insufficient magnesium or manganese to activate the transposase.

[0011] In some embodiments, the method further comprises a wash step after the providing of the first reaction mixture comprising a transposase.

[0012] In some embodiments, the nucleic acid amplification comprises a polymerase chain reaction (PCR) amplification or an isothermal amplification, e.g., using an isothermal amplification polymerase. In some embodiments, the PCR is a high fidelity PCR. In some embodiments, the PCR is a non-hot start PCR. In some embodiments, the nucleic acid amplification comprises an isothermal amplification polymerase. In some embodiments, the reagents for nucleic acid amplification comprises a plurality of primers comprising one or more primers that each hybridize to one or more oligonucleotides.

[0013] In some embodiments, the magnesium or mangagese in the first microdroplet activates the transposase. In some embodiments, the first microdroplet is heated for tagmentation. In some embodiments, the tagmentation occurs at about 37° C, 50° C, or 65° C. In some embodiments, the first microdroplet is heated for protease activity, e.g., digestion. In some embodiments, the protease activity occurs at about 50° C. In some embodiments, the first microdroplet is heated for protease inactivation. In some embodiments, the protease inactivation occurs at about 70° C, 80° C, or 90° C.

[0014] In some embodiments, steps (a) to (e) of the method are performed in order.

[0015] In some embodiments, the target nucleic acid is epigenetically regulated.

[0016] In another aspect, provided herein is a method of analyzing a target nucleic acid in a single cell, the method comprising: a) providing a sample having a plurality of individual cells or cellular nuclei; b) adding a primary detection reagent; c) adding a secondary detection reagent; d) adding a first reaction mixture comprising a transposase; e) forming a first microdroplet comprising the individual cells or cellular nuclei, the first reaction mixture, and a second reaction mixture comprising magnesium or mangagese and a protease, wherein the magnesium or manganese in the first microdroplet activates the transposase; f) forming a second microdroplet comprising components of the first microdroplet and reagents for nucleic acid amplification; g) incubating the microdroplet under conditions allowing for nucleic acid amplification to produce amplification products; and h) sequencing the amplification products.

[0017] In some embodiments, the target nucleic acid is a DNA or an RNA. [0018] In some embodiments, the target nucleic acid is bound to a protein. In some embodiments, the protein is selected from the group consisting of a histone, a chromatinmodifying enzyme, or a transcription factor.

[0019] In some embodiments, the primary detection reagent is an antibody. In some embodiments, the primary detection reagent binds the target nucleic acid.

[0020] In some embodiments, the secondary detection reagent is an antibody. In some embodiments, the secondary detection reagent binds the primary detection reagent.

[0021] In some embodiments, the transposase is a prokaryotic transposase pAG-Tn5. In some embodiments, the transposase binds the secondary detection reagent.

[0022] In some embodiments, the first reaction mixture does not comprise magnesium or manganese.

[0023] In some embodiments, the method further comprises a wash step after the adding the first reaction mixture comprising a transposase.

[0024] In some embodiments, the nucleic acid amplification is a polymerase chain reaction (PCR) amplification or an isothermal amplification. In some embodiments, the PCR is a high fidelity PCR. In some embodiments, the PCR is a hot start PCR. In some embodiments, the PCR is a non-hot start PCR. In some embodiments, the nucleic acid amplification comprises an isothermal amplification polymerase. In some embodiments, the reagents for nucleic acid amplification comprises a plurality of primers comprising one or more primers that each hybridize to one or more oligonucleotides.

[0025] In some embodiments, the first microdroplet is heated for Tagmentation. In some embodiments, the Tagmentation occurs at about 37° C. In some embodiments, a reverse transcriptase or DNA polymerase is added to the first microdroplet for fill-in. In certain embodiments, fill-in occurs at 37° C, 50° C, or 65° C. In some embodiments, a reverse transcriptase or DNA polymerase is added to the first microdroplet for fill-in. In certain embodiments, fill-in occurs at 60° C. In some embodiments, the first microdroplet is heated for protease activity, e.g., digestion. In some embodiments, the protease activity occurs at about 50° C or about 60° C. In some embodiments, the first microdroplet is heated for protease inactivation. In some embodiments, the protease inactivation occurs at about 70° C, 80° C, or 90° C.

[0026] In some embodiments, steps (a) to (h) of the method are performed in order. [0027] In some embodiments, the target nucleic acid is epigenetically regulated.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0028] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

[0029] Figure (FIG.) 1 A depicts an overall system environment including a single cell workflow device and a computing device for conducting single-cell analysis, in accordance with an embodiment.

[0030] FIG. IB shows an embodiment of processing single cells to generate amplified nucleic acid molecules for sequencing, in accordance with an embodiment.

[0031] FIG. 2 shows a flow process of characterizing single cells using sequence reads derived from the cells, in accordance with an embodiment.

[0032] FIGs. 3 A-3B depict an example flow diagram for analyzing a target nucleic acid in single cells, in accordance with an embodiment.

[0033] FIG. 4 depicts an example computing device for implementing system and methods described in reference to FIGs. 1-3.

DETAILED DESCRIPTION

Definitions

[0034] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0035] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate. Thus, for example, reference to “a droplet” includes a plurality of such droplets unless the context clearly dictates otherwise.

[0036] The terms “nucleic acid barcode sequence,” “nucleic acid barcode,” “barcode” and the like as used herein refer to a nucleic acid having a sequence which can be used to identify and/or distinguish one or more first molecules to which the nucleic acid barcode is conjugated from one or more second molecules. Nucleic acid barcode sequences are typically short, e.g., about 5 to 20 bases in length, and may be conjugated to one or more target molecules of interest or amplification products thereof. Nucleic acid barcode sequences may be single or double stranded.

[0037] The terms “nucleic acid,” “nucleic acid molecule,” “oligonucleotide” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The terms encompass, e.g., DNA, RNA and modified forms thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Nonlimiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

[0038] The term “nucleic acid sequence” or “oligonucleotide sequence” refers to a contiguous string of nucleotide bases and in particular contexts also refers to the particular placement of nucleotide bases in relation to each other as they appear in a oligonucleotide. Similarly, the term “polypeptide sequence” or “amino acid sequence” refers to a contiguous string of amino acids and in particular contexts also refers to the particular placement of amino acids in relation to each other as they appear in a polypeptide. A desired nucleic acid for analysis is referred to herein as a “target nucleic acid”. In certain embodiments, a target nucleic acid is a DNA molecule. In certain embodiments, a target nucleic acid is an RNA molecule. In certain embodiments, the target nucleic acid is bound to a protein.

[0039] The term “primary detection reagent” is used herein to describe a molecule capable of binding to a target, e.g., a target protein. In some embodiments, the primary detection reagent is an antibody. In some embodiments, the primary detection reagent is a bead. The term “secondary detection reagent” is used herein to describe a molecule capable of binding to a primary detection reagent. In some embodiments, the secondary detection reagent is an antibody. In some embodiments, the secondary detection reagent is a bead.

[0040] As used herein the term “isolated,” when used in the context of an isolated cell, refers to a cell of interest that is in an environment different from that in which the cell naturally occurs. “Isolated” is meant to include cells that are within samples that are substantially enriched for the cell of interest and/or in which the cell of interest is partially or substantially purified. [0041] The terms “droplets,” “droplet” and the like are used herein to refer to emulsionbased compartments capable of encapsulating and/or containing one or more single cells as described herein and/or one or more barcodes as described herein. Droplets may include a first fluid phase, e.g., an aqueous phase (e.g., water or hydrogel), bounded by a second fluid phase (e.g., oil) which is immiscible with the first fluid phase. In some embodiments, the second fluid phase will be an immiscible phase carrier fluid. Thus droplets according to the present disclosure may be provided as aqueous-in-oil emulsions. Droplets as used or generated in connection with the subject methods, devices, and/or systems may be sphere shaped or they may have any other suitable shape, e.g., an ovular or oblong shape. Droplets as described herein may include a liquid phase and/or a solid phase material. In some embodiments, droplets according to the present disclosure include a gel material. In some embodiments, the subject droplets have a dimension, e.g., a diameter, of or about 1.0 pm to 1000 pm, inclusive, such as 1.0 pm to 750 pm, 1.0 pm to 500 pm, 1.0 pm to 100 pm, 1.0 pm to 10 pm, or 1.0 pm to 5 pm, inclusive. In some embodiments, droplets as described herein have a dimension, e.g., diameter, of or about 1.0 pm to 5 pm, 5 pm to 10 pm, 10 pm to 100 pm, 100 pm to 500 pm, 500 pm to 750 pm, or 750 pm to 1000 pm, inclusive. Furthermore, in some embodiments, droplets as described herein have a volume ranging from about 1 fL to 1 nL, inclusive, such as from 1 fL to 100 pL, 1 fL to 10 pL, 1 fL to 1 pL, 1 fL to 100 fL, or 1 fL to 10 fL, inclusive. In some embodiments, droplets as described herein have a volume of 1 fL to 10 fL, 10 fL to 100 fL, 100 fL to 1 pL, 1 pL to 10 pL, 10 pL to 100 pL or 100 pL to 1 nL, inclusive. In addition, droplets as described herein may have a size and/or shape such that they may be produced in, on, or by a microfluidic device and/or flowed from or applied by a microfluidic device.

[0042] As used herein, the term “carrier fluid” refers to a fluid configured or selected to contain one or more droplets, as described herein. A carrier fluid may include one or more substances and may have one or more properties, e.g., viscosity, which allows it to be flowed through a microfluidic device or a portion thereof. In some embodiments, carrier fluids include, for example: oil or water, and may be in a liquid or gas phase.

[0043] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, primates, canines, felines, and the like), and more preferably include humans. [0044] In some embodiments, a biological sample is obtained from the subject and may be, for example and without limitation, isolated cells (e.g., obtained from a bodily fluid, such as blood, saliva, or urine), or solid tissue (e.g., a solid tumor). In certain embodiments, a solid tissue sample is processed by mechanical maceration and subsequent suspension in a nuclei preparation buffer for analysis. In certain embodiments, a biological sample is not obtained from a subject (e.g., a cell line).

[0045] The terms “treat,” “treating,” or “treatment,” and other grammatical equivalents as used in this disclosure, include alleviating, abating, ameliorating, or preventing a disease, condition or symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition, and can, in selected embodiments, include prophylaxis. The terms further include achieving a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder.

[0046] The term “about” refers to any minimal alteration in the concentration or amount of an agent that does not change the efficacy of the agent in preparation of a formulation and in treatment of a disease or disorder. In certain embodiments, the term “about” may include ±5%, ±10%, or ±15% of a specified numerical value or data point.

[0047] Ranges can be expressed in this disclosure as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it is understood that the particular value forms another aspect. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed in this disclosure, and that each value is also disclosed as “about” that particular value in addition to the value itself. It is also understood that throughout the application, data are provided in a number of different formats and that this data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0048] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0049] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.

Overview

[0050] Described herein are embodiments for performing single-cell analyses for a plurality of cells to characterize individual cells. Generally, the single-cell analysis involves performing DNA-seq to generate sequence reads derived from genomic DNA that are used to determine characteristics of the cell genome. In various embodiments, the single-cell analysis involves performing DNA-seq to generate sequence reads derived from portions of genomic DNA for genome-wide profiling. For example, methods described herein involve single-cell analysis for epigenomic profiling. Therefore, individual cells can be characterized e.g., characterized according to chromatin profile, open chromatin regions, chromatin scaffolding structures, histone characteristics, nucleosome characteristics, presence of transcription factors, presence of polymerases (e.g., RNA polymerase II). Altogether, the single-cell analysis workflow described herein enables chromatin profiling and reveals regulatory information of genomes of individual cells.

[0051] Reference is made to FIG. 1 A, which depicts an overall system environment including a single cell workflow device 100 and a computing device 180 for conducting single-cell analysis, in accordance with an embodiment. A population of cells 110 is obtained. In various embodiments, the cells 110 can be isolated from a test sample obtained from a subject or a patient. In various embodiments, the cells 110 are healthy cells taken from a healthy subject. In various embodiments, the cells 110 include diseased cells taken from a subject. In one embodiment, the cells 110 include cancer cells taken from a subject previously diagnosed with cancer. For example, cancer cells can be tumor cells available in the bloodstream of the subject diagnosed with cancer. As another example, cancer cells can be cells obtained through a tumor biopsy. Thus, single-cell analysis of the tumor cells enables characterization of cells of the subject’s cancer. In various embodiments, the test sample is obtained from a subject following treatment of the subject (e.g., following a therapy such as cancer therapy). Thus, single-cell analysis of the cells enables characterization of cells representing the subject’s response to a therapy. In various embodiments, the number of cells 110 can be 10² cells, 10³ cells, 10⁴ cells, 10⁵ cells, 10⁶ cells, or 10⁷ cells. In various embodiments, the number of cells 110 can be between 10³ cells and 10⁷ cells. In various embodiments, the number of cells 110 can be between 10⁴ cells and 10⁶ cells.

[0052] At step 102, the cells undergo cell preparation. Here, step 102 involves preparing the cells outside of the single cell workflow device 100. In various embodiments, step 102 involves lysing the cells to release cell nuclei of the cells. In various embodiments, the lysing of the cells releases the cell nuclei, but does not release the genomic DNA from its chromatin packaging. In various embodiments, lying the cells involve exposing the cells to one or more lysing agents. Examples of lysing agents include detergents such as Triton X- 100, Nonidet P-40 (NP40) as well as cytotoxins. In some embodiments, the reagents include NP40 detergent which is sufficient to disrupt the cell membrane and cause cell lysis, but does not disrupt chromatin-packaged DNA. In various embodiments, the reagents include 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, or 5.0% NP40 (v/v). In various embodiments, the reagents include at least at least 0.01%, at least 0.05%, 0.1%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, or at least 5% NP40 (v/v).

[0053] In various embodiments, step 102 involves exposing the cell nuclei of the cells to one or more detection reagents. In various embodiments, the detection reagents are antibodies. In various embodiments, varying concentrations of detection reagents are incubated with cells. In various embodiments, for a detection reagent, a concentration of 0.1 nM, 0.5 nM, 1.0 nM, 2.0 nM, 3.0 nM, 4.0 nM, 5.0 nM, 6.0 nM, 7.0 nM, 8.0 nM, 9.0 nM, 10.0 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM of the detection reagent is incubated with cells.

[0054] In various embodiments, step 102 involves exposing the cell nuclei of the cells to two or more detection reagents. For example, the cell nuclei can be exposed to a primary detection reagent and a secondary detection reagent. In various embodiments, the primary detection reagent exhibits binding affinity to a target protein involved in the packing of genomic DNA. Examples of the target protein can include a histone (e.g., including histone modifications of H3K4me3 and H3K27me3) or a transcription factor (e.g., FoxAl). The secondary detection reagent exhibits binding affinity to the primary detection reagent. In various embodiments, step 102 involves exposing the cells or cell nuclei to a reaction mixture that includes a transposase. The transposase can exhibit affinity for the one or more detection reagents. Furthermore, the transposase can be tethered to sequencing adapters, or alternatively, custom adapters or sequences. Such tethering permits for attachment of the barcode during nucleic acid amplification. Generally, the reaction mixture including the transposase does not include magnesium ions (e.g., Mg⁺⁺) or manganese ions (e.g., Mn++), the presence of which would activate the transposase.

[0055] In various embodiments, one or more wash steps can be implemented between the lysing of the cells, exposing of the cells or cell nuclei to the first detection reagent, exposing of the cells or cell nuclei to the second detection reagent, and/or exposing of the cells or cell nuclei to a reaction mixture including a transposase. In various embodiments, step 102 includes one wash step, two wash steps, three wash steps, four wash steps, or five wash steps. In particular embodiments, step 102 includes two wash steps. In particular embodiments, step 102 includes three wash steps.

[0056] In particular embodiments, the cell preparation step of 102 includes: 1) lysing cells to release cell nuclei, 2) adding a primary detection reagent, 3) performing a wash, 4) adding a secondary detection reagent, 5) performing a second wash, 6) adding a reaction mixture including a transposase, and 7) performing a third wash.

[0057] Following cell preparation at step 102, the prepared cells can be loaded onto the single cell workflow device 100. The single cell workflow device 100 refers to a device that processes individuals’ cells to generate nucleic acids for sequencing. In various embodiments, the single cell workflow device 100 can encapsulate individual cell nuclei into emulsions, process the cell nuclei, perform barcoding of nucleic acids in a second emulsion, and perform a nucleic amplification reaction in the second emulsion. Thus, amplified nucleic acids can be collected and sequenced. An exemplary single cell workflow device 100 is the Tapestri® system. In various embodiments, the single cell workflow device 100 further includes a sequencer for sequencing the nucleic acids.

[0058] The computing device 180 is configured to receive the sequenced reads from the single cell workflow device 100. In various embodiments, the computing device 180 is communicatively coupled to the single cell workflow device 100 and therefore, directly receives the sequence reads from the single cell workflow device 100. The computing device 180 analyzes the sequence reads and characterizes the cells 110. In one embodiment, the computing device 180 analyzes the sequence reads to determine epigenomic profiles of the cells 110.

[0059] Reference is now made to FIG. IB, which depicts one embodiment of processing single cells to generate amplified nucleic acid molecules for sequencing. Specifically, FIG. IB depicts a workflow process including the steps of cell nuclei encapsulation 160, nuclei processing 165, barcoding 170, and target amplification 175 of target nucleic acid molecules.

[0060] Generally, the cell nuclei encapsulation step 160 involves encapsulating a single nuclei 105 with a rection mixture 120 into an emulsion. Here, the reaction mixture 120 differs from the reaction mixture described above that was used in step 102 for preparing the cells. In various embodiments, the emulsion is formed by partitioning aqueous fluid containing the cell nuclei and reaction mixture 120 into a carrier fluid (e.g., oil 115), thereby resulting in an aqueous fluid-in-oil emulsion. The emulsion includes encapsulated cell nuclei 125 and the reaction mixture 120. The encapsulated cell nuclei undergoes nuclei processing at step 165. Generally, the reaction mixture 120 includes magnesium ions (Mg⁺⁺) which activates transposases that are bound to detection agents of the cell nuclei. In particular embodiments, the reaction mixture 120 include proteases, such as proteinase K, for releasing the genomic DNA from the chromatin packaging. In various embodiments, the released genomic DNA can further interact with agents in the reaction mixture 120 within the droplet, examples of which include primers in the reaction mixture 120, such as reverse primers. Following step 165, the processed nucleic 130 are in individual droplets and can further undergo barcoding at step 170. [0061] The barcoding step 170 involves encapsulating the processed nuclei 130 into a second droplet a/ong with a barcode 145 and/or reaction mixture 140. In various embodiments, the second droplet is formed by partitioning aqueous fluid containing the processed nuclei 130 into immiscible oil 135. As shown in FIG. IB, the reaction mixture 140 and barcode 145 can be introduced through a separate stream of aqueous fluid, thereby partitioning the reaction mixture 140 and barcode into the second emulsion along with the processed nuclei 130. Here, the reaction mixture 140 differs from the reaction mixture 120 used during step 160, and further differs from the reaction mixture described above at step 102 for preparing the cells.

[0062] Generally, a barcode 145 can label a target analyte to be analyzed (e.g., a target nucleic acid), which enables subsequent identification of the origin of a sequence read that is derived from the target nucleic acid. In various embodiments, multiple barcodes 145 can label multiple target nucleic acid of the processed nuclei, thereby enabling the subsequent identification of the origin of large quantities of sequence reads.

[0063] Generally, the reaction mixture 140 enables the performance of a reaction, such as a nucleic acid amplification reaction. The target amplification step 175 involves amplifying target nucleic acids. For example, target nucleic acids of the processed nuclei undergo amplification using the reaction mixture 140 in the second emulsion, thereby generating amplicons derived from the target nucleic acids. Although FIG. IB depicts cell barcoding 170 and target amplification 175 as two separate steps, in various embodiments, the target nucleic acid is labeled with a barcode 145 through the nucleic acid amplification step.

[0064] As referred herein, the workflow process shown in FIG. IB is a two-step workflow process in which nuclei processing 165 from the cell occurs separate from the steps of cell barcoding 170 and target amplification 175. For example, nuclei processing 165 from a cell occurs within a first emulsion followed by cell barcoding 170 and target amplification 175 in a second emulsion. In various embodiments, alternative workflow processes (e.g., workflow processes other than the two-step workflow process shown in FIG. IB) can be employed. For example, the cell 102, reaction mixture 120, reaction mixture 140, and barcode 145 can be encapsulated in an emulsion. Thus, nuclei processing 165 can occur within the emulsion, followed by cell barcoding 170 and target amplification 175 within the same emulsion. [0065] FIG. 2 shows a flow process of characterizing single cells using sequence reads derived from the cells, in accordance with an embodiment. Specifically, FIG. 2 depicts the steps of pooling amplified nucleic acids at step 205, sequencing the amplified nucleic acids at step 210, and characterizing cells at step 220. Generally, the flow process shown in FIG. 2 is a continuation of the workflow process shown in FIG. IB.

[0066] For example, after target amplification at step 175 of FIG. IB, the amplified nucleic acids 250A, 250B, and 250C are pooled at step 205 shown in FIG. 2. For example, emulsions of amplified nucleic acids are pooled and collected, and the immiscible oil of the emulsions is removed. Thus, amplified nucleic acids from multiple cells can be pooled together. FIG. 2 depicts three amplified nucleic acids 250A, 250B, and 250C but in various embodiments, pooled nucleic acids can include hundreds, thousands, or millions of nucleic acids derived from analytes of multiple cells.

[0067] In various embodiments, each amplified nucleic acid 250 includes at least a sequence of a target nucleic acid 240 and a barcode 230. In various embodiments, an amplified nucleic acid 250 can include additional sequences, such as any of a universal primer sequence (e.g., an oligo-dT sequence), a random primer sequence, a gene specific primer forward sequence, a gene specific primer reverse sequence, or one or more constant regions (e.g., PCR handles).

[0068] In various embodiments, the amplified nucleic acids 250A, 250B, and 250C are derived from the same single cell and therefore, the barcodes 230 A, 230B, and 230C are the same. As such, sequencing of the barcodes 230 enables the determination that the amplified nucleic acids 250 are derived from the same cell. In various embodiments, the amplified nucleic acids 250A, 250B, and 250C are pooled and derived from different cells. Therefore, the barcodes 230 A, 230B, and 230C are different from one another and sequencing of the barcodes 230 enables the determination that the amplified nucleic acids 250 are derived from different cells.

[0069] At step 210, the pooled amplified nucleic acids 250 undergo sequencing to generate sequence reads. For each amplified nucleic acid, the sequence read includes the sequence of the barcode and the target nucleic acid. Sequence reads originating from individual cells are clustered according to the barcode sequences included in the amplified nucleic acids. In various embodiments, one or more sequence reads for each single cell are aligned (e.g., to a reference genome). Aligning the sequence reads to the reference genome enables the determination of where in the genome the sequence read is derived from. For example, multiple sequence reads generated from DNA, when aligned to a position of the genome, can reveal one or more mutations present at or involving the position of the genome. In various embodiments, one or more sequence reads for each single cell do not undergo alignment. For example, sequence reads derived from antibody oligonucleotides need not be aligned to the reference genome, given that the antibody oligonucleotides are not derived from genomic DNA of the cell genome.

[0070] At step 220, aligned sequence reads for a single cell are characterized. For example, sequence reads generated from genomic DNA are analyzed to determine epigenomic profiles of cells. For example, the presence of sequence reads corresponding to a particular genomic DNA segment can be indicative of open chromatin regions at the particular genomic DNA segment. Additionally, the presence of sequence reads corresponding to a particular genomic DNA segment can indicate presence/absence of specific transcription factors, presence/absence of histone modifications, and/or presence/absence of polymerases (e.g., RNA polymerase II) at the particular genomic DNA segment. Altogether, this enables the epigenomic profiling of individual cells.

Methods for Performing Single-Cell Analysis

Encapsulation, Processing, Barcoding, and Amplification

[0071] Embodiments described herein involve encapsulating cell nuclei (e.g., at step 160 in FIG. IB) to perform single-cell analysis on the corresponding cells. In various embodiments, encapsulating a cell nuclei with a reaction mixture (e.g., reaction mixture 120) is accomplished by combining an aqueous phase including the cell and reaction mixture with an immiscible oil phase. In one embodiment, an aqueous phase including the cell and reaction mixture are flowed together with a flowing immiscible oil phase such that water in oil emulsions are formed, where at least one emulsion includes a single cell and the reaction mixture. In various embodiments the immiscible oil phase includes a fluorous oil, a fluorous non-ionic surfactant, or both. In various embodiments, emulsions can have an internal volume of about 0.001 to 1000 picoliters or more and can range from 0.1 to 1000 pm in diameter.

[0072] In various embodiments, the aqueous phase including the cell and reaction mixture need not be simultaneously flowing with the immiscible oil phase. For example, the aqueous phase can be flowed to contact a stationary reservoir of the immiscible oil phase, thereby enabling the budding of water in oil emulsions within the stationary oil reservoir. [0073] In various embodiments, combining the aqueous phase and the immiscible oil phase can be performed in a microfluidic device. For example, the aqueous phase can flow through a microchannel of the microfluidic device to contact the immiscible oil phase, which is simultaneously flowing through a separate microchannel or is held in a stationary reservoir of the microfluidic device. The encapsulated cell and reaction mixture within an emulsion can then be flowed through the microfluidic device to undergo cell lysis.

[0074] Further example embodiments of adding reaction mixture and cells to emulsions can include merging emulsions that separately contain the cells and reaction mixture or picoinjecting reaction mixture into an emulsion. Further description of example embodiments is described in US Application No. 14/420,646, which is hereby incorporated by reference in its entirety.

[0075] The encapsulated cell nuclei in an emulsion is next processed at step 165.

In various embodiments, a cell nuclei is processed through a tagmentation reaction. For example, as described above in reference to the nuclei processing 165, cell nuclei are exposed to one or more detection reagents and transposase, which is linked to sequencing adapters, or alternatively, custom adapters or sequences. Here, the reaction mixture can include magnesium ions (Mg⁺⁺) or manganese ions (e.g., Mn++), thereby activating the transposase and enabling the tagmentation reaction. Such reaction mixture is then incubated at 37 °C for about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about one hour. Thus, sequencing adapters are integrated at specific regions (e.g., integrated at chromatin protein binding sites).

[0076] In various embodiments, the processing of the cell nuclei at step 165 further includes releasing genomic DNA from the chromatin packaging. Here, releasing the genomic DNA can occur after the tagmentation process such that the sequencing adapters are already integrated into the genomic DNA prior to release of the genomic DNA from the chromatin packaging. In various embodiments, the reaction mixture can include a protease for releasing genomic DNA from the chromatin packaging. In various embodiments, the protease is proteinase K.

[0077] In various embodiments, the droplet including the tagmentated genomic

DNA and the protease is exposed to elevated temperatures to enable release of the genomic DNA from chromatin packaging. In various embodiments, the droplet is exposed to a temperature between 40 °C and 60 °C. In various embodiments, the droplet is exposed to a temperature between 45 °C and 55 °C. In various embodiments, the droplet is exposed to a temperature between 48 °C and 52 °C. In various embodiments, the droplet is exposed to a temperature of about 50 °C. In various embodiments, the droplet is exposed to the elevated temperature for about 20 minutes to about one hour. In certain embodiments, the droplet is exposed to the elevated temperature for about one hour.

[0078] In various embodiments, following release of the DNA, e.g., tagmented genomic DNA, from chromatin packaging by the protease, the droplet is further exposed to a yet further elevated temperature to inactivate the protease. In various embodiments, the droplet is exposed to a temperature between 70 °C and 95 °C to inactivate the protease. In various embodiments, the droplet is exposed to a temperature between 75 °C and 85 °C. In various embodiments, the droplet is exposed to a temperature between 78 °C and 82 °C. In various embodiments, the droplet is exposed to a temperature of about 80 °C. In various embodiments, the droplet is exposed to the elevated temperature for about 5 minutes to about 30 minutes. In certain embodiments, the droplet is exposed to the elevated temperature for about 10 minutes. In certain embodiments, the droplet is exposed to the elevated temperature for about 20 minutes. In certain embodiments, the droplet is exposed to the elevated temperature for about 30 minutes.

[0079] In various embodiments, the DNA, e.g., tagmented DNA, can undergo priming within the droplet. In some embodiments, a reverse transcriptase is added to the first droplet for fill-in, wherein the gDNA fragment is extended. In certain embodiments, fill-in occurs at 37 °C. In some embodiments, a reverse transcriptase is added to the first droplet for fill-in. In certain embodiments, fill-in occurs at 60 °C. In various embodiments, reverse primers can hybridize with a portion of the free genomic DNA. As one example, the reverse primer is a gene specific reverse primer that hybridizes with a portion of the free genomic DNA. In some embodiments, an isothermal amplification polymerase is added to the first droplet for fill-in, wherein the gDNA fragment is extended. In certain embodiments, the isothermal amplification polymerase is BST, BSU, or other polymerases as known in the art. [0080] As shown in FIG. IB, the processed nuclei 130 (e.g., nuclei that has undergone tagmentation and/or release of genomic DNA) undergoes barcoding 170 and target amplification 175. In various embodiments, the processed nuclei 130 is encapsulated in a second droplet for barcoding and amplification. Specifically, the step of barcoding 170 in FIG. IB includes encapsulating the processed nuclei 130 with a reaction mixture 140 and a barcode 145. In various embodiments, the reaction mixture 140 includes components for performing a nucleic acid reaction on target nucleic acids (e.g., antibody oligonucleotide and freed genomic DNA). For example, the reaction mixture 140 can include primers, enzymes for performing nucleic acid amplification, and dNTPs or ddNTPs for incorporation into amplified nucleic acids.

[0081] In various embodiments, a processed nuclei is encapsulated with a reaction mixture and a barcode by combining an aqueous phase including the reaction mixture and the barcode with the processed nuclei and an immiscible oil phase. In one embodiment, an aqueous phase including the reaction mixture and the barcode are flowed together with a processed nuclei and a flowing immiscible oil phase such that water in oil emulsions are formed, where at least one emulsion includes a processed cell nuclei, the reaction mixture, and the barcode. In various embodiments the immiscible oil phase includes a fluorous oil, a fluorous non-ionic surfactant, or both. In various embodiments, emulsions can have an internal volume of about 0.001 to 1000 picoliters or more and can range from 0.1 to 1000 pm in diameter.

[0082] In various embodiments, combining the aqueous phase and the immiscible oil phase can be performed in a microfluidic device. For example, the aqueous phase can flow through a microchannel of the microfluidic device to contact the immiscible oil phase, which is simultaneously flowing through a separate microchannel or is held in a stationary reservoir of the microfluidic device. The encapsulated processed nuclei, reaction mixture, and barcode within an emulsion can then be flowed through the microfluidic device to perform amplification of target nucleic acids.

[0083] Further example embodiments of adding reaction mixture and barcodes to emulsions can include merging emulsions that separately contain the processed nuclei and reaction mixture and barcodes or picoinjecting the reaction mixture and/or barcode into an emulsion. Further description of example embodiments of merging emulsions or picoinjecting substances into an emulsion is found in US Application No. 14/420,646, which is hereby incorporated by reference in its entirety.

[0084] Once the reaction mixture and barcode are added to an emulsion, the emulsion may be incubated under conditions that facilitate the nucleic acid amplification reaction. In various embodiments, the emulsion may be incubated on the same microfluidic device as was used to add the reaction mixture and/or barcode, or may be incubated on a separate device, e.g., a thermocycler. In certain embodiments, incubating the emulsion under conditions that facilitates nucleic acid amplification is performed on the same microfluidic device used to encapsulate the cell nuclei and to process the cell nuclei. Incubating the emulsions may take a variety of forms. In certain aspects, the emulsions containing the reaction mix, barcode, and processed nuclei may be flowed through a channel that incubates the emulsions under conditions effective for nucleic acid amplification. Flowing the microdroplets through a channel may involve a channel that snakes over various temperature zones maintained at temperatures effective for PCR. Such channels may, for example, cycle over two or more temperature zones, wherein at least one zone is maintained at about 65 °C. and at least one zone is maintained at about 95 °C. As the drops move through such zones, their temperature cycles, as needed for nucleic acid amplification. The number of zones, and the respective temperature of each zone, may be readily determined by those of skill in the art to achieve the desired nucleic acid amplification.

[0085] In some embodiments, the emulsions are collected, e.g., in PCR tubes, and subsequently thermal cycled on a thermal cycler.

[0086] In various embodiments, following nucleic acid amplification, emulsions containing the amplified nucleic acids are collected. In various embodiments, the emulsions are collected in a well, such as a well of a microfluidic device. In various embodiments, the emulsions are collected in a reservoir or a tube, such as a PCR tube, e.g., an Eppendorf tube. Once collected, the amplified nucleic acids across the different emulsions are pooled. In one embodiment, the emulsions are broken by providing an external stimuli to pool the amplified nucleic acids. In one embodiment, the emulsions naturally aggregate over time given the density differences between the aqueous phase and immiscible oil phase. Thus, the amplified nucleic acids pool in the aqueous phase.

[0087] In some embodiments, the emulsions are combined, e.g., to perform library

PCR.

[0088] In various embodiments, following pooling, the amplified nucleic acids can undergo further preparation for sequencing. For example, sequencing adapters can be added to the pooled nucleic acids. Example sequencing adapters are P5 and P7 sequencing Illumina adapters for Illumina sequencers. The sequencing adapters enable the subsequent sequencing of the nucleic acids on the respective sequencer. Those of skill in the art understand the specific sequencing adapter varies depending on the sequencer to be used.

Exemplary Method for Analyzing a Target Nucleic Acid in Single Cells

[0089] FIGs. 3 A-3B depict an example flow diagram for analyzing a target nucleic acid in single cells. Generally, FIG. 3 A depicts the step of cell preparation 102 (shown in FIG. 1 A) in further detail. FIG. 3B depicts in further detail the steps performed on the single cell workflow device 100. For example, steps 340, 345, and 350 describe the steps of cell nuclei encapsulation and nuclei processing (steps 160 and 165 in FIG. IB), whereas steps 355 and 360 describe the steps of barcoding 170 and target amplification 175 in FIG. IB.

[0090] Referring to FIG. 3A, at step 305, cells are lysed. In particular embodiments, cells are exposed to a detergent, such as NP-40. Here, NP-40 is sufficient to lyse the cells, but does not disrupt the chromatin-DNA packaging. Thus, lysing the cells releases individual cell nuclei.

[0091] At step 310, primary detection agents, such as primary antibodies, are added to the cell nuclei. In various embodiments, the primary detection agents exhibit affinity for any of H3K4me3, H3K27me3, or FoxAl. Thus, the primary detection agents bind to particular regions of genomic DNA with a particular epigenomic profile (e.g, presence of particular histone modification, presence of particular transcription factor). [0092] At step 315, a wash is performed to remove unbound primary detection agents. Secondary detection agents, such as secondary antibodies, are added to the cell nuclei. Here, the secondary antibodies exhibit binding affinity to the primary detection agents. Thus, this increases the concentration of detection agents that are bound to particular regions of genomic DNA.

[0093] At step 320, a wash is performed to remove unbound secondary detection agents. A reaction mixture including transposase is added. In various embodiments, the transposase can be a protein A (pA)-Tn5 transposase. In various embodiments, the transposase can be a pAG-Tn5 transposase. In certain embodiments, the transposase reaction mixture further comprises reagents for the transposase, e.g, HEPES-KOH, NaCl, EDTA, DTT, Triton X-100, Tris-acetate, tris (hydroxymethyl)methylamino]propanesulfonic acid (TAPS), and glycerol. In certain embodiments, the transposase reaction mixture further comprises potassium acetate. Generally, the transposase exhibits affinity for the primary or secondary detection reagents and is tethered to sequencing adapters. In particular embodiments, the reaction mixture does not include magnesium ions (Mg⁺⁺) that would otherwise activate the transposase. Transposase activation may occur between about 1 and about 60 mM Mg++ cation, e.g., about 1 mM to about 5 mM, about 1 mM to about 10 mM, about 1 mM to about 20 mM, about 1 mM to about 30 mM, about 1 mM to about 40 mM, about 1 mM to about 50 mM, and about 1 mM to about 60 mM. Transposase activation may occur between about 1 and about 60 mM Mn++ cation, e.g., about 1 mM to about 5 mM, about 1 mM to about 10 mM, about 1 mM to about 20 mM, about 1 mM to about 30 mM, about 1 mM to about 40 mM, about 1 mM to about 50 mM, and about 1 mM to about 60 mM. Typical ion activation of the transposase, e.g.. Tn5 transposase, is described in Buenrostro et al., Nature, 523(7561): 486-490 and Goryshin and Reznikoff, 273(13):7367- 7364, which are incorporated by reference in their entireties. In certain embodiments, transposase activation occurs at about 10 mM Mg++. In certain embodiments, transposase activation occurs at about 10 mM Mn++. As used herein, Mg++ concentration below 1 mM is referred to as insufficient to activate the transposase, e.g., a Tn5 transposase. As used herein, Mn++ concentration below 1 mM is referred to as insufficient to activate the transposase, e.g., a Tn5 transposase.

[0094] In some embodiments, ethylenediaminetetraacetic acid (EDTA) is added to the reaction mixture. The EDTA does not comprise Mg++ or Mn++. In certain embodiments, the concentration of EDTA is between about 0.1 mM to about 60 mM, e.g., about 0.1 mM to about 1 mM, about 0.1 mM to about 5 mM, about 1 mM to about 5 mM, about 1 mM to about 10 mM, about 1 mM to about 20 mM, about 1 mM to about 30 mM, about 1 mM to about 40 mM, about 1 mM to about 50 mM, and about 1 mM to about 60 mM. In a particular embodiment, the concentration of EDTA is about 0.1 to about 5 mM. In various embodiments, step 320 can further include a wash to remove any unbound transposase from the cell nuclei.

[0095] Referring now to FIG. 3B, it follows after step 320 is performed. At step

340, the cell nuclei is loaded onto a single cell workflow device (e.g., a Tapestri® device) and encapsulated along with a tagmentation mixture into individual droplets. Here, the tagmentation mixture includes magnesium ions (Mg⁺⁺), thereby activating the transposase and integrating sequencing adapters into the genomic DNA. In various embodiments, tagmentation occurs at a temperature of about 37 °C.

[0096] At step 345, the droplets are incubated to cause release of genomic DNA from the chromatin packaging. Here, the mixture may include a protease, such as proteinase K, that is a temperature sensitive protease. Thus, incubating the droplet activates the protease, thereby releasing genomic DNA. For example, the droplets can be incubated to a temperature of about 50 °C. In certain embodiments, the protease digests the transposase, e.g., the Tn5 transposase.

[0097] At step 350, the droplets are further incubated to inactivate the protease.

For example, the droplets can be incubated to a temperature of about 80 °C to inactivate the protease. [0098] At step 355, the processed cell nuclei and a reaction mixture is added to a second droplet. Here, the reaction mixture includes agents for performing a nucleic acid amplification reaction (e.g., primers, polymerases, dNTPs or ddNTPs). In various embodiments, the reaction mixture further includes a barcode correlated to the cell, which enables subsequent identification that a sequenced nucleic acid is derived from a particular cell. In various embodiments, reverse primers can hybridize with a portion of the free genomic DNA. As one example, the reverse primer is a gene specific reverse primer that hybridizes with a portion of the free genomic DNA.

[0099] At step 360, nucleic acid amplification is performed within the second droplets. Thus, the amplicons can be collected and subsequently sequenced (e.g., sequencing at step 210 and subsequent analysis) for determining epigenomic profiling of individual cells.

Nucleic Acid Amplification

[0096] As summarized above, in practicing methods of the present disclosure, a nucleic acid amplification based assay may be used to detect the presence of certain genes of interest, e.g., oncogene(s), present in cells. In various embodiments, such a nucleic amplification reaction is a polymerase chain reaction (PCR). The conditions of such PCR-based assays may vary in one or more ways. In certain embodiments, the PCR reaction is a high fidelity PCR. As used herein, “high-fidelity PCR” refers to a PCR assay that utilizes a DNA polymerase with a low error rate, and consequently results in a high degree of accuracy in the replication of the DN A of interest.

[0097] For example, the number of PCR primers that may be added to a microdroplet may vary. The term “primer” may refer to more than one primer and refers to an oligonucleotide, whether naturally occurring, as in a purified restriction digest, or produced synthetically, which is capable of acting as a point of initiation of synthesis along a complementary strand when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is catalyzed. Such conditions include the presence of four different deoxyribonucleoside triphosphates and a polymerization-inducing agent, such as DNA polymerase or reverse transcriptase, in a suitable buffer (“buffer” includes substituents which are cofactors, or which affect pH, ionic strength, etc.), and at a suitable temperature. The primer is preferably single-stranded for maximum efficiency in amplification. [0098] The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in “antiparallel association.” Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, percent concentration of cytosine and guanine bases in the oligonucleotide, ionic strength, and incidence of mismatched base pairs.

[0099] The number of PCR primers that may be added to a microdroplet may range from about 1 to about 500 or more, e.g., about 2 to 100 primers, about 2 to 10 primers, about 10 to 20 primers, about 20 to 30 primers, about 30 to 40 primers, about 40 to 50 primers, about 50 to 60 primers, about 60 to 70 primers, about 70 to 80 primers, about 80 to 90 primers, about 90 to 100 primers, about 100 to 150 primers, about 150 to 200 primers, about 200 to 250 primers, about 250 to 300 primers, about 300 to 350 primers, about 350 to 400 primers, about 400 to 450 primers, about 450 to 500 primers, or about 500 primers or more.

[0100] These primers may contain primers for one or more genes of interest, e.g. oncogenes. The number of primers for genes of interest that are added may be from about one to 500, e.g., about 1 to 10 primers, about 10 to 20 primers, about 20 to 30 primers, about 30 to 40 primers, about 40 to 50 primers, about 50 to 60 primers, about 60 to 70 primers, about 70 to 80 primers, about 80 to 90 primers, about 90 to 100 primers, about 100 to 150 primers, about 150 to 200 primers, about 200 to 250 primers, about 250 to 300 primers, about 300 to 350 primers, about 350 to 400 primers, about 400 to 450 primers, about 450 to 500 primers, or about 500 primers or more.

[0101] Such primers and/or reagents may be added to a microdroplet in one step, or in more than one step. For instance, the primers may be added in two or more steps, three or more steps, four or more steps, or five or more steps. Regardless of whether the primers are added in one step or in more than one step, they may be added after the addition of a lysing agent, prior to the addition of a lysing agent, or concurrently with the addition of a lysing agent. When added before or after the addition of a lysing agent, the PCR primers may be added in a separate step from the addition of a lysing agent.

[0102] Once primers have been added to a microdroplet, the microdroplet may be incubated under conditions allowing for PCR. The microdroplet may be incubated on the same microfluidic device as was used to add the primer(s), or may be incubated on a separate device. In certain embodiments, the incubating the microdroplet under conditions allowing for PCR amplification is performed on the same microfluidic device used to encapsulate the cell nuclei and to process the cell nuclei. Incubating the microdroplets may take a variety of forms. In certain embodiments, the drops containing the PCR mix may be flowed through a channel that incubates the droplets under conditions effective for PCR. Flowing the microdroplets through a channel may involve a channel that snakes over various temperature zones maintained at temperatures effective for PCR. Such channels may, for example, cycle over two or more temperature zones, wherein at least one zone is maintained at about 65 °C and at least one zone is maintained at about 95° C. As the drops move through such zones, their temperature cycles, as needed for PCR. The precise number of zones, and the respective temperature of each zone, may be readily determined by those of skill in the art to achieve the desired PCR amplification.

[0103] In other embodiments, incubating the microdroplets may involve the use of a device of the general types referred to herein as a “Megadroplet Array.” In such a device, an array of hundreds, thousands, or millions of traps indented into a channel (e.g., a PDMS channel) sit above a thermal system. The channel may be pressurized, thereby preventing gas from escaping. The height of the microfluidic channel is smaller than the diameter of the drops, causing drops to adopt a flattened pancake shape. When a drop flows over an unoccupied indentation, it adopts a lower, more energetically favorable, radius of curvature, leading to a force that pulls the drop entirely into the trap. By flowing drops as a close pack, it is ensured that all traps on the array are occupied. The entire device may then be thermal cycled using a heater. In certain embodiments, the heater includes a Peltier plate, heat sink, and control computer. The Peltier plate allows for the heating or cooling of the chip above or below room temperature by controlling the applied current. To ensure controlled and reproducible temperature, a computer may monitor the temperature of the array using integrated temperature probes, and may adjust the applied current to heat and cool as needed. A metallic (e.g. copper) plate allows for uniform application of heat and dissipation of excess heat during cooling cycles, enabling cooling from about 95 °C to about 60 °C in less than about one minute.

[0104] Methods of the present disclosure may also include introducing one or more probes into the microdroplet. As used herein with respect to nucleic acids, the term “probe” refers to a labeled oligonucleotide which forms a duplex structure with a sequence in the target nucleic acid, due to complementarity of at least one sequence in the probe with a sequence in the target region. The probe, preferably, does not contain a sequence complementary to sequence(s) used to prime the polymerase chain reaction. The number of probes that are added may be from about one to 500, e.g., about 1 to 10 probes, about 10 to 20 probes, about 20 to 30 probes, about 30 to 40 probes, about 40 to 50 probes, about 50 to 60 probes, about 60 to 70 probes, about 70 to 80 probes, about 80 to 90 probes, about 90 to 100 probes, about 100 to 150 probes, about 150 to 200 probes, about 200 to 250 probes, about 250 to 300 probes, about 300 to 350 probes, about 350 to 400 probes, about 400 to 450 probes, about 450 to 500 probes, or about 500 probes or more. The probe(s) may be introduced into the microdroplet prior to, subsequent with, or after the addition of the one or more primer(s). Probes of interest include, but are not limited to, TaqMan® probes e.g., as described in Holland, P. M.; Abramson, R. D.; Watson, R.; Gelfand, D. H. (1991). “Detection of specific polymerase chain reaction product by utilizing the 5 '-3' exonuclease activity of Thermus aquaticus DNA polymerase”. PNAS, 88 (16): 7276-7280).

[0105] In certain embodiments, an RT-PCR based assay may be used to detect the presence of certain transcripts of interest, e.g., oncogene(s), present in cells. In such embodiments, reverse transcriptase and any other reagents necessary for cDNA synthesis are added to the microdroplet in addition to the reagents used to carry out PCR as described herein (collectively referred to as the “RT-PCR reagents”). The RT-PCR reagents are added to the microdroplet using any of the methods described herein. Once reagents for RT-PCR have been added to a microdroplet, the microdroplet may be incubated under conditions allowing for reverse transcription, followed by conditions allowing for PCR as described herein. The microdroplet may be incubated on the same microfluidic device as was used to add the RT-PCR reagents, or may be incubated on a separate device. In certain embodiments, incubating the microdroplet under conditions allowing for RT-PCR is performed on the same microfluidic device used to encapsulate the cell nuclei.

[0106] In certain embodiments, the reagents added to the microdroplet for RT-PCR or PCR further includes a fluorescent DNA probe capable of detecting real-time RT-PCR or PCR products. Any suitable fluorescent DNA probe can be used including, without limitation, SYBR Green, TaqMan®, Molecular Beacons and Scorpion probes. In certain embodiments, the reagents added to the microdroplet include more than one DNA probe, e.g., two fluorescent DNA probes, three fluorescent DNA probes, four fluorescent DNA probes, or five or more fluorescent DNA probes. The use of multiple fluorescent DNA probes allows for the concurrent measurement of RT-PCR or PCR products in a single reaction.

[0107] To amplify rare transcripts, a microdroplet that has undergone a first-step RT-PCR or PCR reaction as described herein may be further subjected to a second step PCR reaction. In some embodiments, a portion of a microdroplet that has undergone a first-step RT-PCR or PCR reaction is extracted from the microdroplet and coalesced with a droplet containing additional PCR reagents, including, but not limited to enzymes (e.g. DNA polymerase), DNA probes (e.g. fluorescent DNA probes) and primers. In certain embodiments, the droplet containing the additional PCR reagents is larger than the microdroplet that has undergone the first step RT-PCR or PCR reaction. This may be beneficial, for example, because it allows for the dilution of cellular components that may be inhibitory to the second step PCR. The second step PCR reaction may be carried out on the same microfluidic device used to carry out the first-step reaction or on a different microfluidic device.

[0108] In some embodiments, the primers used in the second step PCR reaction are the same primers used in the first step RT-PCR or PCR reaction. In other embodiments, the primers used in the second step PCR reaction are different than the primers used in the first step reaction.

Adding Reagents to Microdroplets

[0109] In practicing the subject methods, a number of reagents may need to be added to the microdroplets, in one or more steps (e.g., 2, 3, 4, or 5 or more steps). The means of adding reagents to the microdroplets may vary in a number of ways. Approaches of interest include, but are not limited to, those described by Ahn, et al., Appl. Phys. Lett. 88, 264105 (2006); Priest, et al., Appl. Phys. Lett. 89, 134101 (2006); Abate, et al., PNAS, Nov. 9, 2010 vol. 107 no. 45 19163-19166; and Song, et al., Anal. Chem., 2006, 78 (14), pp 4839-4849; the disclosures of which are incorporated herein by reference in their entireties.

[0110] For instance, a reagent may be added to a microdroplet by a method involving merging a microdroplet with a second microdroplet that contains the reagent(s). The reagent(s) that are contained in the second microdroplet may be added by any convenient means known in the art, specifically including those described herein. This droplet may be merged with the first microdroplet to create a microdroplet that includes the contents of both the first microdroplet and the second microdroplet. [0111] One or more reagents may also, or instead, be added using techniques such as droplet coalescence, or picoinjection. In droplet coalescence, a target drop i.e., the microdroplet) may be flowed alongside a microdroplet containing the reagent(s) to be added to the microdroplet. The two microdroplets may be flowed such that they are in contact with each other, but not touching other microdroplets. These drops may then be passed through electrodes or other means of applying an electrical field, wherein the electric field may destabilize the microdroplets such that they are merged together.

[0112] Reagents may also, or instead, be added using picoinjection. In this approach, a target drop (i.e., the microdroplet) may be flowed past a channel containing the reagent(s) to be added, wherein the reagent(s) are at an elevated pressure. Due to the presence of the surfactants, however, in the absence of an electric field, the microdroplet will flow past without being injected, because surfactants coating the microdroplet may prevent the fluid(s) from entering. However, if an electric field is applied to the microdroplet as it passes the injector, fluid containing the reagent(s) will be injected into the microdroplet. The amount of reagent added to the microdroplet may be controlled by several different parameters, such as by adjusting the injection pressure and the velocity of the flowing drops, by switching the electric field on and off, and the like.

[0113] In other aspects, one or more reagents may also, or instead, be added to a microdroplet by a method that does not rely on merging two droplets together or on injecting liquid into a drop. Rather, one or more reagents may be added to a microdroplet by a method involving the steps of emulsifying a reagent into a stream of very small drops, and merging these small drops with a target microdroplet. Such methods shall be referred to herein as “reagent addition through multiple-drop coalescence.” These methods take advantage of the fact that due to the small size of the drops to be added compared to that of the target drops, the small drops will flow faster than the target drops and collect behind them. The collection can then be merged by, for example, applying an electric field. This approach can also, or instead, be used to add multiple reagents to a microdroplet by using several co-flowing streams of small drops of different fluids. To enable effective merger of the tiny and target drops, it is important to make the tiny drops smaller than the channel containing the target drops, and also to make the distance between the channel injecting the target drops from the electrodes applying the electric field sufficiently long so as to give the tiny drops time to “catch up” to the target drops. If this channel is too short, not all tiny drops will merge with the target drop and adding less reagent than desired. To a certain degree, this can be compensated for by increasing the magnitude of the electric field, which tends to allow drops that are farther apart to merge. In addition to making the tiny drops on the same microfluidic device, they can also, or instead, be made offline using another microfluidic drop maker or through homogenization and then injecting them into the device containing the target drops.

[0114] Accordingly, in certain aspects a reagent is added to a microdroplet by a method involving emulsifying the reagent into a stream of droplets, wherein the droplets are smaller than the size of the microdroplet, flowing the droplets together with the microdroplet, and merging a droplet with the microdroplet. The diameter of the droplets contained in the stream of droplets may vary ranging from about 75% or less than that of the diameter of the microdroplet, e.g., the diameter of the flowing droplets is about 75% or less than that of the diameter of the microdroplet, about 50% or less than that of the diameter of the microdroplet, about 25% or less than that of the diameter of the microdroplet, about 15% or less than that of the diameter of the microdroplet, about 10% or less than that of the diameter of the microdroplet, about 5% or less than that of the diameter of the microdroplet, or about 2% or less than that of the diameter of the microdroplet. In certain aspects, a plurality of flowing droplets may be merged with the microdroplet, such as 2 or more droplets, 3 or more, 4 or more, or 5 or more. Such merging may be achieved by any convenient means, including but not limited to, by applying an electric field, wherein the electric field is effective to merge the flowing droplet with the microdroplet.

[0115] In some embodiments of the above-described methods, the fluids may be jetting. That is, rather than emulsifying the fluid to be added into flowing droplets, a long jet of this fluid can be formed and flowed alongside the target microdroplet. These two fluids can then be merged by, for example, applying an electric field. The result is a jet with bulges where the microdroplets are, which may naturally break apart into microdroplets of roughly the size of the target microdroplets before the merger, due to the Rayleigh plateau instability. A number of variants are contemplated. For instance, one or more agents may be added to the jetting fluid to make it easier to jet, such as gelling agents and/or surfactants. Moreover, the viscosity of the continuous fluid may be adjusted to enable jetting, such as that described by Utada, et al.. Phys. Rev. Lett. 99, 094502 (2007), the disclosure of which is incorporated herein by reference in its entirety.

[0116] In other aspects, one or more reagents may be added using a method that uses the injection fluid itself as an electrode, by exploiting dissolved electrolytes in solution. [0117] In another aspect, a reagent is added to a drop (e.g., a microdroplet) formed at an earlier time by enveloping the drop to which the reagent is be added (i.e., the “target drop”) inside a drop containing the reagent to be added (the “target reagent”). In certain embodiments such a method is carried out by first encapsulating the target drop in a shell of a suitable hydrophobic phase, e.g., an oil, to form a double emulsion. The double emulsion is then encapsulated by a drop containing the target reagent to form a triple emulsion. To combine the target drop with the drop containing the target reagent, the double emulsion is then burst open using any suitable method, including, but not limited to, applying an electric field, adding chemicals that destabilizes the droplet interface, flowing the triple emulsion through constrictions and other microfluidic geometries, applying mechanical agitation or ultrasound, increasing or reducing temperature, or by encapsulating magnetic particles in the drops that can rupture the double emulsion interface when pulled by a magnetic field.

Detecting PCR Products

[0118] In practicing the subject methods, the manner in which PCR products may be detected may vary. For example, if the goal is simply to count the number of a particular cell type, e.g., tumor cells, present in a population, this may be achieved by using a simple binary assay in which SybrGreen, or any other stain and/or intercalating stain, is added to each microdroplet so that in the event a characterizing gene, e.g., an oncogene, is present and PCR products are produced, the drop will become fluorescent. The change in fluorescence may be due to fluorescence polarization. The detection component may include the use of an intercalating stain (e.g., SybrGreen).

[0119] A variety of different detection components may be used in practicing the subject methods, including using fluorescent dyes known in the art. Fluorescent dyes may typically be divided into families, such as fluorescein and its derivatives; rhodamine and its derivatives; cyanine and its derivatives; coumarin and its derivatives; Cascade Blue and its derivatives; Lucifer Yellow and its derivatives; BODIPY and its derivatives; and the like. Exemplary fluorophores include indocarbocyanine (C3), indodicarbocyanine (C5), Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red, Pacific Blue, Oregon Green 488, Alexa fluor-355, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor-555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, JOE, Lissamine, Rhodamine Green, BODIPY, fluorescein isothiocyanate (FITC), carboxy-fluorescein (FAM), phycoerythrin, rhodamine, dichlororhodamine (dRhodamine), carboxy tetramethylrhodamine (TAMRA), carboxy-X-rhodamine (ROX), LIZ, VIC, NED, PET, SYBR, PicoGreen, RiboGreen, and the like. Descriptions of fluorophores and their use, can be found in, for example, R. Haugland, Handbook of Fluorescent Probes and Research Products, 9th ed. (2002), Molecular Probes, Eugene, Oreg.; M. Schena, Microarray Analysis (2003), John Wiley & Sons, Hoboken, N.J.; Synthetic Medicinal Chemistry 2003/2004 Catalog, Berry and Associates, Ann Arbor, Mich.; G. Hermanson, Bioconjugate Techniques, Academic Press (1996); and Glen Research 2002 Catalog, Sterling, Va.

[0120] In other aspects, particularly if a goal is to further characterize the oncogenes present, additional testing may be undertaken. For instance, in the case of the multiplex assays described herein, this may be achieved by having optical outputs that relate which of the gene(s) amplified in the drop. An alternative approach would be to use a binary output, for example, with an intercalated stain, to simply determine which droplets have any oncogenes. These can then be sorted to recover these drops so that they could be analyzed in greater detail to determine which oncogenes they contain. To determine the oncogenes present in such a drop, microfluidic techniques or nonmicrofluidic techniques could be used. Using non-microfluidic techniques, a droplet identified as containing an oncogene can be placed into a well on a wellplate where will be diluted into a larger volume, releasing all of the PCR products that were created during the multiplexed PCR reaction. Samples from this well can then be transferred into other wells, into each of which would be added primers for one of the oncogenes. These wells would then be temperature-cycled to initiate PCR, at which point an intercalating stain would be added to cause wells that have matching oncogenes and primers to light up.

[0121] In practicing the subject methods, therefore, a component may be detected based upon, for example, a change in fluorescence. In certain aspects, the change in fluorescence is due to fluorescence resonance energy transfer (FRET). In this approach, a special set of primers may be used in which the 5' primer has a quencher dye and the 3' primer has a fluorescent dye. These dyes can be arranged anywhere on the primers, either on the ends or in the middles. Because the primers are complementary, they will exist as duplexes in solution, so that the emission of the fluorescent dye will be quenched by the quencher dye, since they will be in close proximity to one another, causing the solution to appear dark. After PCR, these primers will be incorporated into the long PCR products, and will therefore be far apart from one another. This will allow the fluorescent dye to emit light, causing the solution to become fluorescent. Hence, to detect if a particular oncogene is present, one may measure the intensity of the droplet at the wavelength of the fluorescent dye. To detect if different oncogenes are present, this would be done with different colored dyes for the different primers. This would cause the droplet to become fluorescent at all wavelengths corresponding to the primers of the oncogenes present in the cell.

[0122] In practicing the methods of the present disclosure, one or more sorting steps may be employed. For example, after nucleic acid amplification, droplets can be sorted based on presence or absence of a target amplicon. Sorting approaches of interest include, by are not necessarily limited to, approaches that involve the use of membrane valves, bifurcating channels, surface acoustic waves, and/or dielectrophoresis. Sorting approaches of interest further include those described by Agresti, et al., PNAS vol. 107, no 9, 4004-4009; the disclosure of which is incorporated herein by reference in its entirety. A population may be enriched by sorting, in that a population containing a mix of members having or not having a desired property may be enriched by removing those members that do not have the desired property, thereby producing an enriched population having the desired property.

[0123] Sorting may be applied before or after any of the steps described herein. Moreover, two or more sorting steps may be applied to a population of microdroplets, e.g., 2 or more sorting steps, 3 or more, 4 or more, or 5 or more, etc. When a plurality of sorting steps is applied, the steps may be substantially identical or different in one or more ways (e.g., sorting based upon a different property, sorting using a different technique, and the like).

[0124] Moreover, droplets may be purified prior to, or after, any sorting step. That is, a majority of the fluid in the drop is replaced it with a purified solution, without removing any discrete reagents that may be encapsulated in the drop, such a cells or beads. The microdroplet is first injected with a solution to dilute any impurities within it. The diluted microdroplet is then flowed through a microfluidic channel on which an electric field is being applied using electrodes. Due to the di electrophoretic forces generated by the field, as the cells or other discrete reagents pass through the field they will be displaced in the flow. The drops are then split, so that all the objects end up in one microdroplet. Accordingly, the initial microdroplet has been purified, in that the contaminants may be removed while the presence and/or concentration of discrete reagents, such as beads or cells, that may be encapsulated within the droplet are maintained in the resulting microdroplet.

[0125] Microdroplets may be sorted based on one or more properties. Properties of interest include, but are not limited to, the size, viscosity, mass, buoyancy, surface tension, electrical conductivity, charge, magnetism, and/or presence or absence of one or more components. In certain aspects, sorting may be based at least in part upon the presence or absence of a cell in the microdroplet. In certain aspects, sorting may be based at least in part based upon the detection of the presence or absence of PCR amplification products.

[0126] Microdroplet sorting may be employed, for example, to remove microdroplets in which no cells are present. Encapsulation may result in one or more microdroplets, including a majority of the microdroplets, in which no cell is present. If such empty drops were left in the system, they would be processed as any other drop, during which reagents and time would be wasted. To achieve the highest speed and efficiency, these empty drops may be removed with droplet sorting. For example, a drop maker may operate close to the dripping- to-jetting transition such that, in the absence of a cell, 8 pm drops are formed; by contrast, when a cell is present the disturbance created in the flow will trigger the breakup of the jet, forming drops 25 pm in diameter. The device may thus produce a bi-disperse population of empty 8 pm drops and single-cell containing 25 pm drops, which may then be sorted by size using, e.g., a hydrodynamic sorter to recover only the larger, single-cell containing drops.

[0127] Passive sorters of interest include hydrodynamic sorters, which sort microdroplets into different channels according to size, based on the different ways in which small and large drops travel through the microfluidic channels. Also of interest are bulk sorters, a simple example of which is a tube containing drops of different mass in a gravitational field. By centrifuging, agitating, and/or shaking the tube, lighter drops that are more buoyant will naturally migrate to the top of the container. Drops that have magnetic properties could be sorted in a similar process, except by applying a magnetic field to the container, towards which drops with magnetic properties will naturally migrate according to the magnitude of those properties. A passive sorter as used in the subject methods may also involve relatively large channels that will sort large numbers of drops simultaneously based on their flow properties.

[0128] Picoinjection can also be used to change the electrical properties of the drops. This could be used, for example, to change the conductivity of the drops by adding ions, which could then be used to sort them, for example, using dielectrophoresis. Alternatively, picoinjection can be used to charge the drops. This could be achieved by injecting a fluid into the drops that is charged, so that after injection, the drops would be charged. This would produce a collection of drops in which some were charged and others not, and the charged drops could then be extracted by flowing them through a region of electric field, which will deflect them based on their charge amount. By injecting different amounts of liquid by modulating the picoinjection, or by modulating the voltage to inject different charges for affixed injection volume, the final charge on the drops could be adjusted, to produce drops with different charge. These would then be deflected by different amounts in the electric field region, allowing them to be sorted into different containers.

[0129] Methods according to the present invention also involve methods for detecting cancer. Such methods may include encapsulating in a microdroplet oligonucleotides obtained from a biological sample from the subject, wherein at least one oligonucleotide is present in the microdroplet; introducing polymerase chain reaction (PCR) reagents, a detection component, and a plurality of PCR primers into the microdroplet and incubating the microdroplet under conditions allowing for PCR amplification to produce PCR amplification products, wherein the plurality of PCR primers include one or more primers that each hybridize to one or more oncogenes; and detecting the presence or absence of the PCR amplification products by detection of the detection component, wherein detection of the detection component indicates the presence of the PCR amplification products.

[0130] Detection of one or more PCR amplification products corresponding to one or more oncogenes may be indicative that the subject has cancer. The specific oncogenes that are added to the microdroplet may vary. In certain aspects, the oncogene(s) may be specific for a particular type of cancer, e.g., breast cancer, colon cancer, and the like.

[0131] Moreover, in practicing the subject methods the biological sample from which the components are to be detected may vary, and may be based at least in part on the particular type of cancer for which detection is sought. For instance, breast tissue may be used as the biological sample in certain instances, if it is desired to determine whether the subject has breast cancer, and the like.

[0132] In practicing the methods for detecting cancer, any variants to the general steps described herein, such as the number of primers that may be added, the manner in which reagents are added, suitable subjects, and the like, may be made.

Fragmenting and tagging nucleic acids

[0133] The disclosed methods may include a step of fragmenting the genomic DNA or RNA, e.g., to a length that permits their sequencing with existing sequencing platforms, which often have limited read length. Fragmentation can be achieved in a variety of ways and can be applied to either amplified or non-amplified nucleic acid targets. For example, enzymes capable of fragmenting DNA such as Fragmentase® or other nucleases can be introduced into a droplet as described herein and subjected to conditions sufficient for fragmentation. Suitable enzymes capable of fragmenting DNA may include, e.g., DNase I, micrococcal nuclease, DNase III, and any other nuclease that results in fragmented DNA, including nucleases with sequence specific catalysis. Alternatively, chemical methods can be used, such as the inclusion of acids, reactive oxygen species, etc. Organisms that degrade DNA can also be used by including them in the droplet with the nucleic acids. Physical methods, such as shear generated by flow of the nucleic acids, in the droplet, can also be used. Other methods can also be used that perform multiple operations on the nucleic acids including fragmentation. For example, transposons can be used to insert or attach sequences into the nucleic acids, often fragmenting them in the process.

[0134] Accordingly, in some embodiments, the fragmented genomic DNA or RNA may be size selected for nucleic acid fragments in the 200-600 bp range. For example, the fragmented nucleic acid may be size selected in the 50-750 bp range, 75-725 bp range, 100- 700 bp range, 125-675 bp range, 150-650 bp range, 175-625 bp range, or any range bound between two of the following sizes: 25 bp, 50 bp, 75 bp, 100 bp, 125 bp, 150 bp, 175 bp, 200 bp, 225 bp, 250 bp, 275 bp, 300 bp, 325 bp, 350 bp, 375 bp, 400 bp, 425 bp, 450 bp, 475 bp, 500 bp, 525 bp, 550 bp, 575 bp, 600 bp, 625 bp, 650 bp, 675 bp, 700 bp, 725 bp, 750 bp, 775 bp, 800 bp or more. Size selection of the fragmented nucleic acid can be performed by any method known in the art, for example, using agarose gel electrophoresis, solid phase reversible immobilization beads (e.g., AMPure XP beads), microfluidic instruments (e.g., Caliper Labchip XT), commercially available library construction kits (e.g., Sage Science Pippin Prep), etc. Size selection of fragmented genomic DNA or RNA may occur after fragmented sample is obtained, after the fragmented sample is tagged, or after the tagged, fragmented sample is barcoded.

[0135] In some embodiments, the population of droplets including purified genomic DNA or RNA is re-encapsulated before the step of fragmenting the purified genomic DNA or RNA. Accordingly, in some embodiments the present disclosure provides a method for sequencing single cell genomic DNA or RNA including purifying genomic DNA or RNA from cells contained within a population of droplets in bulk to provide a population of droplets including purified genomic DNA or RNA, encapsulating the population of droplets including purified genomic DNA into droplets to provide a population of purified genomic DNA or RNA -containing droplets, and fragmenting the purified genomic DNA or RNA to provide a population of fragmented genomic DNA or RNA -containing droplets.

[0136] In some embodiments, encapsulating the population of droplets including purified genomic DNA or RNA into droplets to provide a population of purified genomic DNA or RNA-containing droplets includes encapsulating the droplets with reagents for use in fragmentation and tagging of the purified genomic DNA or RNA. In some embodiments, fragmentation and tagging of genomic DNA or RNA occurs simultaneously, e.g., in a tagmentation step, and encapsulating the droplets with reagents for use in fragmentation and tagging of the purified genomic DNA or RNA includes encapsulating the droplets with tagmentation reagents, e.g., a complex including a transposase and a transposon. For example, in some embodiments, each of the members of the population of purified genomic DNA or RNA-containing droplets includes a complex including a transposase and a transposon. In certain embodiments, the transposase is pAG-Tn5.

[0137] In some embodiments, a method for sequencing single cell genomic DNA or RNA includes purifying genomic DNA or RNA from cells contained within a population of droplets in bulk to provide a population including purified genomic DNA or RNA. In some embodiments, the purified genomic DNA or RNA is subject to conditions that fragment the purified genomic DNA or RNA to provide a population of droplets including fragmented genomic DNA or RNA. In some embodiments, the fragmented genomic DNA or RNA is optionally tagged with a common adapter sequence. In some embodiments, fragmentation and tagging of genomic DNA or RNA occurs simultaneously.

[0138] In some embodiments, fragmentation of genomic DNA or RNA can be achieved using Fragmentase® (NEB), Transposon Insertion (Nextera), non-specific DNA endonuclease such as DNase, or incorporation of modified bases during amplification and cleavage using DNA repair enzymes, such as dUTP incorporation during amplification and specific cleavage using EndoV and uracil glycosylase. Hydrodynamic shearing can also be used to fragment DNA or RNA.

[0139] In some embodiments, the method includes fragmenting the purified genomic DNA or RNA via transposon insertion, e.g., using Tn5 transposon, Mu transposon, or any other suitable transposon known in the art. In such embodiments, the method includes contacting the purified genomic DNA or RNA with a complex including a transposase and a transposon. In some embodiments, the complex includes a transposon that includes an adapter sequence. Contacting the purified genomic DNA or RNA with the complex results in fragmented genomic DNA or RNA including the adapter sequence. In certain embodiments, because of the dimeric nature of transposases, the fragmented genomic DNA or RNA remains intact as a macromolecular complex and continues to be retained within the population of droplets. Accordingly, a population of droplets including fragmented genomic DNA or RNA optionally including a common adapter sequence is obtained.

Barcoding fragmented nucleic acids

[0140] The disclosed methods may include a step of barcoding a population of droplets including fragmented genomic DNA or RNA optionally including a common adapter sequence. Barcoding is performed such that the fragmented genomic DNA or RNA of each individual single cell is associated with an identifying barcode sequence, e.g., a single unique barcode sequence. In some embodiments, barcoding of the fragmented genomic DNA or RNA can be performed in a single step, for example, by incorporating the barcode sequences using a transposase, or in two steps, in which barcode sequences are added to the fragmented genomic DNA or RNA with, for example, ligase or overlap extension PCR.

[0141] In some embodiments, a population of droplets including fragmented genomic DNA or RNA can be merged together with a library of barcode sequences, wherein each identifying barcode sequence (or population of an identifying barcode sequence), e.g., each unique barcode sequence (or population of a unique barcode sequence) of the library of barcode sequences is separately encapsulated in a droplet. Accordingly, in some embodiments, a method of sequencing single cell genomic DNA or RNA includes encapsulating the population of droplets including fragmented genomic DNA or RNA into droplets to provide a population of fragmented genomic DNA or RNA -containing droplets. The population of fragmented genomic DNA or RNA-containing droplets may then be merged with a library of barcode sequence containing droplets such that each fragmented genomic DNA or RNA-containing droplet is merged with an identifying barcode sequence (or population of an identifying barcode sequence), e.g., a unique barcode sequence (or population of a unique barcode sequence) containing the droplet. The method may further include subjecting the population of droplets containing both the fragmented genomic DNA or RNA and barcode sequence to conditions sufficient for enzymatic incorporation of the barcode sequence into the fragmented genomic DNA or RNA. [0142] One approach for incorporating a barcode sequence into fragmented genomic DNA or RNA is to use primers that are complementary to the adapter sequences and the barcode sequences, such that the product amplicons of both fragmented genomic DNA or RNA and barcodes can anneal to one another and, via an extension reaction such as DNA polymerization, be extended onto one another, generating a double stranded product including the fragmented genomic DNA or RNA attached to the barcode sequence.

[0143] Alternatively or additionally, the primers that amplify the target can themselves be barcoded so that, upon annealing and extending onto the target, the amplicon produced has the barcode sequence incorporated into it. This can be applied with a number of amplification strategies, including specific amplification with PCR or non-specific amplification with, for example, multiple displacement amplification (MDA).

[0144] An alternative or additional enzymatic reaction that can be used to attach barcodes to fragmented genomic DNA or RNA is ligation, including blunt or sticky end ligation. In this approach, the DNA barcodes are incubated with the fragmented genomic DNA or RNA and ligase enzyme, resulting in the ligation of the barcode to the targets. The ends of the fragmented genomic DNA or RNA can be modified as needed for ligation by a number of techniques, including by using adaptors introduced with ligase or fragments to enable greater control over the number of barcodes added to the end of the molecule.

[0145] Yet another approach for adding the barcodes to the fragmented genomic DNA or RNA is to introduce them directly with a transposase or with a combination of enzymes, such as a non-specific endonuclease or combination of non-specific endonucleases (e.g., Fragmentase®) and ligase. For example, in this approach, barcodes can be synthesized that are compatible with a transposase. The transposase can then fragment the purified genomic DNA or RNA and add the barcodes to the ends of the fragment molecules, performing all steps of the reaction in one reaction. A combination of Fragmentase® and ligase can also be used, wherein the Fragmentase® is used to fragment the nucleic acids to a size suitable for sequencing, and the ligase is used to attach the barcodes to the fragment ends.

[0146] In some embodiments, upon obtaining a population of barcoded, fragmented genomic DNA or RNA -containing droplets, the emulsion including the population of droplets is broken and the barcoded, fragmented DNA or RNA is purified to provide purified, barcoded, fragmented genomic DNA or RNA. An optional size selection step may occur to select for purified, barcoded, genomic DNA or RNA fragments of a certain size that permits their sequencing with existing sequencing platforms. Additional disclosure with respect to barcoding nucleic acids in droplets is provided in International Patent Application Publication No. WO2016/126871, the disclosure of which is incorporated by reference herein in its entirety.

[0147] As an alternative to tagmentation/fragmentation, purified single-cell genomes in hydrogels can be subjected to a MALBAC (Multiple Annealing and Looping Based Amplification Cycles) amplification reaction in droplets by co-flowing the droplets with amplification reagents in a microfluidic dropmaker. The MALBAC reaction is described generally in Zong el al. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell, Science, 2012, the disclosure of which is incorporated by reference herein in its entirety. Briefly, in a MALBAC reaction, degenerate primers anneal to genomic DNA and extend. In cycles 2 and later, hairpin loops form after extension and denaturation. These hairpins do not participate in the later cycles of the reaction as they are in a looped conformation. Following this “quasi-linear” amplification (6-10+ cycles), PCR with a single primer is used to amplify the looped products exponentially (10+ cycles).

Sequencing and Read Alignment

[0148] The methods described herein may include a step of sequencing the purified, barcoded, fragmented genomic DNA or RNA. DNA or RNA sequencing can be achieved with commercially available next generation sequencing (NGS) platforms, including platforms that perform sequencing by synthesis, sequencing by ligation, pyrosequencing, using reversible terminator chemistry, using phospholinked fluorescent nucleotides, or realtime sequencing. For example, the purified, barcoded, fragmented genomic DNA may be sequenced on an Illumina MiSeq platform using a custom index primer.

[0149] Amplified nucleic acids (e.g., amplicons) are sequenced to obtain sequence reads for generating a sequencing library. Sequence reads can be achieved with commercially available next generation sequencing (NGS) platforms, including platforms that perform any of sequencing by synthesis, sequencing by ligation, pyrosequencing, using reversible terminator chemistry, using phospholinked fluorescent nucleotides, or real-time sequencing. As an example, amplified nucleic acids may be sequenced on an Illumina MiSeq platform.

[0150] When pyrosequencing libraries of NGS fragments are cloned in-situ amplified by capture of one matrix molecule using granules coated with oligonucleotides complementary to adapters. Each granule containing a matrix of the same type is placed in a microbubble of the “water in oil” type and the matrix is cloned amplified using a method called emulsion PCR. After amplification, the emulsion is destroyed and the granules are stacked in separate wells of a titration picoplate acting as a flow cell during sequencing reactions. The ordered multiple administration of each of the four dNTP reagents into the flow cell occurs in the presence of sequencing enzymes and a luminescent reporter, such as luciferase. In the case where a suitable dNTP is added to the 3 ' end of the sequencing primer, the resulting ATP produces a flash of luminescence within the well, which is recorded using a CCD camera. It is possible to achieve a read length of more than or equal to 400 bases, and it is possible to obtain 10⁶ readings of the sequence, resulting in up to 500 million base pairs (megabytes) of the sequence. Additional details for pyrosequencing are described in Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; US patent No. 6,210,891; US patent No. 6,258,568; each of which is hereby incorporated by reference in its entirety.

[0151] On the Solexa / Illumina platform, sequencing data is produced in the form of short readings. In this method, fragments of a library of NGS fragments are captured on the surface of a flow cell that is coated with oligonucleotide anchor molecules. An anchor molecule is used as a PCR primer, but due to the length of the matrix and its proximity to other nearby anchor oligonucleotides, elongation by PCR leads to the formation of a “vault” of the molecule with its hybridization with the neighboring anchor oligonucleotide and the formation of a bridging structure on the surface of the flow cell . These DNA loops are denatured and cleaved. Straight chains are then sequenced using reversibly stained terminators. The nucleotides included in the sequence are determined by detecting fluorescence after inclusion, where each fluorescent and blocking agent is removed prior to the next dNTP addition cycle. Additional details for sequencing using the Illumina platform are found in Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; US patent No. 6,833,246; US patent No. 7,115,400; US patent No. 6,969,488; each of which is hereby incorporated by reference in its entirety.

[0152] Sequencing of nucleic acid molecules using SOLiD technology includes clonal amplification of the library of NGS fragments using emulsion PCR. After that, the granules containing the matrix are immobilized on the derivatized surface of the glass flow cell and annealed with a primer complementary to the adapter oligonucleotide. However, instead of using the indicated primer for 3 'extension, it is used to obtain a 5' phosphate group for ligation for test probes containing two probe-specific bases followed by 6 degenerate bases and one of four fluorescent labels. In the SOLiD system, test probes have 16 possible combinations of two bases at the 3 'end of each probe and one of four fluorescent dyes at the 5' end. The color of the fluorescent dye and, thus, the identity of each probe, corresponds to a certain color space coding scheme. After many cycles of alignment of the probe, ligation of the probe and detection of a fluorescent signal, denaturation followed by a second sequencing cycle using a primer that is shifted by one base compared to the original primer. In this way, the sequence of the matrix can be reconstructed by calculation; matrix bases are checked twice, which leads to increased accuracy. Additional details for sequencing using SOLiD technology are found in Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; US patent No. 5,912,148; US patent No. 6,130,073; each of which is incorporated by reference in its entirety.

[0153] In particular embodiments, HeliScope from Helicos BioSciences is used. Sequencing is achieved by the addition of polymerase and serial additions of fluorescently- labeled dNTP reagents. Switching on leads to the appearance of a fluorescent signal corresponding to dNTP, and the specified signal is captured by the CCD camera before each dNTP addition cycle. The reading length of the sequence varies from 25-50 nucleotides with a total yield exceeding 1 billion nucleotide pairs per analytical work cycle. Additional details for performing sequencing using HeliScope are found in Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; US Patent No. 7,169,560; US patent No. 7,282,337; US patent No. 7,482,120; US patent No. 7,501,245; US patent No. 6,818,395; US patent No. 6,911,345; US patent No. 7,501,245; each of which is incorporated by reference in its entirety.

[0154] In some embodiments, a Roche sequencing system 454 is used. Sequencing 454 involves two steps. In the first step, DNA is cut into fragments of approximately 300-800 base pairs, and these fragments have blunt ends. Oligonucleotide adapters are then ligated to the ends of the fragments. The adapter serves as primers for amplification and sequencing of fragments. Fragments can be attached to DNA-capture beads, for example, streptavidin- coated beads, using, for example, an adapter that contains a 5'-biotin tag. Fragments attached to the granules are amplified by PCR within the droplets of an oil-water emulsion. The result is multiple copies of cloned amplified DNA fragments on each bead. At the second stage, the granules are captured in wells (several picoliters in volume). Pyrosequencing is carried out on each DNA fragment in parallel. Adding one or more nucleotides leads to the generation of a light signal, which is recorded on the CCD camera of the sequencing instrument. The signal intensity is proportional to the number of nucleotides included. Pyrosequencing uses pyrophosphate (PPi), which is released upon the addition of a nucleotide. PPi is converted to ATP using ATP sulfurylase in the presence of adenosine 5 'phosphosulfate. Luciferase uses ATP to convert luciferin to oxyluciferin, and as a result of this reaction, light is generated that is detected and analyzed. Additional details for performing sequencing 454 are found in Margulies et al. (2005) Nature 437: 376-380, which is hereby incorporated by reference in its entirety.

[0155] Ion Torrent technology is a DNA sequencing method based on the detection of hydrogen ions that are released during DNA polymerization. The microwell contains a fragment of a library of NGS fragments to be sequenced. Under the microwell layer is the hypersensitive ion sensor ISFET. All layers are contained within a semiconductor CMOS chip, similar to the chip used in the electronics industry. When dNTP is incorporated into a growing complementary chain, a hydrogen ion is released that excites a hypersensitive ion sensor. If homopolymer repeats are present in the sequence of the template, multiple dNTP molecules will be included in one cycle. This results in a corresponding amount of hydrogen atoms being released and in proportion to a higher electrical signal. This technology is different from other sequencing technologies that do not use modified nucleotides or optical devices. Additional details for Ion Torrent Technology is found in Science 327 (5970): 1190 (2010); US Patent Application Publication Nos. 20090026082, 20090127589, 20100301398, 20100197507, 20100188073, and 20100137143, each of which is incorporated by reference in its entirety.

[0156] In various embodiments, sequencing reads obtained from the NGS methods can be filtered by quality and grouped by barcode sequence using any algorithms known in the art, e.g., Python script barcodeCleanup.py . In some embodiments, a given sequencing read may be discarded if more than about 20% of its bases have a quality score (Q-score) less than Q20, indicating a base call accuracy of about 99%. In some embodiments, a given sequencing read may be discarded if more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30% have a Q-score less than Q10, Q20, Q30, Q40, Q50, Q60, or more, indicating a base call accuracy of about 90%, about 99%, about 99.9%, about 99.99%, about 99.999%, about 99.9999%, or more, respectively.

[0157] In some embodiments, sequencing reads associated with a barcode containing less than 50 reads may be discarded to ensure that all barcode groups, representing single cells, contain a sufficient number of high-quality reads. In some embodiments, all sequencing reads associated with a barcode containing less than 30, less than 40, less than 50, less than 60, less than 70, less than 80, less than 90, less than 100 or more may be discarded to ensure the quality of the barcode groups representing single cells.

[0158] In various embodiments, sequence reads with common barcode sequences (e.g., meaning that sequence reads originated from the same cell) may be aligned to a reference genome using known methods in the art to determine alignment position information. For example, sequence reads derived from genomic DNA can be aligned to a range of positions of a reference genome. In various embodiments, sequence reads derived from genomic DNA can align with a range of positions corresponding to a gene of the reference genome. The alignment position information may indicate a beginning position and an end position of a region in the reference genome that corresponds to a beginning nucleotide base and end nucleotide base of a given sequence read. A region in the reference genome may be associated with a target gene or a segment of a gene. Further details for aligning sequence reads to reference sequences is described in US Application No. 16/279,315, which is hereby incorporated by reference in its entirety. In various embodiments, an output file having SAM (sequence alignment map) format or BAM (binary alignment map) format may be generated and output for subsequent analysis.

[0159] Once the raw sequencing reads are filtered by quality and grouped by barcode sequence, the sequences may be exported to a table, e.g., a table in a relational database, e.g., a SQLite database, including fields pertinent to identifying sequencing reads that are obtained from a single cell. In some embodiments, the sequences may be exported to a table in a relational database, e.g., a SQLite database including fields containing the barcode sequence, barcode group size, a unique read ID number, and read sequence. In some embodiments, e.g., in the case of analyzing a synthetic cell population, sequencing reads may be aligned using any suitable available software program, e.g., bowtie2 v2.2.9, and the SQLite table may be updated with relevant alignment information for each sequencing read. In some embodiments, e.g., when analyzing environmental samples, the sequencing reads may be classified by taxonomy using any suitable available software program, e.g., Kraken v0.10.5, and the SQLite table may be updated with relevant taxonomic information for each sequencing read. In some embodiments, barcode group purity may be calculated from reference alignment data or phylogenetic labels using any suitable available script, e.g.

Python script purity.py. Example System and/or Computer Embodiments

[0160] Additionally described herein are systems and computer embodiments for performing the single cell analysis described above. An example system can include a single cell workflow device and a computing device, such as single cell workflow device 100 and computing device 180 shown in FIG. 1A. In various embodiments, the single cell workflow device 100 is configured to perform the steps of cell nuclei encapsulation 160, nuclei processing 165, barcoding 170, target amplification 175, nucleic acid pooling 205, and sequencing 210. In various embodiments, the computing device 180 is configured to perform the in silico steps of read alignment 215 and characterizing 220 the cells.

[0161] In various embodiments, a single cell workflow device 100 includes at least a microfluidic device that is configured to encapsulate cell nuclei with a reaction mixture, encapsulate processed nuclei with a nucleic acid amplification reaction mixture, and perform nucleic acid amplification reactions. For example, the microfluidic device can include one or more fluidic channels that are fluidically connected. Therefore, the combining of an aqueous fluid through a first channel and a carrier fluid through a second channel results in the generation of emulsion droplets. In certain embodiments, for example, microfluidics devices have at least one “micro” channel. Such channels may have at least one cross-sectional dimension on the order of a millimeter or smaller (e.g., less than or equal to about 1 millimeter). Obviously for certain applications, this dimension may be adjusted; in some embodiments the at least one cross-sectional dimension is about 500 micrometers or less. In some embodiments, again as applications permit, the cross-sectional dimension is about 100 micrometers or less (or even about 10 micrometers or less — sometimes even about 1 micrometer or less). A cross-sectional dimension is one that is generally perpendicular to the direction of centerline flow, although it should be understood that when encountering flow through elbows or other features that tend to change flow direction, the cross-sectional dimension in play need not be strictly perpendicular to flow. It should also be understood that in some embodiments, a micro-channel may have two or more cross-sectional dimensions such as the height and width of a rectangular cross-section or the major and minor axes of an elliptical cross-section. Either of these dimensions may be compared against sizes presented here. Note that micro-channels employed in this invention may have two dimensions that are grossly disproportionate — e.g., a rectangular cross-section having a height of about 100-200 micrometers and a width on the order or a centimeter or more. Of course, certain devices may employ channels in which the two or more axes are very similar or even identical in size (e.g., channels having a square or circular cross-section). Additional details of microchannel design and dimensions is described in International Patent Application No.

PCT/US2016/016444 and US Patent Application No. 14/420,646, each of which is hereby incorporated by reference in its entirety.

[0162] In various embodiments, the single cell workflow device 100 may also include one or more of: (a) a temperature control module for controlling the temperature of one or more portions of the subject devices and/or droplets therein and which is operably connected to the microfluidic device(s), (b) a detection module, i.e., a detector, e.g., an optical imager, operably connected to the microfluidic device(s), (c) an incubator, e.g., a cell incubator, operably connected to the microfluidic device(s), and (d) a sequencer operably connected to the microfluidic device(s). The one or more temperature and/or pressure control modules provide control over the temperature and/or pressure of a carrier fluid in one or more flow channels of a device. As an example, a temperature control module may be one or more thermal cycler that regulates the temperature for performing nucleic acid amplification. The one or more detection modules i.e., a detector, e.g., an optical imager, are configured for detecting the presence of one or more droplets, or one or more characteristics thereof, including their composition. In some embodiments, detector modules are configured to recognize one or more components of one or more droplets, in one or more flow channel. The sequencer is a hardware device configured to perform sequencing, such as next generation sequencing. Examples of sequencers include Illumina sequencers (e.g., MiniSeq™, MiSeq™, NextSeq™ 550 Series, or NextSeq™ 2000), Roche sequencing system 454, and Thermo Fisher Scientific sequencers (e.g., Ion GeneStudio S5 system, Ion Torrent Genexus System).

[0163] FIG. 4 depicts an example computing device for implementing system and methods described in reference to FIGs. 1-3. For example, the example computing device 180 is configured to perform the in silico steps of read alignment 215 and cell characterization 220. Examples of a computing device can include a personal computer, desktop computer laptop, server computer, a computing node within a cluster, message processors, hand-held devices, multi-processor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, mobile telephones, PDAs, tablets, pagers, routers, switches, and the like. [0164] In some embodiments, the computing device 180 includes at least one processor 402 coupled to a chipset 404. The chipset 404 includes a memory controller hub 420 and an input/output (I/O) controller hub 422. A memory 406 and a graphics adapter 412 are coupled to the memory controller hub 420, and a display 418 is coupled to the graphics adapter 412. A storage device 408, an input interface 414, and network adapter 416 are coupled to the I/O controller hub 422. Other embodiments of the computing device 180 have different architectures.

[0165] The storage device 408 is a non-transitory computer-readable storage medium such as a hard drive, compact disk read-only memory (CD-ROM), DVD, or a solid-state memory device. The memory 406 holds instructions and data used by the processor 402. The input interface 414 is a touch-screen interface, a mouse, track ball, or other type of input interface, a keyboard, or some combination thereof, and is used to input data into the computing device 180. In some embodiments, the computing device 180 may be configured to receive input (e.g., commands) from the input interface 414 via gestures from the user. The graphics adapter 412 displays images and other information on the display 418. The network adapter 416 couples the computing device 180 to one or more computer networks.

[0166] The computing device 180 is adapted to execute computer program modules for providing functionality described herein. As used herein, the term “module” refers to computer program logic used to provide the specified functionality. Thus, a module can be implemented in hardware, firmware, and/or software. In one embodiment, program modules are stored on the storage device 408, loaded into the memory 406, and executed by the processor 402.

[0167] The types of computing devices 180 can vary from the embodiments described herein. For example, the computing device 180 can lack some of the components described above, such as graphics adapters 412, input interface 414, and displays 418. In some embodiments, a computing device 180 can include a processor 402 for executing instructions stored on a memory 406.

[0168] In various embodiments, methods described herein, such as methods of aligning sequence reads, methods of determining cellular genotypes and phenotypes, and/or methods of analyzing cells using cellular genotypes and phenotypes can be implemented in hardware or software, or a combination of both. In one embodiment, a non-transitory machine- readable storage medium, such as one described above, is provided, the medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying any of the datasets and execution and results described herein. Such data can be used for a variety of purposes, such as patient monitoring, treatment considerations, and the like. Embodiments of the methods described above can be implemented in computer programs executing on programmable computers, comprising a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), a graphics adapter, an input interface, a network adapter, at least one input device, and at least one output device. A display is coupled to the graphics adapter. Program code is applied to input data to perform the functions described above and generate output information. The output information is applied to one or more output devices, in known fashion. The computer can be, for example, a personal computer, microcomputer, or workstation of conventional design.

[0169] Each program can be implemented in a high level procedural or object oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. In any case, the language can be a compiled or interpreted language. Each such computer program is preferably stored on a storage media or device (e.g., ROM or magnetic diskette) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein. The system can also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.

[0170] The signature patterns and databases thereof can be provided in a variety of media to facilitate their use. “Media” refers to a manufacture that contains the signature pattern information of the present invention. The databases of the present invention can be recorded on computer readable media, e.g. any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. One of skill in the art can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising a recording of the present database information. "Recorded" refers to a process for storing information on computer readable medium, using any such methods as known in the art. Any convenient data storage structure can be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g. word processing text file, database format, etc.

EXAMPLES

[0171] The disclosure now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the scope of the disclosure in any way.

Example 1: Example Single Cell Analysis Workflow

[0172] A suspension of cells is obtained. The cells are gently lysed using a non-ionic detergent NP-40 (e.g., between 0.05-5% NP-40). The cell nuclei are suspended and a primary antibody is added and incubated. The primary antibody exhibits binding affinity for H3K4me3, H3K27me3, or FoxAl. The cell nuclei are washed to remove unbound primary antibody.

[0173] Secondary antibody is added and incubated. The secondary antibody exhibits binding affinity for the primary antibody. The cell nuclei are washed to remove unbound secondary antibody. Reaction mixture including pAG-Tn5 transposase is added to the cell nuclei and incubated. The transposase includes sequencing adapters, e.g., custom sequencing adapters, and exhibits affinity to the primary or secondary antibodies. The reaction mixture does not include magnesium ions (Mg⁺⁺). The cell nuclei are washed to remove excess transposase.

[0174] The cell nuclei are loaded on Tapestri® to perform single cell analysis. Individual cell nuclei are encapsulated with a tagmentation reaction mixture which include magnesium ions (Mg⁺⁺) and proteinase K. Droplets are incubated at 37 °C which enables activation of the transposase and subsequent tagmentation.

[0175] Droplets are incubated at 50 °C to activate proteinase K and release genomic DNA from chromatin packaging. Droplets are incubated with Bst 3.0 DNA polymerase or a reverse transcriptase for DNA fill-in at 65 °C. Droplets are next incubated at 80 °C to inactivate proteinase K. [0176] Processed cell nuclei are encapsulated in a second droplet with nucleic acid amplification reaction mixture. The reaction mixture includes the NEB (New England Biolabs) Q5, High-Fidelity PCR Master Mix, or an alternate barcoding mastermix. Additionally, a barcoded bead is added to the second droplet.

[0177] In the second droplet, cell barcodes are attached through nucleic acid amplification. These droplets are then broken, the amplicons processed into sequencing libraries, and the libraries are sequenced..

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of analyzing a target nucleic acid in a single cell, the method comprising: a) providing a sample comprising a plurality of individual cells or cellular nuclei, a primary detection reagent, a secondary detection reagent, and a first reaction mixture comprising a transposase; b) forming a first microdroplet comprising a single cell or cellular nucleus isolated within the first microdroplet from the plurality of individual cells or cellular nuclei, the first reaction mixture, and a second reaction mixture comprising magnesium or manganese and a protease; c) forming a second microdroplet comprising components of the first microdroplet and reagents for nucleic acid amplification; d) incubating the microdroplet under conditions allowing for nucleic acid amplification to produce amplification products; and e) sequencing the amplification products.

2. The method of claim 1, wherein the target nucleic acid is a DNA or an RNA.

3. The method of claim 1 or 2, wherein the target nucleic acid is bound to a protein.

4. The method of claim 3, wherein the protein is selected from the group consisting of a histone, a chromatin-modifying enzyme, or a transcription factor.

5. The method of any one of claims 1-4, wherein the primary detection reagent is an antibody.

6. The method of any one of claims 1-5, wherein the primary detection reagent binds the target nucleic acid.

7. The method of any one of claims 1-6, wherein the secondary detection reagent is an antibody.

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8. The method of any one of claims 1-7, wherein the secondary detection reagent binds the primary detection reagent.

9. The method of any one of claims 1-8, wherein the transposase is a prokaryotic transposase pAG-Tn5.

10. The method of any one of claims 1-9, wherein the transposase binds the secondary detection reagent.

11. The method of any one of claims 1-10, wherein the first reaction mixture does not comprise magnesium or manganese.

12. The method of any one of claims 1-10, wherein the first reaction mixture comprises insufficient magnesium or manganese to activate the transposase.

13. The method of any one of claims 1-12, further comprising a wash step after the providing of the first reaction mixture comprising a transposase.

14. The method of any one of claims 1-13, wherein the nucleic acid amplification comprises a polymerase chain reaction (PCR) amplification or an isothermal amplification.

15. The method of any one of claims 1-14, wherein the PCR is a high fidelity PCR.

16. The method of any one of claims 1-15, wherein the reagents for nucleic acid amplification comprises a plurality of primers comprising one or more primers that hybridize to one or more oligonucleotides suspected to be present in the target nucleic acid, if present in the target nucleic acid.

17. The method of any one of claims 1-16, wherein the magnesium or the manganese in the first microdroplet activates the transposase.

18. The method of any one of claims 1-17, wherein the first microdroplet is heated for Tagmentation.

19. The method of claim 18, wherein the Tagmentation occurs at about 37° C.

50

20. The method of any one of claims 1-19, further comprising adding a reverse transcriptase or a DNA polymerase to the first microdroplet for fill-in.

21. The method of claim 20, wherein the fill-in occurs at 37° C, 50° C, or 65° C.

22. The method of any one of claims 1-19, further comprising adding a reverse transcriptase or a DNA polymerase to the first microdroplet for fill-in.

23. The method of claim 22, wherein the fill-in occurs at 60° C.

24. The method of any one of claims 1-19, wherein the first microdroplet is heated for protease activity.

25. The method of claim 24, wherein the protease activity occurs at about 50° C or about 60° C.

26. The method of any one of claims 1-25, wherein the first microdroplet is heated for protease inactivation.

27. The method of claim 26, wherein the protease inactivation occurs at about 70° C, 80° C, or 90° C.

28. The method of any one of claims 1-27, wherein steps (a) to (e) are performed in order.

29. The method of any one of claims 1-28, wherein the target nucleic acid is epigenetically regulated.

30. A method of analyzing a target nucleic acid in a single cell, the method comprising: a) providing a sample having a plurality of individual cells or cellular nuclei; b) adding a primary detection reagent; c) adding a secondary detection reagent; d) adding a first reaction mixture comprising a transposase;

51 e) forming a first microdroplet comprising the individual cells or cellular nuclei, the first reaction mixture, and a second reaction mixture comprising magnesium or manganese and a protease, wherein the magnesium or manganese in the first microdroplet activates the transposase; f) forming a second microdroplet comprising components of the first microdroplet and reagents for nucleic acid amplification; g) incubating the microdroplet under conditions allowing for nucleic acid amplification to produce amplification products; and h) sequencing the amplification products.

31. The method of claim 30, wherein the target nucleic acid is a DNA or an RNA.

32. The method of claim 30 or 31, wherein the target nucleic acid is bound to a protein.

33. The method of claim 32, wherein the protein is selected from the group consisting of a histone, a chromatin-modifying enzyme, or a transcription factor.

34. The method of any one of claims 30-33, wherein the primary detection reagent is an antibody.

35. The method of any one of claims 30-34, wherein the primary detection reagent binds the target nucleic acid.

36. The method of any one of claims 30-35, wherein the secondary detection reagent is an antibody.

37. The method of any one of claims 30-36, wherein the secondary detection reagent binds the primary detection reagent.

38. The method of any one of claims 30-37, wherein the transposase is a prokaryotic transposase pAG-Tn5.

39. The method of any one of claims 30-38, wherein the transposase binds the secondary detection reagent.

52

40. The method of any one of claims 30-39, wherein the first reaction mixture does not comprise magnesium or manganese.

41. The method of any one of claims 30-39, wherein the first reaction mixture comprises insufficient magnesium or manganese to activate the transposase.

42. The method of any one of claims 30-41, further comprising a wash step after the adding the first reaction mixture comprising a transposase.

43. The method of any one of claims 30-42, wherein the nucleic acid amplification is a polymerase chain reaction (PCR) amplification or an isothermal amplification.

44. The method of any one of claims 30-43, wherein the PCR is a high fidelity PCR.

45. The method of any one of claims 30-44, wherein the reagents for nucleic acid amplification comprises a plurality of primers comprising one or more primers that each hybridize to one or more oligonucleotides.

46. The method of any one of claims 30-45, wherein the first microdroplet is heated for Tagmentation.

47. The method of claim 46, wherein the Tagmentation occurs at about 37° C.

48. The method of any one of claims 26-47, further comprising adding a reverse transcriptase or a DNA polymerase to the first microdroplet for fill-in.

49. The method of claim 48, wherein the fill-in occurs at 37° C, 50° C, or 65° C.

50. The method of any one of claims 26-47, further comprising adding a reverse transcriptase or a DNA polymerase to the first microdroplet for fill-in.

51. The method of claim 50, wherein the fill-in occurs at 60° C.

52. The method of any one of claims 1-47, wherein the first microdroplet is heated for protease activity.

53. The method of claim 52, wherein the protease activity occurs at about 50° C or about 60° C.

54. The method of any one of claims 30-53, wherein the first microdroplet is heated for protease inactivation.

55. The method of claim 54, wherein the protease inactivation occurs at about 70° C, 80° C, or 90° C.

56. The method of any one of claims 30-55, wherein steps (a) to (h) are performed in order.

57. The method of any one of claims 30-56, wherein the target nucleic acid is epigenetically regulated.