RU2835967C2 - Genotyping kits - Google Patents

Abstract

FIELD: chemistry; biotechnology.

SUBSTANCE: group of inventions relates to chemistry and molecular biology. Object 1 is a kit for producing a monoclonal cluster of amplicons with a unique target locus for genotyping, comprising a flow cell comprising a substrate and first and second capture primers attached to the substrate; and a genotyping probe fluid comprising a liquid carrier and a genotyping oligonucleotide in a liquid carrier, wherein the genotyping oligonucleotide comprises a first primer sequence, a portion of an index sequence directly attached to the first primer sequence, a portion of the priming site directly attached to the portion of the index sequence, a probe sequence directly attached to the portion of the priming site, wherein the probe sequence is a target genotyping locus having a nucleic base of interest, a restriction endonuclease site and a second primer sequence that is at least partially complementary to the second capture primer. Object 2 is a method of producing a monoclonal cluster of amplicons with a unique target locus for genotyping.

EFFECT: providing genotyping of several different target loci on any surface of a flow cell, which uses clonally amplified clusters, without the need to add additional immobilisation components.

19 cl, 4 dwg, 2 ex

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 62/981,866, filed February 26, 2020, the contents of which are incorporated herein by reference in their entirety.

LINK TO SEQUENCE LISTING

[0002] The sequence listing submitted via EFS-Web is incorporated herein by reference in its entirety. The file name is ILI184BPCT_IP-1897-PCT_Sequence_Listing_ST25.txt, the file size is 788 bytes, and the file creation date is December 29, 2020.

PREREQUISITES FOR THE CREATION OF THE INVENTION

[0003] Detection of specific nucleic acids can be used for research in diagnostic medicine and molecular biology. Gene probe assays can be useful, for example, in identifying infectious organisms such as bacteria and viruses; in probing the expression of normal and mutant genes and identifying mutant genes such as oncogenes; in tissue typing for compatibility prior to tissue transplantation; in comparing tissue or blood samples in forensic science; and in studying homology between genes from different species.

ESSENCE OF THE INVENTION

[0004] Described herein are genotyping oligonucleotides that include specific sections of a nucleotide sequence designed to perform a specific function in cluster formation, linearization, target locus identification, and/or sequencing. The genotyping oligonucleotides allow genotyping to be performed on unstructured or structured flow cells and do not involve additional immobilization components.

[0005] The first aspect described herein is a kit comprising: a flow cell comprising: a support; and first and second capture primers attached to the support; and a genotyping probe fluid comprising: a liquid carrier; and a genotyping oligonucleotide in the liquid carrier, the genotyping oligonucleotide comprising: a first primer sequence; a probe sequence representing a genotyping target locus; a restriction endonuclease site; and a second primer sequence that is at least partially complementary to the second capture primer.

[0006] It should be understood that any features of the kit described herein can be combined in any desired manner and/or in any desired configuration to achieve the advantages disclosed herein, including, for example, to provide genotyping in a flow cell.

[0007] A second aspect described herein is a method comprising: introducing a genotyping probe fluid into a flow cell comprising first and second capture primers, the genotyping probe fluid comprising a plurality of genotyping oligonucleotides, wherein each of the genotyping oligonucleotides comprises: a first primer sequence; a probe sequence representing a corresponding genotyping target locus; a restriction endonuclease site; and a second primer sequence that is at least partially complementary to the second capture primer; thereby causing the corresponding genotyping oligonucleotide to react to form corresponding clonal populations of amplicons from the corresponding genotyping oligonucleotide; linearizing the amplicons to obtain probe templates; sequencing at least one probe-detecting portion of the probe templates to detect each of the probe sequences; removing at least the corresponding nascent chains from the probe templates, thereby exposing the 3' OH group at the end of the probe templates; hybridizing the corresponding samples to the probe templates; and performing corresponding genotyping reactions on the samples on the accessible 3' OH groups.

[0008] It should be understood that any features of the method can be combined together in any desired manner. Furthermore, it should be understood that any combination of features of the method and/or kit can be used together and/or in combination with any of the examples described herein to achieve the advantages disclosed herein, including, for example, achieving genotyping in a flow cell.

BRIEF DESCRIPTION OF DRAWINGS

[0009] The features according to the examples of the present description will become apparent upon reading the following detailed description and the drawings, in which like reference numerals correspond to similar, although possibly non-identical, components. For brevity, reference numerals or features having a previously described function may or may not be described with respect to other drawings in which they appear.

[0010] Fig. 1A is a schematic representation of an example of a genotyping oligonucleotide described herein;

[0011] Fig. 1B is a schematic representation of another example of a genotyping oligonucleotide described herein;

[0012] Fig. 2A shows a top view of an example of a flow cell;

[0013] Fig. 2B shows an enlarged partial cross-sectional view of an example of a flow channel of a flow cell including recesses formed along the flow channel;

[0014] Fig. 3A-3H schematically illustrates an example of the method described in this document; and

[0015] Fig. 4A-4H schematically illustrates another example of the method described herein.

DETAILED DESCRIPTION OF THE INVENTION

[0016] Described herein are genotyping oligonucleotides that include specific sections of a nucleotide sequence. Each section of the nucleotide sequence of the genotyping oligonucleotide is designed to perform a designated function in clustering, linearization, target locus identification, and/or sequencing.

[0017] Genotyping oligonucleotides can be used in an unstructured flow cell having capture primers on its surface, or in a structured flow cell including wells having capture primers. In different regions on the surface of the unstructured flow cell or in corresponding wells of the structured flow cell, monoclonal populations (clusters) of amplicons can be generated from the corresponding genotyping oligonucleotides. The amplicons in a particular region or well may be different from the amplicons in each of the other regions or wells, and thus each monoclonal population of amplicons can represent a unique target locus for genotyping.

[0018] The genotyping oligonucleotides described herein allow genotyping to be performed on unstructured flow cells or structured flow cells and do not include additional immobilizing components such as solid phase beads. In addition, the genotyping oligonucleotides described herein may be suitable for use with any flow cell surface that utilizes clonally amplified cassettes.

[0019] The present document also describes methods for producing a target genotyping locus that can be used with genotyping oligonucleotides. These methods are amplification methods that allow the generation of genomic DNA (gDNA) fragments without the need to introduce a cleavage site and perform fragmentation.

[0020] Definitions

[0021] Unless otherwise specified, it is understood that the terms used in this document have their ordinary meaning in the relevant field. Below are a number of terms used in this document and their meanings.

[0022] As used herein, the singular forms "a", "an", and "an" include both singular and plural forms unless the context clearly dictates otherwise. As used herein, the term "comprising" is synonymous with "including", "including", or "characterized by" and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0023] References in the present description to "one example," "another example," "an example," and the like mean that a particular element (e.g., a feature, structure, composition, configuration, and/or characteristic) described with respect to an example is included in at least one example described herein, and may or may not be present in other examples. In addition, it should be understood that the described elements of any example may be combined in any suitable manner with different examples, unless the context clearly indicates otherwise.

[0024] The terms "substantially" and "about" used throughout this specification, including the claims, are used to describe and account for small variations, such as due to processing variations. For example, these terms may refer to equal to ±10% or less of a stated value, such as equal to ±5% or less of a stated value, such as equal to ±2% or less of a stated value, such as equal to ±1% or less of a stated value, such as equal to ±0.5% or less of a stated value, such as equal to ±0.2% or less of a stated value, such as equal to ±0.1% or less, such as equal to ±0.05% or less of a stated value.

[0025] Furthermore, it should be understood that the ranges recited herein include the stated range and any value or subrange within the stated range, as if such ranges were expressly stated. For example, a range represented by boundaries from about 2 mm to about 300 mm should be interpreted as including not only the expressly recited limits from about 2 mm to about 300 mm, but also including individual values such as about 15 mm, 22.5 mm, 245 mm, etc., and subranges such as from about 20 mm to about 225 mm, etc.

[0026] Attached. The state of two components attached, fastened, glued, connected, or linked to each other directly or indirectly. For example, a nucleic acid can be attached to a functionalized polymer via a covalent or non-covalent bond. A covalent bond is characterized by the presence of electron pairs between the atoms. A non-covalent bond is a chemical bond that does not involve the sharing of electron pairs, but may include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, or hydrophobic interactions. When two components are directly attached, there is no intermediate component. For example, in some examples of a genotyping oligonucleotide, the first capture primer and the probe sequence are directly covalently linked to each other. When two components are indirectly attached, there is some intermediate component. For example, the first capture primer and probe sequence in some examples of the genotyping oligonucleotide are indirectly linked to each other via an index sequence region and a priming site region.

[0027] Application. Any suitable application technique, which may be manual or automated and in some cases results in a modification of the surface properties. In general, application may be accomplished using vapor deposition techniques, coating techniques, surface grafting techniques, or the like. Some specific examples include chemical vapor deposition (CVD), spray coating (e.g., ultrasonic spray coating), spin coating, dip coating, immersion coating, knife coating, curtain coating, flow coating, aerosol printing, screen printing, microcontact printing, inkjet printing, or the like.

[0028] Recess. A discrete concave element on a substrate or profiled resin having an inlet opening on the surface that is at least partially surrounded by intermediate region(s) of the substrate or profiled resin. Recesses may have any of a variety of different shapes of inlet opening on the surface, including, for example, a circular, elliptical, square, polygonal, star-shaped (with any number of vertices), etc. The cross-section of the recess in a plane perpendicular to the surface may be curved, square, polygonal, hyperbolic, conical, angular, etc. As examples, the recess may be a dimple or two dimples connected to each other. The recess may also have a more complex architecture, including ribs, stepped elements, etc.

[0029] Each. As used herein, the term "each" with respect to a collection of elements is intended to refer to an individual element in that collection, but does not necessarily refer to every element in that collection. Exceptions may arise where explicit description or context clearly indicates otherwise.

[0030] Flow cell. A vessel having a chamber (e.g., a flow channel) in which a reaction can occur, an inlet for introducing reagent(s) into the chamber, and an outlet for removing the reagent(s) from the chamber. In some examples, the chamber allows for detection of a reaction occurring in the chamber. For example, the chamber may include one or more transparent surfaces that allow for optical detection of arrays, optically labeled molecules, or the like.

[0031] Genomic DNA (gDNA). One or more chromosomal polymeric deoxyribonucleotide molecules naturally occurring in the nucleus of a eukaryotic cell or in a prokaryote, virus, mitochondrion, or chloroplast and containing sequences that are naturally transcribed into RNA as well as sequences that are not naturally transcribed into RNA by the cell. Eukaryotic cell gDNA contains at least one centromere, two telomeres, one origin of replication, and one sequence that is not transcribed into RNA by the eukaryotic cell, including, for example, an intron or a transcription promoter. Prokaryotic cell gDNA contains at least one origin of replication and one sequence that is not transcribed into RNA by the prokaryotic cell, including, for example, a transcription promoter. Eukaryotic genomic DNA can be distinguished from prokaryotic, viral, or organellar genomic DNA, for example, by the presence of introns in eukaryotic genomic DNA and the absence of introns in the gDNA of other organisms.

[0032] Genotyping oligonucleotide. A single-stranded deoxyribonucleic acid sequence that includes defined sections of the nucleotide sequence, one of which is a probe sequence representing the target locus for genotyping. The genotyping oligonucleotide can act as a template for generating clusters.

[0033] Sequence index region. A short chain in the range of 10 to 30 nucleotide bases that defines the sequence of the probe in some examples of a genotyping oligonucleotide.

[0034] Locus or loci. A sequence-specific location within a nucleic acid sample. The term may include predetermined or predicted nucleotide sequences that are expected to be present in the isolated nucleic acid molecules. These predetermined or predicted nucleotide sequences may be referred to herein as a "genotyping target locus" and may be the region(s) of interest for analysis. The term is intended to include single nucleotide polymorphisms (SNPs), mutations, variable number of tandem repeats (VNTRs) and single tandem repeats (SNRs), other polymorphisms, insertions, deletions, splice variants, or any other known genetic markers.

[0035] Nucleic acid. An oligomeric or polymeric form of nucleotides of any length and may include deoxyribonucleotides, their analogs, or mixtures thereof. The term may refer to single-stranded or double-stranded oligonucleotides or polynucleotides.

[0036] Nucleotide. A nitrogen-containing heterocyclic base (a nucleotide base), a sugar, and one or more phosphate groups. Nucleotides are the monomeric units of a nucleotide sequence. In ribonucleotides (RNA), the sugar is ribose, and in deoxyribonucleotides (DNA), the sugar is deoxyribose, i.e., a sugar that lacks the hydroxyl group that is present at the 2' position in ribose. The nitrogen-containing heterocyclic base (i.e., a nucleotide base) can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof. The C-1 atom of deoxyribose is linked to N-1 of pyrimidine or N-9 of purine. Naturally occurring nucleotides typically have a backbone containing phosphodiester bonds. A nucleic acid analogue may have any altered phosphate backbone, sugar or nitrogenous base. Examples of nucleic acid analogues include, for example, universal bases or phosphate-sugar backbone analogues such as peptide nucleic acid (PNA).

[0037] Nucleotide Sequence Section. Genotyping Oligonucleotide Region. Each genotyping oligonucleotide region may be designed to participate in a particular process(es) in the method(s) described herein.

[0038] Primer. A single-stranded deoxyribonucleic acid sequence that can hybridize to a specific sequence. One example of a primer is a "capture primer." The capture primer may be present in the wells of a flow cell and may hybridize to the primer sequence of a genotyping oligonucleotide or its amplicon. The capture primer may serve as a starting point for cluster amplification and cluster generation. Another example of a primer is a "sequencing primer." The sequencing primer may hybridize to a portion of a single-stranded probe template to initiate synthesis of at least a portion of the single-stranded probe template. Other primers, such as random primers, may be used in a random primer amplification reaction to generate gDNA fragments, such as a genotyping target locus. Any primer may include any combination of nucleotides or their analogs. The primer length may be any number of bases and may include various non-natural nucleotides. In an example, the capture primer or sequencing primer is a short strand in the range of 10 to 60 nucleotide bases or 20 to 40 nucleotide bases.

[0039] Priming sequence region. A priming site for sequencing an index sequence region, both of which are included in some examples of a genotyping oligonucleotide.

[0040] Probe sequence. A specific segment of a genotyping oligonucleotide that includes a nucleotide sequence that represents a target locus for genotyping. The probe sequence or its amplicon includes 25 to 50 nucleotide bases that have a specific order that is complementary to the target locus sequence and can thus hybridize with it.

[0041] Single-stranded probe templates. A single-stranded deoxyribonucleic acid sequence that is generated during clustering. The genotyping oligonucleotide serves as a template for generating clusters, and thus single-stranded probe templates are amplicons or portions of amplicons of the genotyping oligonucleotide.

[0042] Genotyping oligonucleotides

[0043] Fig. 1A and Fig. 1B schematically depict two different examples of genotyping oligonucleotides 10, 10'. Each of the genotyping oligonucleotides 10, 10' includes specific sections of the nucleotide sequence, which include a first primer sequence 12, a probe sequence 14, a restriction endonuclease site 16 or 16', and a second primer sequence 18 or 18'.

[0044] An example of a genotyping oligonucleotide 10 shown in Fig. 1A includes a first primer sequence 12 directly and covalently attached to a probe sequence 14, which is directly and covalently attached to a restriction endonuclease site 16, which is directly and covalently attached to a second primer sequence 18. An example of a method including these genotyping oligonucleotides 10 is shown and described with reference to Figs. 3A-3H.

[0045] The first primer sequence 12 of the genotyping oligonucleotide 10 may have the same polarity and the same sequence as the first capture primer 22 (see Fig. 2B), which is present on the surface of the flow cell 20 (see Fig. 2A and Fig. 2B). Thus, the number, order and type of nucleotide bases in at least a portion of the first primer sequence 12 depends on the number, order and type of nucleotide bases in the first capture primer 22. During clustering, amplicons of the genotyping oligonucleotides 10 are generated that include a sequence complementary to the first primer sequence 12 (e.g., P5'). This complementary region (shown in Fig. 3B as C ₁₂ ) can hybridize with the first capture primer 22.

[0046] In an example, the first capture primer 22 has a universal sequence for capture and/or amplification purposes. An example of the first capture primer 22 includes P5 primers, examples of which are used on the surface of commercial flow cells sold by Illumina Inc. for sequencing, such as on HISEQ™, HISEQX™, MISEQ™ MISEQDX™ MINISEQ™ NEXTSEQ™ NEXTSEQDX™ NOVASEQ™ ISEQ™, GENOME ANALYZER™ and other instrument platforms. In another example, the first capture primer 22 includes the following:

[0047] The first primer sequence 12 may have the same sequence as the first capture primer 22. Although an example is provided, it should be understood that other sequences may be used for the first primer sequence 12 and the first capture primer 22. The first primer sequence 12 may also be at least partially the same as the first capture primer 22. The expression "at least partially the same" means that in the first primer sequence 12 and in the first capture primer 22 a sufficient number of nucleotide bases are the same so that hybridization can occur between the two 12, 22. The first primer sequence 12 of the genotyping oligonucleotide 10 is directly attached to the probe sequence 14. The probe sequence 14 is the target locus of genotyping. Thus, during genotyping, the probe sequence 14 is able to hybridize with the target locus sequence.

[0048] The restriction endonuclease site 16 of the genotyping oligonucleotide 10 is directly attached to the probe sequence 14. This restriction endonuclease site 16 provides the genotyping oligonucleotide 10 with a cleavage site. In an example, the restriction endonuclease site 16 is sensitive to a restriction enzyme selected from the group consisting of a 4-base restriction endonuclease, a 5-base restriction endonuclease, a 6-base restriction endonuclease. Some examples of a 4-base restriction endonuclease include DpnII, FatI, MluCI, BfuCI, MboI, Sau3AI, BfaI, BstUI, PmII, Kasl, and others that are commercially available from, for example, New England BioLabs Inc., ThermoFisher Scientific, etc. Some examples of 5-base restriction endonuclease include BssKI, StyD41, MaeIII, PspGI, Ddel, Fmul, PspGI, Tfil, etc., which are commercially available from, for example, New England BioLabs Inc., ThermoFisher Scientific, etc. Some examples of 6-base restriction endonuclease include AcII, Afel, Nspl, HaeII, Tatl, etc., which are commercially available from, for example, New England BioLabs Inc., ThermoFisher Scientific, etc. The 16-base restriction endonuclease site is positioned such that after restriction enzyme digestion/cleavage, the last base of the 14-base probe sequence is the last 3' OH that is accessible for the sequencing/genotyping reaction.

[0049] The second primer sequence 18 of the genotyping oligonucleotide 10 is at least partially complementary to the second capture primer 24 (see Fig. 2B), which is present on the surface of the flow cell 20. The expression "at least partially complementary" means that in the second primer sequence 18 and in the second capture primer 24, a sufficient number of nucleotide bases are complementary such that hybridization can occur between the two 18, 24. Thus, the number, order and type of nucleotide bases in the second primer sequence 18 depends on the number, order and type of nucleotide bases in the second capture primer 24.

[0050] In an example, the second capture primer 24 has a universal sequence for capture and/or amplification purposes. An example of the second capture primer 24 includes P7 primers, examples of which are used on the surface of commercial flow cells sold by Illumina Inc. for sequencing, such as on the HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MFNISEQ™, NEXTSEQ™, NEXTSEQDX™, NOVASEQ™, ISEQ™, GENOME ANALYZER™ and other instrument platforms. In another example, the second capture primer 24 includes the following:

The second primer sequence 18 is at least partially complementary to the sequence of the second capture primer 24. In this example, the second primer sequence 18 may include the following:

Although an example has been provided, it should be understood that other sequences may be used for the second primer sequence 18 and the second capture primer 24.

[0051] The second capture primer 24 on the flow cell 20 also includes a cleavage site (see item number 46 in Fig. 3A). The cleavage site can be used to linearize the bridging probe templates after cluster formation (described in more detail with reference to Fig. 3E). The chemistry of the cleavage site of the second capture primer 24 can be different from the chemistry of the restriction endonuclease site 16 of the genotyping nucleotide 10 so that premature cleavage of the restriction endonuclease site 16 does not occur. The cleavage site and the restriction endonuclease site 16 have separate and specific cleavage reactions. These reactions can be considered orthogonal in the sense that one reaction does not initiate, affect, or otherwise interfere with the other reaction. Examples of suitable cleavage sites for the second capture primer 24 include enzymatically cleavable nucleotide bases or chemically cleavable nucleotide bases, modified nucleotide bases, or linkers (e.g., attached between nucleotide bases). An enzymatically cleavable nucleotide base may be susceptible to cleavage by reaction with a glycosylase and endonuclease or with an exonuclease. One specific example of a cleavable nucleotide base is deoxyuracil (dU), which can be targeted by a uracil-specific excision reagent (USER) enzyme. In an example, the uracil base can be inserted at the 7th base position from the 3' end of the second capture primer 24. Other non-base sites can also be used. Examples of chemically cleavable nucleobases, modified nucleobases, or linkers include 8-oxoguanine, vicinal diol, disulfide, silane, azobenzene, photocleavable group, allyl-T (a nucleotide analog of thymine having an allyl functional group), allyl ethers, or an azide functional ether.

[0052] Fig. 1B schematically depicts another example of a genotyping oligonucleotide 10'. The example of a genotyping oligonucleotide 10' shown in Fig. 1B includes more nucleotide sequence sections than the genotyping oligonucleotide 10 shown in Fig. 1A. In particular, the genotyping oligonucleotide 10' further comprises an index sequence region 26 and a priming site region 28. In this example, the genotyping oligonucleotide 10' includes a first primer sequence 12 directly and covalently attached to an index sequence region 26, which is directly and covalently attached to a priming site region 28, which is directly and covalently attached to a probe sequence 14, which is attached to a second primer sequence 18'. In this example, the 16' restriction endonuclease site may be located between the 14 probe sequence and the second 18' primer sequence or may be inserted into the second 18' primer sequence. An example of a method incorporating these 10' genotyping oligonucleotides is shown and described with reference to Figs. 4A-4H.

[0053] The first sequence 12 of the genotyping oligonucleotide primer 10' may be any example described herein for the genotyping oligonucleotide 10.

[0054] The first sequence 12 of the primer of the genotyping oligonucleotide 10' is directly attached to the region 26 of the index sequence. The region 26 of the index sequence is unique to the sequence 14 of the probe in the genotyping oligonucleotide 10'. Thus, the region 26 of the index sequence provides an identifier or a separate barcode that can be used to determine the sequence of the target locus represented by the sequence 14 of the probe.

[0055] The index sequence region 26 is directly attached to the priming site region 28. The priming site region 28 can hybridize with a sequencing primer that initiates the introduction of nucleotides along the index sequence region 26 in a template-dependent manner, one nucleotide base at a time.

[0056] The 28 region of the priming site of the genotyping oligonucleotide 10' is directly attached to the 14 probe sequence. As described herein, during genotyping, the 14 probe sequence is capable of hybridizing to the sequence of the target locus.

[0057] In the genotyping oligonucleotide, the 10' probe sequence 14 is attached to a second 18' primer sequence. In some examples, the 14 probe sequence is indirectly attached to the second 18' primer sequence, and the 16' restriction endonuclease site is located between the two 14, 18' stretches. In other examples, the 14 probe sequence is directly attached to the second 18 primer sequence, and the 16' restriction endonuclease site is inserted into the second 18' primer sequence. In any of these examples, the 16' restriction endonuclease site may be sensitive to a type IIS restriction enzyme. The type IIS restriction enzyme may be methyl sensitive or may not be methyl insensitive. Type IIS restriction enzymes are a specific group of enzymes that recognize asymmetric DNA sequences (e.g., the 16' restriction endonuclease site) and cause cleavage at a certain distance (e.g., from 1 nucleotide to about 20 nucleotides) beyond the recognition sequence. Some examples of type IIS restriction enzymes that are methyl-averse are selected from the group consisting of BvI (BseXI), BmrI, Bvel (BspMI), etc. Some type IIS restriction enzymes that are methyl-sensitive are selected from the group consisting of SfaNI, FoKI, HGAI, etc. Although some examples are given, any suitable type IIS restriction enzyme can be used depending on the recognition sequence of the 16' restriction endonuclease site. Methyl-sensitive and methyl-insensitive enzymes are commercially available from, for example, New England BioLabs Inc., ThermoFisher Scientific, etc.

[0058] When the 16' restriction endonuclease site is attached at the end of the second 18' primer sequence, any example of the second 18' primer sequence can be used. When the 16' restriction endonuclease site is integrated into the second 18' primer sequence, the 16' restriction endonuclease site can be introduced at any desired position along the length of the second 18' primer sequence. The position can depend on the position of the second restriction endonuclease site along the second 24' capture primer, the desired cleavage distance of the type IIS restriction enzyme to be used, and the position at which cleavage is desired along the 10' genotyping oligonucleotide (or amplicon thereof). This cleavage occurs prior to the genotyping reaction (during linearization, as further described herein) and makes the genotyping oligonucleotide 10' or its amplicon ready for analysis of the target genotyping locus at the nucleotide base of interest. For example, during the genotyping reaction, a single-stranded DNA sample, such as the target genotyping locus, is hybridized to, for example, the amplicon and a single sequencing by synthesis reaction is performed to identify the nucleotide base of interest. To perform this analysis, the amplicon must be cleaved at a specific position where the next nucleotide base to be sequenced is complementary to the nucleotide base of interest. Essentially, the 16' restriction endonuclease site is positioned such that after restriction enzyme cleavage/digestion, the last base of the 14 probe sequence is the terminal 3' OH, which is accessible to the sequencing/genotyping reaction. In one example, the 16' restriction endonuclease site can be integrated anywhere from the 7th base position to the 15th base position from the 3' end of the second 18' primer sequence.

[0059] In the case of the genotyping oligonucleotide 10', the second sequence of the 18' primer is complementary to the second capture primer 24 (see Fig. 2B), which is present on the surface of the flow cell 20. As such, the genotyping oligonucleotide 10' can hybridize to the second capture primer 24' on the flow cell 20.

[0060] The second 24' capture primer may also include a second restriction endonuclease site (see reference numeral 46' in Fig. 4A), wherein the second restriction endonuclease site is complementary to the 16' restriction endonuclease site of the genotyping oligonucleotide 10'. Asymmetric sequences may be sensitive to type IIS restriction endonucleases. During linearization, cleavage is performed within a certain predetermined distance from the hybridized 16', 46' restriction endonuclease sites (as mentioned above and further described with reference to Fig. 4F). In this way, strands that are not subject to genotyping are removed. The position of the second 46' restriction endonuclease site may be at the end of the second 24' capture primer or integrated into it, provided that the two 16', 46' restriction endonuclease sites can hybridize and form a type IIS restriction endonuclease recognition site.

[0061] Any example of a genotyping oligonucleotide 10, 10' can be obtained using oligonucleotide synthesis processes.

[0062] Flow cells

[0063] The genotyping oligonucleotides 10, 10' can be used with any structured flow cell 20. Although not shown, it should be understood that in the examples described herein, in an alternative embodiment, other unstructured flow cells (e.g., that do not contain indentations) can be used that utilize the clustering chemistry described herein. When unstructured flow cells are used, clusters can be determined using software.

[0064] An example of a structured flow cell 20 is shown in Fig. 2A, and an example of a structured structure inside the flow cell 20 is shown in Fig. 2B. The flow cell 20 includes a substrate 30 that at least partially forms a track or flow channel 32.

[0065] The substrate 30 may be a single layer/material. Examples of suitable monolayer substrates include epoxy siloxane, glass, modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene (e.g., TEFLON® from Chemours), cyclic olefins/cycloolefin polymers (COPs) (such as ZEONOR® from Zeon), polyimides, etc.), nylon (polyamides), ceramics/ceramic oxides, silica, fused silica or silica-based materials, aluminum silicate, silicon and modified silicon (e.g., boron-doped silicon p+), silicon nitride _{( Si3N4} ), silicon oxide ( _SiO2 ) _, tantalum pentoxide ( _Ta2O5 ) or other tantalum oxides (TaO _x ), hafnium oxide (HfO ₂ ), carbon, metals, inorganic glasses, etc.

[0066] If the substrate 30 is a single layer, recesses 38 are formed in the single layer (see Fig. 2B).

[0067] As shown in Fig. 2B, the substrate 30 may also be a multilayer substrate 30'. Some examples of the multilayer substrate 30' include glass or silicon with a coating layer of tantalum oxide or other oxide ceramic on the surface. Other examples of the multilayer substrate 30' may include a silicon-on-insulator (SOI) substrate. In the example shown in Fig. 2B, the multilayer substrate 30' includes an underlying substrate 34 (e.g., glass or silicon) and a structured material 36 located on the substrate 34.

[0068] The structured material 36 defines recesses 38 that are separated by intermediate regions 40. The recesses 38 are located within each of the flow channels 32.

[0069] It should be understood that for the structured material 36, any material can be used that can be selectively applied or applied and structured to form recesses 38 and intermediate regions 40.

[0070] As one example, the inorganic oxide can be selectively applied to the substrate 34 by vapor deposition, aerosol printing, or inkjet printing. Examples of suitable inorganic oxides include tantalum oxide (e.g., Ta ₂ O ₅ ), aluminum oxide (e.g., Al ₂ O ₃ ), silicon oxide (e.g., SiO ₂ ), hafnium oxide (e.g., HfO ₂ ), etc.

[0071] As another example, a resin can be applied to the substrate 34 and then a pattern can be applied. Suitable application techniques include chemical vapor deposition, dip coating, dip coating, spin coating, spray coating, curtain coating, ultrasonic spray coating, doctor blade coating, spray printing, screen printing, microcontact printing, etc. Suitable patterning technologies include photolithography, nanoimprint lithography (NIL), stamping techniques, embossing techniques, casting techniques, microetching techniques, printing techniques, etc. Some examples of suitable resins include a polyhedral oligomeric silsesquioxane resin (e.g., POSS® manufactured by Hybrid Plastics), a non-polyhedral oligomeric silsesquioxane epoxy resin, a poly(ethylene glycol) resin, a polyester resin (e.g., ring-opening epoxy resins), an acrylic resin, an acrylate resin, a methacrylate resin, an amorphous fluoropolymer resin (e.g., CYTOP® manufactured by Bellex), and combinations thereof.

[0072] As used herein, the term "polyhedral oligomeric silsesquioxane" refers to a chemical composition that is a hybrid intermediate (e.g., RSiO _1,5 ) between silica (SiO ₂ ) and silicone (R ₂ SiO). An example of a polyhedral oligomeric silsesquioxane is described in Kehagias et al., Microelectronics Engineering 86 (2009), pp. 776-778, which is incorporated herein by reference in its entirety. In the example, the composition is an organosilicon compound with the chemical formula [RSiO _3/2 ] _n , wherein the R groups may be the same or different. Examples of R groups for the polyhedral oligomeric silsesquioxane include epoxy, azide/azido, thiol, poly(ethylene glycol), norbornene, tetrazine, acrylates and/or methacrylates, or additionally, for example, an alkyl, aryl, alkoxy and/or haloalkyl group. The resin composition described herein may contain one or more other framework or core structures compared to the monomer units.

[0073] In an example, the substrate 30, 30' can be produced using a circular plate having a diameter in the range of about 2 mm to about 300 mm, or from a rectangular sheet or panel, the largest dimension of which is up to about 10 feet (- 3 meters). In an example, the substrate 30, 30' is produced using a circular plate with a diameter in the range of about 200 mm to about 300 mm. In another example, a panel can be used, which is a rectangular substrate with a larger surface area than a 300 mm circular plate. Plates, panels and other large substrate materials can be separated into individual substrates 30, 30' of the flow cell. In another example, the substrate 30, 30' is a crystal having a width in the range of about 0.1 mm to about 10 mm. Although sample sizes are provided, it should be understood that any acceptable size can be used to make a 30, 30' backing.

[0074] The flow cell 20 also includes flow channels 32. Although several flow channels 32 are shown in Fig. 2A, it should be understood that any number of channels 32 (e.g., one channel 32, four channels 32, etc.) can be included in the flow cell 20. Each flow channel 32 is a region formed between two connected components (e.g., a substrate 30, 30' and a lid or two substrates 30, 30') that can contain fluids introduced into and removed from them. Each flow channel 32 may be isolated from each other flow channel 32 such that a fluid introduced into any particular flow channel 32 does not flow into any adjacent flow channel 32. Some examples of fluids introduced into the flow channels 32 may include reaction components (e.g., fragments from a library of target loci for genotyping of a polymerase, polymerases, etc.), wash solutions, etc.

[0075] As indicated above, the flow channel 32 is formed between the substrate 30, 30' and the cover (not shown), or between the substrate 30, 30' and another substrate (not shown, but similar to the substrate 30, 30').

[0076] In an example, the cover or additional substrate may be bonded to at least a portion of the substrate 30, 30', such as in some intermediate regions 40. The bond that is formed between the cover or additional substrate and the substrate 30, 30' may be a chemical bond or a mechanical bond (e.g., using a fastener, etc.).

[0077] The lid may be of any material that is transparent to excitation light that is directed toward the substrate 30, 30'. As examples, the lid may be glass (e.g., borosilicate glass, fused silica, etc.), plastic, or the like. A commercially available example of a suitable borosilicate glass is D 263® from Schott North America, Inc. Commercially available examples of suitable plastic materials, namely, cycloolefin polymers, are ZEONOR® products from Zeon Chemicals L.P.

[0078] The lid or additional substrate may be bonded to the substrate 30, 30' using any suitable method, such as laser soldering, diffusion soldering, anodic soldering, eutectic alloy soldering, plasma activation soldering, glass-ceramic soldering, or other methods known in the art. In an example, a release layer may be used to bond the lid or additional substrate to the substrate 30, 30'. The release layer may be any material that will hermetically bond at least a portion of the substrate 30, 30' and the lid or additional substrate. In some examples, the release layer may be a radiation absorbing material that facilitates bonding.

[0079] In an example, the flow channel 32 has a rectangular configuration. The length and width of the flow channel 32 may be less than the length and width of the substrate 30, 30', respectively, so that a portion of the surface of the substrate surrounding the flow channel 32 is available for attachment to a lid (not shown) or another substrate 30, 30'. In some cases, the width of each flow channel 32 may be at least about 1 mm, at least about 2.5 mm, at least about 5 mm, at least about 7 mm, at least about 10 mm, or more. In some cases, the length of each track/flow channel 32 may be at least about 10 mm, at least about 25 mm, at least about 50 mm, at least about 100 mm, or more. The width and/or length of each flow channel 32 may be greater than, less than, or an intermediate value. In another example, the flow channel 32 has a square shape (e.g. 10 mm × 10 mm).

[0080] The depth of each flow channel 32 may be only one monolayer thick, for example, when microcontact, aerosol or inkjet printing is used to apply the separation layer that defines the walls of the flow channel. The depth of the flow channel 32 may be greater, for example, when the flow channel 32 is partially formed in the substrate 30, 30' (for example, by etching, lithography, etc.), so that a portion of the substrate and the separation layer form the walls of the flow channel. In other examples, the depth of each flow channel 32 may be about 1 μm, about 10 μm, about 50 μm, about 100 μm or more. In an example, the depth may be in the range of about 10 μm to about 100 μm. In another example, the depth may be in the range of about 10 μm to about 30 μm. In yet another example, the depth is about 5 μm or less. It should be understood that the depth of each flow channel 32 may be greater, less than the above values, or may have an intermediate value.

[0081] In particular, Fig. 2B shows an example of the structure inside one of the flow channels 32 of the flow cell 20.

[0082] As shown in Fig. 2B, the structured material 36 includes depressions 38 formed therein and intermediate regions 40 separating adjacent depressions 38. A variety of different arrangements of depressions 38 can be provided, including regular, repeating, and irregular patterns. In an example, the depressions 38 are arranged in a hexagonal grid to provide dense packing and increased density. Other arrangements can include, for example, rectangular arrangements, triangular arrangements, and the like. In some examples, the arrangement or pattern can be in an x-y format of depressions 38 that are arranged in rows and columns. In some other examples, the arrangement or pattern may be a repeating arrangement of depressions 38 and/or intermediate regions 40. In still other examples, the arrangement or pattern may be a random arrangement of depressions 38 and/or intermediate regions 40. The structure may include stripes, swirls, lines, triangles, rectangles, circles, arcs, check marks, diagonal lines, arrows, squares, and/or cross hatching.

[0083] The arrangement or pattern of the depressions 38 can be characterized by the density of the depressions 38 (the number of depressions 38) in a certain area. For example, the depressions 38 can be arranged with a density of about 2 million per mm ² . The density ^{can be adjusted to various densities, including, for example, a density of about 100 per mm 2 ,} about 1000 per mm ² , about 0.1 million per mm ² , about 1 million per mm ² , about 2 million per mm ² , about 5 million per mm ² , about 10 million per mm 2 , about 50 million per mm ² , or more or less. In addition, it should be understood that the density of the depressions 38 in the structured material 36 can be between one of the lower values and one of the upper values selected from the above ranges. For example, a high density array may be characterized by having pits 38 separated by less than about 100 nm, a medium density array may be characterized by having pits 38 separated by from about 400 nm to about 1 μm, and a low density array may be characterized by having pits 38 separated by more than about 1 μm. Although examples of densities are provided, it should be understood that any suitable density values can be used. The density of the pits 38 may depend in part on the depth of the pits 38. In some cases, it may be desirable for the distance between the pits 38 to be even greater than in the examples listed herein.

[0084] The arrangement or pattern of the recesses 38 may also or in an alternative embodiment be characterized in terms of the average pitch or distance from the center of the recess 38 to the center of the adjacent recess 38 (center-to-center distance) or from the left edge of one recess 38 to the right edge of the adjacent recess 38 (edge-to-edge distance). The pattern may be regular, so that the coefficient of variation relative to the average pitch is small, or the pattern may be irregular, in which case the coefficient of variation may be relatively large. In any case, the average pitch may be, for example, about 50 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm, or more or less. The average pitch for a particular pattern of recesses 38 may be in the range from one of the lower values to one of the upper values selected from the ranges given above. In the example, the recesses 38 have a pitch (center-to-center distance) of about 1.5 µm. Although examples of average pitch values are provided, it should be understood that other average pitch values can be used.

[0085] The size of each recess 38 may be characterized by volume, opening area, depth and/or diameter.

[0086] Each well 38 may have any volume capable of holding a fluid. The minimum or maximum volume may be selected, for example, to match the throughput (e.g., multiplexity), resolution, nucleotides, or analyte reactivity expected in subsequent applications of the flow cell 20. For example, the volume may be at least about 1×10 ^-3 μm ³ , at least about 1×10 ^-2 μm ³ , at least about 0.1 μm ³ , at least about 1 μm ³ , at least about 10 μm ³ , at least about 100 μm ³ , or more. In an alternative or additional embodiment, the volume may be no more than about 1×10 ^{4 μm3} , no more than about 1×10 ^{3 μm3} , no more than about 100 ^μm3 , no more than about 10 ^μm3 , no more than about 1 ^μm3 , no more than about 0.1 ^μm3 , or less.

[0087] The area occupied by each opening of the recess may be selected based on criteria similar to those described above for the volume. For example, the area for each opening of the recess may be at least about 1×10 ^-3 μm ² , at least about 1×10 ^-2 μm ² , at least about 0.1 μm ² , at least about 1 μm ² , at least about 100 μm ² , or more. In an alternative or additional embodiment, the area may be no more than about ¹ ×10 ³ μm ² , no more than about 100 μm ² , no more than about 10 μm ² , no more than about 1 μm ² , no more than about 0.1 μm ² , no more than about 1×10 ^-2 μm ² , or less. The area occupied by each recess opening may be greater or less than the values specified, or may be between the values specified above.

[0088] The depth of each recess 38 may be large enough to accommodate a certain amount of the polymer hydrogel 42. In an example, the depth may be at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, at least about 100 μm, or more. In an alternative or additional embodiment, the depth may be no more than about 1×10 ^{3 μm} , no more than about 100 μm, no more than about 10 μm, or less. In some examples, the depth is about 0.4 μm. The depth of each recess 38 may be greater than, less than, or between the values specified above.

[0089] In some cases, the diameter or the length and width of each recess 38 can be at least about 50 nm, at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, at least about 100 μm, or more. In an alternative or additional embodiment, the diameter or the length and width can be no more than about 1×10 ³ μm, no more than about 100 μm, no more than about 10 μm, no more than about 1 μm, no more than about 0.5 μm, no more than about 0.1 μm, or less (e.g., about 50 nm). In some examples, the diameter or the length and width is about 0.4 μm. The diameter or the length and width of each recess 38 can be greater than, less than, or between the values specified above.

[0090] In the example shown in Fig. 2B, a polymer hydrogel 42 is located within each of the recesses 38. An example of a polymer hydrogel 42 includes an acrylamide copolymer, such as poly(N-(5-azidoacetamidylpentyl)acrylamide and acrylamide (PAZAM). PAZAM and certain other forms of acrylamide copolymer are represented by the following structure (I):

Where:

R ^A is selected from the group consisting of azido, optionally substituted amino, optionally substituted alkenyl, optionally substituted alkyne, halogen, optionally substituted hydrazone, optionally substituted hydrazine, carboxyl, hydroxy, optionally substituted tetrazole, optionally substituted tetrazine, nitrile oxide, nitrone, sulfate, and thiol;

R ^B represents H or optionally substituted alkyl;

each of R ^C , R ^D and R ^E is independently selected from the group consisting of H and optionally substituted alkyl;

each of -(CH ₂ ) _p - may be optionally substituted;

p is an integer in the range 1 to 50;

n is an integer in the range 1 to 50000; and

m is an integer in the range 1 to 100000.

[0091] One skilled in the art will appreciate that the configuration of the repeating units n and m in structure (I) is typical, and the monomeric subunits may be present in any order in the polymer structure (e.g., random, block, structured form, or a combination thereof).

[0092] The molecular weight of PAZAM and other forms of acrylamide copolymer may range from about 5 kDa to about 1500 kDa, or from about 10 kDa to about 1000 kDa, or may be about 312 kDa in a particular example.

[0093] In some examples, PAZAM and other forms of acrylamide copolymer are linear polymers. In some other examples, PAZAM and other forms of acrylamide copolymer are weakly crosslinked polymers.

[0094] In other examples, the polymer hydrogel 42 may be a variant of structure (I). In one example, the acrylamide unit may be replaced with N,N-dimethylacrylamide In this example, the acrylamide unit in structure (I) can be replaced by , where each of R ^D , R ^E , R ^F is H or C1-C6 alkyl, and each of R ^G and R ^H is C1-C6 alkyl (instead of H, as in the case of acrylamide). In this example, q can be an integer in the range from 1 to 100,000. In another example, N,N-dimethylacrylamide can be used in addition to the acrylamide unit. In this example, structure (I) can include in addition to the repeating units num, wherein each of R ^D , R ^E , and R ^F is H or C1-C6 alkyl, and each of R ^G and R ^H is C1-C6 alkyl. In this example, q may be an integer in the range of 1 to 100,000.

[0095] As another example of polymer hydrogel 42, the repeating element n in structure (I) can be replaced by a monomer comprising a heterocyclic azide group having the structure (II):

wherein R ¹ is H or C1-C6 alkyl; R ² is H or C1-C6 alkyl; L is a linker comprising a linear chain of 2-20 atoms selected from the group consisting of carbon, oxygen and nitrogen and 10 optional substituents at carbon and any nitrogen atoms in the chain; E is a linear chain comprising 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and optional substituents at carbon and any nitrogen atoms in the chain; A is an N-substituted amide having H or C1-C4 alkyl attached to N; and Z is a nitrogen-containing heterocycle. Examples of Z include 5-10 ring members present as a single cyclic structure or a fused structure. Some specific examples of Z include pyrrolidinyl, pyridinyl or pyrimidinyl.

[0096] As another example, the polymer hydrogel 42 may include a repeating unit of each of structures (III) and (IV):

wherein each of R ^1a , R ^2a , R ^1b and R ^2b is independently selected from hydrogen, optionally substituted alkyl or optionally substituted phenyl; each of R ^3a and R ^3b is independently selected from hydrogen, optionally substituted alkyl, optionally substituted phenyl or optionally substituted C7-C14 aralkyl; and each of and is independently selected from an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker.

[0097] It should be understood that other molecules can be used to form the polymer hydrogel 42, provided that they are functionalized to graft the capture primers 22, 24 or 22, 24' thereto. Other examples of suitable polymer layers include those having a colloidal structure, such as agarose; or a polymeric network structure, such as gelatin; or a cross-linked polymer structure, such as polyacrylamide polymers and copolymers, silane-free acrylamide (SFA) or an azidolized version of SFA. Examples of suitable polyacrylamide polymers can be synthesized from acrylamide and acrylic acid or acrylic acid containing a vinyl group, or from monomers that undergo [2+2] photocycloaddition reactions. Other examples of suitable polymer hydrogels 42 include mixed copolymers of acrylamides and acrylates. The examples described herein may utilize various polymer architectures containing acrylic monomers (e.g., acrylamides, acrylates, etc.), such as branched polymers, including star polymers, star or star block polymers, dendrimers, etc. For example, monomers (e.g., acrylamide, etc.) may be incorporated, either randomly or en bloc, into branches (fragments) of a star polymer.

[0098] To introduce the polymer hydrogel 42 into the flow channel 32, a mixture of the polymer hydrogel 42 can be created and then applied to the substrate 30, 30' (including the recesses 38). In one example, the polymer hydrogel 42 can be present in a mixture (e.g., with water or with ethanol and water). The mixture can then be applied to the surfaces of the substrate (including the recesses 38) using spin coating, dip coating, immersion coating, spray coating, positive or negative pressure flow coating, or another suitable technique. Such types of techniques provide for the application of a continuous layer of the polymer hydrogel 42 on the substrate 30, 30' (e.g., in the recesses 38 and in the intermediate regions 40 surrounding the recesses 38). For the specific application of the polymer hydrogel 42 in the recesses 38, and not in the intermediate regions 40, other methods of selective deposition can also be used (for example, using a stencil, controlled printing techniques, etc.).

[0099] In some examples, the surface of the substrate (including the portion exposed in the recesses 38) can be activated and then the mixture (including the polymer hydrogel 42) can be applied thereto. In one example, a silane or silane derivative (e.g., norbornene silane) can be applied to the surface of the substrate using vapor deposition, spin coating, or other deposition methods. In another example, the surface of the substrate can be plasma ashing to form a surface activating agent(s) (e.g., OH groups) that can be attached to the polymer hydrogel 42.

[00100] Depending on the polymer hydrogel 42, the applied mixture can be subjected to a curing process. In an example, curing can occur at a temperature in the range of room temperature (for example, from about 18 °C to about 25 °C) to about 95 °C for a time in the range of about 1 millisecond to about several days.

[00101] In some examples, polishing may then be performed to remove the polymer hydrogel 42 from the intermediate regions 40 around the perimeter of the recesses 38 while leaving the polymer hydrogel 42 on the surface of the recesses 38 at least substantially intact.

[00102] The flow cell 20 also includes first and second capture primers 22, 24 or 22, 24'. Any example of the capture primers 22, 24 or 22, 24' described herein can be used. The set of capture primers 22, 24 or 22, 24' that is selected for a particular flow cell 20 may depend in part on which genotyping oligonucleotide 10, 10' is to be used with them.

[00103] A grafting process can be implemented to graft the first and second capture primers 22, 24 or 22, 24' into the polymer hydrogel 42 in the recesses 38. In an example, the first and second capture primers 22, 24 or 22, 24' can be immobilized into the polymer hydrogel 42 by a single-point covalent attachment at or near the 5'-ends of each of the first and second capture primers 22, 24 or 22, 24'. As a result of this attachment, i) the primer sequence-specific region of the capture primers 22, 24 or 22, 24' remains free for annealing to its cognate sequence 12 primer or the copied sequence C ₁₈ , C _18' primer, and ii) a free 3' hydroxyl (OH) group for completion of the capture primer. Any suitable covalent attachment can be used to attach the first and second capture primers 22, 24 or 22, 24' to the polymer hydrogel 42. Examples of primers with a terminal group that can be used include primers with a terminal alkyne group that can be attached to the azide functional group of the polymer hydrogel 42. As mentioned, specific examples of suitable capture primers 22, 24 include primers P5 and P7, and a specific example of a suitable capture primer 24' is P7 modified to include a second restriction endonuclease site.

[00104] In an example, grafting may include flow coating (e.g., using a temporarily bonded or permanently attached lid or additional substrate), dip coating, spray coating, pour coating, or other appropriate method in which the capture primer(s) 22, 24 or 22, 24' will be attached to the polymer hydrogel 42. Each of these example techniques may utilize a primer solution or mixture that may include the capture primer(s) 22, 24 or 22, 24', water, a buffer, and a catalyst. In any of the grafting methods, the capture primer(s) 22, 24 or 22, 24' react with the reactive groups of the polymer hydrogel 42 in the recesses 38 and have no affinity for the surrounding intermediate regions 40. Thus, the capture primer(s) 22, 24 or 22, 24' are selectively grafted onto the polymer hydrogel 42 in the recesses 38.

[00105] Methods involving genotyping oligonucleotides

[00106] Examples of the method described herein essentially comprise introducing a genotyping probe fluid into a flow cell 20 comprising individual wells 38 and first and second capture primers 22, 24 or 22, 24' in each of the individual wells 38, the genotyping probe fluid comprising a plurality of genotyping oligonucleotides 10 or 10', whereby a corresponding genotyping oligonucleotide 10 or 10' reacts in at least some of the individual wells 38 to produce corresponding clonal populations of amplicons from the corresponding genotyping oligonucleotide 10 or 10'; linearizing the amplicons to produce probe templates; sequencing at least one probe-detecting portion of the probe templates to detect each of the probe sequences; removing at least the corresponding nascent chains from the probe templates, thereby exposing the 3' OH group at the end of the probe templates; hybridizing the corresponding samples to the probe templates; and performing corresponding genotyping reactions on the samples on the accessible 3' OH groups.

[00107] In examples of the method, the flow cell 20 may be introduced into a system (not shown) where it is in fluid communication with a fluid control system (e.g., pumps, valves, etc.) and is in optical communication with a lighting system and a detection system.

[00108] Figs. 3A-3H collectively show an example of a method involving genotyping oligonucleotide 10.

[00109] Fig. 3A shows one well 38 of the flow cell 20 that includes a polymer hydrogel 42 with first and second capture primers 22, 24 attached thereto. As described herein, the second capture primer 24 includes a cleavage site 46.

[00110] In Fig. 3B, a genotyping probe fluid (not shown) is introduced into the flow cell 20 (e.g., into each flow channel 32). In this example, the genotyping probe fluid includes a liquid carrier and a genotyping oligonucleotide 10 in the liquid carrier. The liquid carrier of the genotyping probe fluid can be any suitable hybridization buffer, such as Tris-HCl buffer or 0.5x sodium citrate buffered saline (SSC). In some examples, the genotyping probe fluid includes a plurality of genotyping oligonucleotides 10, where each genotyping oligonucleotide 10 includes a probe sequence 14 that is different from the sequence of each other genotyping oligonucleotide 10. Using this fluid, different genotyping oligonucleotides 10 (each having a unique probe sequence 14) can be delivered to different wells 38.

[00111] As shown in Fig. 3B, one genotyping oligonucleotide 10 is seeded into well 38. More specifically, the second primer sequence 18 of one genotyping oligonucleotide 10 hybridizes with one of the second capture primers 24 in well 38.

[00112] Upon seeding of a single genotyping oligonucleotide 10, cluster formation may begin immediately. In other examples, separate hybridization (seeding) and cluster generation may be performed. The processes involved in cluster formation are shown in Fig. 3B, Fig. 3C, Fig. 3D, and Fig. 3E.

[00113] As shown by the arrow in Fig. 3B, the genotyping oligonucleotide 10 is copied from the hybridized primers by 3'-end extension using DNA polymerase. As a result, the amplicon 44A is obtained, which is attached to the surface of the flow cell by the second capture primer 24. The sections of the amplicon 44A designated _C16 , _C14 , _C12 are complementary copies of the restriction endonuclease site 16, the probe sequence 14 and the first primer sequence 12, respectively.

[00114] The original genotyping oligonucleotide 10 is denatured, leaving the amplicon 44A immobilized in the well 38 by the second capture primer 24. The single-stranded amplicon 44A is flipped and bridged, for example, by hybridization of a copy of the C ₁₂ first primer sequence to the adjacent complementary first capture primer 22. This is shown in Fig. 3C. As shown by the arrow in Fig. 3C, the hybridized primer (first capture primer 22) is then extended by the polymerase(s) to form another amplicon 44B. The _CC16 , _CC14 regions of the 44B amplicon are complementary copies of the _C16 , _C14 regions and thus have the same sequences as the original restriction endonuclease site 16 and the probe sequence 14, respectively. The _C24 region of the 44B amplicon is a complementary copy of the second capture primer 24 and thus has the same sequence as the second primer sequence 18. As shown in Fig. 3C, the formation of the 44B amplicon generates a double-stranded bridge comprising the amplicons 44A and 44B.

[00115] The double-stranded bridge is then denatured as shown in Fig. 3D. This results in two copies (amplicons 44A and 44B) covalently linked to the flow cell 20. Isothermal bridge amplification or some other form of amplification amplifies the immobilized copies. For example, the copied templates form a loop and hybridize to the adjacent complementary capture primer 22,24, and the polymerase copies the copied templates to form double-stranded bridges that are denatured to form two single-stranded strands. These two strands form a loop and hybridize to the adjacent complementary capture primers 22,24, and then are extended again to form two new double-stranded loops. The process is repeated on each copy of the template in cycles of isothermal denaturation and amplification to produce dense clonal clusters of double-stranded bridges. A simplified cluster including two double-stranded bridges is shown in Fig. 3E.

[00116] It should be understood that the seeding of the respective genotyping oligonucleotides 10 in the respective wells 38 and the amplification of such genotyping oligonucleotides 10 in the respective wells 38 can occur under conditions in which the rate of amplification exceeds the rate of seeding. Thus, the relatively high rate of obtaining copies (amplicons 44A, 44B) within the well 38 seeded with one genotyping oligonucleotide 10 effectively excludes seeding of this well 38 with a second genotyping oligonucleotide 10 for amplification. Thus, in each of the wells 38, a different genotyping oligonucleotide 10 (with a unique probe sequence 14) can be captured and amplified, which allows for the simultaneous analysis on the flow cell 20 of a plurality of different genotyping target loci.

[00117] Linearization of the bridge amplicons 44A, 44B can be accomplished by cleaving the amplicons attached to the second capture primers (e.g., amplicons 44A) at the corresponding cleavage sites 46 of the second capture primers 24; and denaturing the cleaved regions of the amplicons (e.g., amplicons 44A) attached to the second capture primers 24 to yield probe templates 48 (Fig. 3F).

[00118] To initiate cleavage, a cleaving agent may be introduced into the flow cell 20, such as through an inlet (not shown). The cleaving agent selected will depend on the cleavage site 46 of the second capture primer 24. Depending on the cleavage site 46, the chemical cleavage agent may be a chemical cleavage agent or an enzymatic cleavage agent. Cleavage at the cleavage site 46 separates the amplicons 44A between the second capture primers 24 and the remainder of the amplicon sequence (which includes stretches _C16 , _C14 , and _C12 or stretches _C16 , _C14 , and _C22 (which is a complementary copy of the capture primer 22)).

[00119] As a result of the cleavage, the _C16 , _C14 , and _C12 stretches and the _C16 , _C14 , and _C22 stretches are no longer attached to the surface of the flow cell via the primer 24 and can thus be removed by denaturation. The denaturation can occur using any suitable conditions. Upon removal of the _C16 , _C14 , and _C12 stretches and the _C16 , _C14 , and _C22 stretches, the amplicons 44B remain attached to the surface of the flow cell via the first capture primer 22 and hybridized to the second capture primers 24 (via the _C24 stretch). In this example, the accessible single-stranded portion of the amplicons 44B, in particular the _CC16 and _CC14 stretches, form the probe template 48. The CC ₁₄ region is a complementary copy of the C ₁₄ region and thus has the same sequence as the 14 probe sequence. Sequencing this CC ₁₄ region allows the original 14 probe sequence to be determined/decoded. In some cases, a portion of the CC ₁₄ region can be sequenced to determine or decode the original probe sequence. The CC ₁₆ region is a complementary copy of the C ₁₆ region and thus has the same sequence as the 16 site of the restriction enzyme. When sequenced, the double-stranded region comprising CC ₁₆ and N ₁₆ (Fig. 3G) provides a support for restriction enzyme cleavage.

[00120] After digestion and denaturation, each second capture primer 24 may have a 3'-phosphate at its end that should be removed before further processing. The 3'-phosphate can be removed by introducing a kinase that removes the protecting groups of the second capture primer 24, making it suitable for use as a sequencing primer (as described with reference to Fig. 3G).

[00121] Fig. 3G shows the sequencing of probe array 48, including stretches CC ₁₆ and CC ₁₄ , the latter of which is at least one probe-defining stretch.

[00122] In the example shown, sequencing the probe template 48 includes using the second capture primer 24 as a sequencing primer and performing a base extension reaction (one base at a time) along the probe template 48.

[00123] In another example, the C ₂₄ stretch and the second capture primer 24 can be denatured and a separate sequencing primer (which is in solution) can be added. The separate sequencing primer can hybridize to the C ₂₄ stretch and a base extension reaction (one base at a time) will occur along the probe template 48.

[00124] The underlying chemical process for sequencing may be polymerization (e.g., catalyzed by a polymerase enzyme). In a particular polymerase-based process, fluorescently labeled nucleotides are added to the second capture primer 24 in a template-dependent manner such that the order and type of nucleotides added to the second capture primer 24 can be determined. This allows the sequence of a probe-determining stretch, such as the CC stretch ₁₄ , to be known, which can be used to decode the original probe sequence 14 .

[00125] To initiate the first sequencing cycle, one or more labeled nucleotides, DNA polymerase, etc. can be delivered to or through the flow cell 20, etc., wherein extension of the sequencing primer results in incorporation of the labeled nucleotide into the probe template 48. Such incorporation can be detected using an imaging event. During the imaging event, the illumination system can provide excitation light to the flow cell 20.

[00126] In some examples, the fluorescently labeled nucleotides may further have a reversible termination property that stops further primer extension after the nucleotide has been added to the template 48. For example, a nucleotide analog having a functional group, a reversible terminator, may be added to the template 48 such that further extension will not occur until a deblocking agent is supplied to remove the functional group. Thus, for examples in which reversible termination is used, a deblocking reagent, etc., may be supplied to the flow cell 20 (after detection).

[00127] Wash(s) may occur between the various fluid feed steps. The sequencing cycle may then be repeated n times to extend the 48 template by n nucleotides to form a nascent strand comprising nascent stretch _N16 (complementary to stretch _CC16 ) and nascent stretch _N14 (complementary to stretch _CC14 ).

[00128] Sequencing of the CC ₁₄ region provides the N ₁₄ region of the nascent strand that can be used to decode the original sequence 14 of the genotyping oligonucleotide probe 10. This information allows the user to determine the target genotyping locus to be analyzed in a particular well 38 (since all arrays 48 in well 38 have the same probe-defining region, e.g., CC ₁₄ ).

[00129] Sequencing of the CC ₁₆ stretch provides the N ₁₆ stretch of the nascent strand. Thus, in this sequencing example, a double-stranded stretch is also generated that includes both CC ₁₆ and N ₁₆ , which provides a support for restriction enzyme cleavage.

[00130] After sequencing the probe(s) defining section(s), the method further comprises removing at least the corresponding nascent strands (which include sections _N14 and _N16 ) from the probe templates 48, thereby exposing the 3' OH group at the end of the probe templates 48. In this example, the removal comprises more than removing the nascent strands _N14 and _N16 . In this example, the removal comprises cleaving restriction endonuclease sites, such as sections _CC16 and _N16 ; and denaturing the remaining nascent strand, including section _N14 , from the probe templates 48.

[00131] The probe template 48 includes a CC ₁₆ stretch (which has the same sequence as a restriction endonuclease site 16), and the nascent strand includes a N ₁₆ stretch (which is complementary to a restriction endonuclease site 16). This double-stranded region (CC ₁₆ and N ₆ stretches) creates a support for the corresponding restriction endonuclease (restriction enzyme). Thus, when a restriction endonuclease is introduced, the restriction endonuclease sites, in this example, the CC ₁₆ stretch and the N ₁₆ stretch, can be cleaved. As mentioned herein, the restriction enzyme may be a 4-base cleaving restriction endonuclease, a 5-base cleaving restriction endonuclease, a 6-base cleaving restriction endonuclease, or the like. The restriction enzyme cleaves the _CC16 , _N16 stretches at specific nucleotides, which are schematically indicated by asterisks in Fig. 3G. In this example, the restriction endonuclease site extends from the second capture primer 24, and the first capture primer 22 has the _CC14 stretch attached thereto and the nascent _N14 strand hybridized to the _CC14 stretch. The nascent _N14 strand is then denatured from the _CC14 stretch. The cleavage and denaturation allow the removal of the CC ₁₆ segment and the forming chains N ₁₄ , N ₁₆ from the well 38 and the flow cell 20, for example, by means of a washing step.

[00132] Cleavage and denaturation expose the 3' OH at the end of what remains of the probe template 48. The remaining probe template is shown with reference to Fig. 3H at 52. The remaining probe template 52 includes a first capture primer 22 and a CC stretch ₁₄ that is similar to the original probe sequence 14 and thus, in this example, is complementary to the DNA sample having the genotyping target locus 54.

[00133] A sample of denatured DNA fragments, including a target genotyping locus 54, is introduced into a flow cell 20. The target genotyping locus 54 may be included in a library fluid that includes a plurality of target genotyping loci, at least some of which have different genotypable loci. The target genotyping loci may be obtained from a larger DNA sample using any genotyping library preparation technique. Some genotyping library preparation techniques include amplification and fragmentation. Others include amplification without fragmentation (examples of which are described below in this document). Thus, multiple copies of the target genotyping locus 54 of any type may be present in the library fluid.

[00134] After introduction into the flow cell 20, the target genotyping loci 54 hybridize with the corresponding complementary segments CC ₁₄ of the remaining probe matrices 52 in the recess 38.

[00135] A genotyping reaction can then be performed. In this reaction, the remaining probe template 52 is used as a sequencing primer to perform one round of sequencing, as described herein. As shown in Fig. 3H, one labeled nucleotide 57, which is complementary to the nucleotide base of interest at the genotyping target locus 54, is incorporated into the remaining probe template 52.

[00136] Although the description provided herein relates to a single well 38 and thus genotyping of a single genotyping target locus 54, it should be understood that each well 38 includes different probe arrays 52 with different CC ₁₄ stretches. Thus, using this method, hundreds to thousands, up to millions (depending on the number of wells in the flow cell 20) of different loci can be genotyped simultaneously.

[00137] Fig. 4A-4H together show another example of a method comprising a 10' genotyping oligonucleotide.

[00138] Fig. 4A shows one well 38 of the flow cell 20 that includes a polymer hydrogel 42 with first and second capture primers 22,24' attached thereto. As described herein, the second capture primer 24' includes a second restriction endonuclease site 46'.

[00139] In Fig. 4B, a genotyping probe fluid (not shown) is introduced into the flow cell 20 (e.g., into each flow channel 32). In this example, the genotyping probe fluid includes a liquid carrier and a genotyping oligonucleotide 10' in the liquid carrier. The liquid carrier may be any of the examples described herein. In some examples, the genotyping probe fluid includes a plurality of genotyping oligonucleotides 10', wherein each genotyping oligonucleotide 10' includes a probe sequence 14 that is different from each other genotyping oligonucleotide 10'. Using this fluid, different genotyping oligonucleotides 10' (each having a unique probe sequence 14) can be delivered to different wells 38.

[00140] As shown in Fig. 4B, one genotyping oligonucleotide 10' is seeded into well 38. More specifically, the second primer sequence 18' and the restriction endonuclease site 16' of one genotyping oligonucleotide 10' respectively hybridize with one of the second capture primers 24' and its restriction endonuclease site 46' in well 38.

[00141] By seeding a single genotyping oligonucleotide 10', cluster generation can be immediately initiated. In other examples, separate hybridization (seeding) and cluster generation can be performed. The processes involved in cluster formation are shown in Fig. 4B, Fig. 4C, Fig. 4D, and Fig. 4E.

[00142] As indicated by the arrow in Fig. 4B, the genotyping oligonucleotide 10' is copied from the hybridized primers by extension at the 3' end using a high-fidelity DNA polymerase. This results in amplicon 44C, which is attached to the surface of the flow cell via a second capture primer 24'. The portions of the amplicon 44C designated _C14 , _C28 , _C26 , and _C12 are complementary copies of the probe sequence 14, the priming site region 28, the index sequence region 26, and the first primer sequence 12, respectively.

[00143] The initial genotyping oligonucleotide 10' is denatured, leaving the amplicon 44C immobilized in the well 38 by the second capture primer 24' and the second restriction endonuclease site 46'. The single-stranded amplicon 44C is flipped and bridged, for example, by hybridization of the _C12 copy of the first primer sequence to the adjacent complementary first capture primer 22. This is shown in Fig. 4C.

[00144] As indicated by the arrow in Fig. 4C, the hybridized primer is then extended by the polymerase(s) to form another amplicon 44D. The regions _CC14 , _CC26 , _CC28 of the 44D amplicon are complementary copies of the regions _C14 , _C26 , _C28 and thus have the same sequences as the original sequence 14 of the probe, the region 26 of the index sequence and the region 28 of the priming site, respectively. The region _C46' of the 44D amplicon is a complementary copy of the second restriction endonuclease site 46' and thus has the same sequence as the restriction endonuclease site 16' of the genotyping oligonucleotide 10'. The _24' C section of the 44D amplicon is a complementary copy of the second 24' capture primer and thus has the same sequence as the second 18' primer sequence. As shown in Fig. 4C, formation of the 44D amplicon generates a double-stranded bridge comprising the 44C and 44D amplicons covalently linked to the flow cell 20.

[00145] The double-stranded bridge is then denatured as shown in Fig. 4D. This results in two copies (amplicons 44C and 44D) covalently linked to the flow cell 20. Isothermal bridge amplification or some other form of amplification amplifies the immobilized copies. For example, the copied templates form a loop and hybridize to the adjacent complementary capture primer 22, 24', and the polymerase copies the copied templates to form double-stranded bridges that are denatured to form two single-stranded strands. These two strands form a loop and hybridize to the adjacent complementary capture primers 22, 24, and then are again extended to form two new double-stranded loops. The process is repeated on each copy of the template in cycles of isothermal denaturation and amplification to produce dense clonal clusters of double-stranded bridges. A simplified cluster including two double-stranded bridges is shown in Fig. 4E.

[00146] It should be understood that the seeding of the respective genotyping oligonucleotides 10' into the respective wells 38 and the amplification of such genotyping oligonucleotides 10' in the respective wells 38 can occur under conditions in which the rate of amplification exceeds the rate of seeding. Thus, the relatively high rate of obtaining copies (amplicons 44C, 44D) within the well 38 seeded with one genotyping oligonucleotide 10' effectively excludes seeding of this well 38 with a second genotyping oligonucleotide 10' for amplification. Thus, in each of the wells 38, a different genotyping oligonucleotide 10' (with a unique probe sequence 14) can be captured and amplified, which allows for the simultaneous analysis on the flow cell 20 of a plurality of different genotyping target loci.

[00147] In this example, amplification can be performed using methylated dCTP (deoxycytidine triphosphate) to protect the 16' restriction endonuclease site and complementary copies C46' of the second 46' restriction endonuclease site. This modification will fully methylate the amplicons 44C, 44D and leave the capture primers 22, 24' (including the second 46' restriction endonuclease site) hemimethylated.

[00148] The double-stranded bridges are then linearized. The linearization is described with reference to Fig. 4E and Fig. 4F. In this example of the method, the linearization of the bridge amplicons 44C, 44D can be accomplished by cleaving the restriction endonuclease region 56 of the amplicons 44C, 44D to leave the second capture primers 24' in the wells 38; and denaturing the remaining portion of the amplicons 44C, 44D to yield single-stranded probe templates 49 comprising the first capture primers 22 and at least one probe-detecting region in the wells 38. The result of the linearization process is shown in Fig. 4F.

[00149] In this example, each of the second 24' capture primers further comprises a second 46' restriction endonuclease site, wherein the second 46' restriction endonuclease site is complementary to the 16' restriction endonuclease site of the 10' genotyping oligonucleotide and, therefore, is also complementary to the C46' stretch. As shown in Fig. 4E, the double-stranded bridges comprise hybridized 46', C46' stretches that provide appropriate supports for restriction enzyme cleavage. More specifically, the hybridized C46' and 46' stretches of the 44C, 44D bridge amplicons form a restriction endonuclease site 56 that is recognized by a type IIS restriction enzyme. In this example, cleavage of site 56 by restriction endonuclease is accomplished by introducing a type IIS restriction enzyme. The type IIS restriction enzyme may be methyl sensitive or may not be methyl sensitive. Methylation protocols will protect sites within the _C14 , _CC14 copies of the probe sequence and/or may protect symmetric breaks. The type IIS restriction enzyme will recognize asymmetric sequences of site 56 DNA and cleave at a certain distance (e.g., from 1 nucleotide to about 20 nucleotides) beyond site 56. Upon cleavage, the second 24' capture primers remain attached to the flow cell 20 and the 44C amplicon is cut at the desired position for genotyping.

[00150] The remaining portions of the amplicons 44C, 44D can then be denatured, leaving the single-stranded probe templates 49 attached to the flow cell 20. As shown in Fig. 4F, the single-stranded probe templates 49 include first capture primers 22 and at least one probe-detecting region, which in this example includes all or part of the CC ₁₄ region, as well as the CC ₂₈ and CC ₂₆ regions.

[00151] The method may further include blocking the second 24' capture primers prior to sequencing along at least one probe-determining section of each of the first single-stranded probe templates 49. A blocking group (e.g., 3'-phosphate) may be added that is attached to the accessible 3'-ends of the second 24' capture primers to prevent unwanted extension on these 24' primers.

[00152] With reference to Fig. 4F, sequencing of at least one probe-determining section of single-stranded probe templates 49 is shown. In this example, the at least one probe-determining section is section CC ₂₆ , which is identical to index sequencing region 26.

[00153] Sequencing at least one probe-defining region comprises introducing a sequencing primer 50 that hybridizes to a region CC ₂₈ identical to the region 28 of the priming site. This sequencing primer 50 conditions the readiness for sequencing of at least one probe-defining region, for example CC ₂₆ , of the single-stranded probe template 49. A base extension reaction is then performed along the region CC ₂₆ .

[00154] The primary chemical process for sequencing may be polymerization (e.g., catalyzed by a polymerase enzyme), as described herein with reference to Fig. 3G.

[00155] To initiate the first sequencing cycle, one or more labeled nucleotides, DNA polymerase, etc. can be delivered into or through the flow cell 20, etc., whereby extension of the sequencing primer results in the incorporation of the labeled nucleotide into the nascent strand N ₂₆ formed along the CC segment ₂₆ of the single-stranded probe template 49. Such incorporation can be detected using a visualization event.

[00156] In some examples, the fluorescently labeled nucleotides may additionally have a reversible termination property that stops further primer extension after the nucleotide has been added to the nascent N strand _26. Thus, for examples in which reversible termination is used, an unblocking reagent, etc., may be supplied to the flow cell 20 (after detection).

[00157] Wash(s) may occur between the various fluid feed steps. The sequencing cycle may then be repeated n times to extend the single-stranded probe template 49 by n nucleotides to form a nascent strand including nascent stretch N ₂₆ (which is complementary to stretch CC ₂₆ , which has the same sequence as the sequencing index region 26).

[00158] Sequencing of the nascent strand N ₂₆ can be used to determine the CC ₂₆ stretch, and thus the initial sequencing index region 26, which is unique to the probe sequence 14 (and its complementary copy C ₁₄ ). This information allows the user to determine the target genotyping locus to be analyzed in a particular well 38 (since all templates 49 in well 38 have the same probe-defining stretch, e.g., CC ₂₆ , and probe sequence stretch, e.g., CC ₁₄ ).

[00159] After sequencing the probe defining section(s), the method further comprises removing at least the corresponding nascent strands (including section _N26 ) from the single stranded probe templates 49. In this example, the removal comprises denaturing the sequencing primer 50 and the nascent strand _N26 .

[00160] A sample of single-stranded DNA fragments, including a target genotyping locus 54, is introduced into a flow cell 20, as shown in Fig. 4H. The target genotyping locus 54 may be included in a library fluid that includes a plurality of target genotyping loci, at least some of which have different genotypable loci. The target genotyping loci may be obtained from a larger DNA sample using any genotyping library preparation technique. Some genotyping library preparation techniques include amplification and fragmentation. Others include amplification without fragmentation, as described below herein. Thus, multiple copies of any type of target genotyping locus 54 may be present in the library fluid.

[00161] After introduction into the flow cell 20, the target genotyping loci 54 hybridize with the corresponding complementary segments CC ₁₄ of the single-stranded probe templates 49 in the recess 38.

[00162] A genotyping reaction can then be performed. In this reaction, the single-stranded probe template 49 is used as a sequencing primer to perform one round of sequencing, as described herein. As shown in Fig. 4H, one labeled nucleotide 57, which is complementary to the nucleotide base of interest at the genotyping target locus 54, is incorporated into the single-stranded probe template 49.

[00163] Although the description provided herein relates to a single well 38 and thus genotyping of a single genotyping target locus 54, it should be understood that each well 38 includes different single-stranded probe templates 49 with different CC ₁₄ stretches. Thus, using this method, hundreds to thousands, up to millions (depending on the number of wells in the flow cell 20) of different loci can be genotyped simultaneously.

[00164] In the examples described herein, instead of blocking the second capture primers 24 or 24' during sequencing and/or genotyping, these primers 24 or 24' can be cleaved after the decoding reactions using a cleavage process that will not have a detrimental effect on the templates 48 or 49 to be genotyped.

[00165] Methodology for preparing a genotyping library

[00166] Any suitable genotyping library preparation technique can be used to obtain the target genotyping locus 54.

[00167] The genotyping target locus 54 can be obtained from genomic DNA. The genomic DNA can be isolated from one or more cells, bodily fluids, or tissues. Any suitable method can be used to obtain a biological fluid (e.g., blood, sweat, tears, lymph, urine, saliva, semen, cerebrospinal fluid, feces, or amniotic fluid). Some specific examples include a buccal swab, an oral swab, a surgical removal, an aspiration biopsy, or the like. The genomic DNA can also be obtained from one or more cells or tissues in a primary culture, an expanded cell line, a fixed archival sample, a forensic sample, or an archaeological sample.

[00168] gDNA can be obtained by lysing a cell that contains DNA. The cell can be lysed under conditions that substantially preserve the integrity of the gDNA of the cell. In one particular example, thermal lysis can be used to lyse the cell. In another particular example, exposing the cell to an alkaline pH can be used to lyse the cell, resulting in relatively little damage to the gDNA. Any of a variety of basic compounds can be used for lysing, including, for example, potassium hydroxide, sodium hydroxide, and the like. Additionally, relatively intact gDNA can be obtained from a cell lysed with an enzyme that cleaves a cell wall. Cells that lack a cell wall naturally or due to enzymatic removal can also be lysed by exposing the cell to osmotic stress. Other conditions that can be used to lyse the cell include surfactants, mechanical disruption, sonication, pressure differentials such as a French press or Dounce homogenization. Agents that stabilize the gDNA can be added to the cell lysate or isolated gDNA sample, including, for example, nuclease inhibitors, chelating agents, salts, buffers, etc.

[00169] In some examples, a crude cell lysate containing gDNA can be directly amplified without further isolation of the gDNA. For example, a blood sample can be heat-lysed and then the crude cell lysate can be amplified by any suitable method, including the methods described herein. In an alternative embodiment, gDNA can be further isolated from other cellular components prior to amplification. Accordingly, amplification can be performed on purified or partially purified gDNA. Genomic DNA can be isolated using known methods, including, for example, liquid phase extraction, precipitation, solid phase extraction, chromatography, and the like.

[00170] An amplified representative population of genomic fragments (genotyping target loci 54) can be produced by amplifying the native genome under conditions in which the genomic DNA (gDNA) template is replicated to produce one or more copies in which the relative proportion of each copied sequence is substantially the same as its proportion in the original gDNA. Any of a variety of methods that replicate genomic DNA in a sequence-independent manner can be used to produce the genotyping target loci 54. In the examples described herein, double-stranded genomic DNA can be denatured to form single-stranded genomic DNA templates that can be used in amplification processes.

[00171] In one specific example, the amplification method comprises contacting a single-stranded genomic DNA template with a low processivity polymerase, a plurality of primers, and free nucleotides, thereby generating complementary fragments of the single-stranded genomic DNA template; and displacing the complementary fragments from the single-stranded genomic DNA template, thereby generating at least some of the corresponding samples.

[00172] A low processivity polymerase can synthesize short chains as they naturally fall off from a single-stranded genomic DNA template before the entire strand is replicated. Some examples of a low processivity polymerase can synthesize less than 100 bases per polymerization event. Shorter fragments can be produced using a polymerase that synthesizes 50, 40, 30, 20, 10, or 5 bases per polymerization event under amplification conditions. The low processivity polymerase is selected from the group consisting of T4 DNA polymerase, T7 DNA polymerase, Taq polymerase, Stoffel fragment (a fragment of Taq DNA polymerase), Klenow fragment (a large fragment of Escherichia coli DNA polymerase I), Bsu DNA polymerase, Bst DNA polymerase, and an engineered polymerase. In this exemplary method, the term "engineered polymerase" is meant to refer to any synthetic polymerase that is designed to synthesize less than 100 bases per polymerization event. Other suitable low processivity polymerases include monomeric polymerase III (Pol III) (lacking the beta subunit) of E. coli or polymerase I (Pol I) of E. coli.

[00173] The processivity of some polymerases can be altered by the processing conditions used. For example, processivity can be altered by the reaction temperature, the level (e.g., ionic strength) of salt (e.g., Mg2 ⁺ ) in the reaction, pH, buffer composition (e.g., creatine kinase or cAMP [cyclic adenosine monophosphate]), or combinations thereof. As one example, T7 DNA polymerase has low processivity at temperatures below 37°C, as well as at high ionic strength, such as greater than about 100 mM NaCl; however, it is otherwise highly processive. As another example, Taq polymerase has high processivity at temperatures of about 70°C when reacted with DNA samples in 10-fold molar excess and randomly selected primers. In another example, polymerization of the Klenow fragment of Escherichia coli DNA polymerase can be slowed by decreasing the pH to below 6.2. In another example, the efficiency of Bacillus stearothermophilus DNA polymerase (BST pol), a family A DNA pol, can be altered several-fold by substituting metal cofactors such as Mg++ and Cd++.

[00174] The primers may be random primers. The population of random primers may be synthesized such that it has an increased content of guanine (G) and/or cytosine (C) nucleotides compared to adenine (A) and thymidine (T) nucleotides. The resulting population of random primers will be GC-rich and will therefore have a higher probability of hybridizing to regions of the genome with high GC content, such as gene-coding regions of the human genome, which typically have a higher GC content than non-coding regions of gDNA. The primers in the population of random primers may also have a region of identical sequence, such as a universal tail. The universal tail may include a universal priming site for amplification.

[00175] In this illustrative method, free nucleotides may include any naturally occurring nucleotide, such as deoxyadenine triphosphate, deoxythymine triphosphate, deoxyguanine triphosphate, and deoxycytosine triphosphate.

[00176] Contacting a single-stranded genomic DNA template with a low processivity polymerase, a plurality of primers, and free nucleotides may involve mixing the various components and subjecting them to appropriate amplification conditions for the low processivity polymerase used. During the amplification process, the primers are attached to various regions of the single-stranded genomic DNA template, and the low processivity polymerase introduces complementary free nucleotides into the template strand according to the sequence of the template strand. The low processivity polymerase naturally drops out of the template strand, typically after 100 bases or less have been replicated. The replicated complementary fragments may be displaced using, for example, denaturation. The method may involve repeating both the contacting and the displacement for a predetermined number of cycles, producing additional complementary fragments in each of the predetermined number of cycles.

[00177] Below are examples of this method.

[00178] T4 DNA polymerase can be used to amplify single-stranded or denatured gDNA, for example, in about 50 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) (pH 7.5), about 50 mM Tris-HCl (pH 8.6), or about 50 mM glycinate (pH 9.7). Examples of reaction mixtures may also include about 50 mM KCl, about 5 mM _MgCl2 , about 5 mM dithiothreitol (DTT), about 40 μg/mL gDNA, about 0.2 mM of each deoxynucleoside triphosphate (dNTP), about 50 μg/mL bovine serum albumin (BSA), about 100 μM of a randomly selected primer (n=6), and about 10 units of T4 DNA polymerase incubated at 37°C for at least one hour. Temperature cycling may be used to bias the replicated strands for multiple amplification cycles.

[00179] Taq polymerase has low processivity at temperatures below 70°C. Accordingly, small gDNA fragments can be produced using Taq polymerase at low temperature or under another condition in which Taq has low processivity. In another example, the Stoffel fragment, which lacks the 289 N-terminal amino acid residues of Taq polymerase and has low processivity at 70°C, can be used to produce relatively small gDNA fragments. Taq or the Stoffel fragment can be used to amplify single-stranded or denatured DNA templates, and temperature cycling can be used to displace replicated strands over multiple amplification cycles.

[00180] The Klenow fragment can be used for isothermal amplification of the genome to produce small fragments of genomic DNA, for example, in a low salt reaction (I=0.085) incubated at a temperature of from about 5°C to 37°C. Examples of buffers and pH conditions that can be used to amplify gDNA with the Klenow fragment include, for example, about 50 mM Tris-HCl (pH 7.5), about 5 mM _MgCl2 , about 50 mM NaCl, about 50 μg/mL bovine serum albumin (BSA), about 0.2 mM of each dNTP, about 2 μg of randomly selected primer (n=6), about 10 ng of gDNA template, and about 5 units of the Klenow fragment incubated at 37°C for about 16 hours. Similar reactions can be carried out when one or more reaction components are omitted or substituted. For example, the buffer can be replaced with about 50 mM phosphate (pH 7.4) or other pH values in the range of about 7.0 to 7.8 can be used. In another example, amplification conditions using the Klenow fragment can include, for example, about 10 ng of gDNA template, about 2 mM dNTPs, about 10 mM MgCl ₂ , about 0.5 U/μl (microliter) of polymerase, about 50 μM (micromolar) random primer (n=6), and isothermal incubation at 37°C for 16 hours.

[00181] Another example of an amplification method described herein comprises contacting a single-stranded genomic DNA template with a polymerase, a plurality of primers, and a mixture of free nucleotides, including natural nucleotides and dideoxythymidine triphosphate (ddTTP), thereby forming truncated complementary fragments of the single-stranded genomic DNA template; and displacing the truncated complementary fragments from the single-stranded genomic DNA template, thereby forming at least some of the corresponding samples.

[00182] In this example, any suitable polymerase can be used. ddTTP acts as a truncating agent, and thus, a polymerase with high processivity can be used. In this exemplary method, any of the polymerases described herein can be used, as long as the processing conditions can be varied such that the polymerase exhibits high processivity (e.g., can synthesize more than 100 bases per polymerization event). In one example, the polymerase with high processivity is selected from the group consisting of T4 DNA polymerase, T7 DNA polymerase, Taq polymerase, Stoffel fragment, Klenow fragment, Bsu DNA polymerase, Bst DNA polymerase, and an engineered polymerase. In this exemplary method, the term "engineered polymerase" refers to any synthetic polymerase that is designed to synthesize more than 100 bases per polymerization event. Highly processive polymerases produce fragments of 10 kb (kilobases) to 20 kb in length. Other suitable highly processive polymerases include F29 polymerase.

[00183] Any of the randomly selected primers described herein can be used in this example.

[00184] In this exemplary method, the free nucleotides in the mixture include naturally occurring nucleotides and dideoxythymidine triphosphate (ddTTP). Dideoxythymidine triphosphate serves as a nucleotide synthesis terminating or truncating agent. In an example, the naturally occurring nucleotides include deoxyadenine triphosphate, deoxythymine triphosphate, deoxyguanine triphosphate, and deoxycytosine triphosphate. In an example, the mixture of free nucleotides includes deoxythymine triphosphate (dTTP) and dideoxythymidine triphosphate (ddTTP) in a ratio ranging from about 10:5 to about 10:0.01. A ratio within this range helps ensure that the desired amount of deoxythymine triphosphate is incorporated into the resulting fragments before amplification with dideoxythymidine triphosphate is terminated. In one specific example, the ratio of dTTP to ddTTP is about 10:1.

[00185] Bringing a single-stranded genomic DNA template into contact with a polymerase, a plurality of primers, and a mixture of free nucleotides may involve mixing the various components and exposing them to appropriate amplification conditions for the polymerase used. During amplification, primers are attached to various sites on the single-stranded genomic DNA template, and the polymerase introduces complementary natural nucleotides into the template strand according to the sequence of the template strand. By introducing dideoxythymidine triphosphate (ddTTP) instead of deoxythymidine triphosphate (dTTP), amplification of a particular strand is terminated. Thus, ddTTP truncates the replicated DNA fragment. The ratio of dTTP to ddTTP in the mixture helps ensure that the generated fragments are of sufficient length for subsequent analysis.

[00186] The replicated truncated complementary fragments can be displaced, for example, using denaturation. The method can include repeating a predetermined number of cycles of both contacting and displacing to produce additional truncated complementary fragments in each of a predetermined number of cycles.

[00187] In this illustrative method, any of the buffers (e.g., HEPES, Tris-HCl, glycinate, etc.), salts (e.g., KCl, MgCb, etc.), redox reagents (e.g., dithiothreitol), and/or stabilizers (e.g., bovine serum albumin [BSA]) can be used in suitable amounts for high processivity.

[00188] In one specific example, T7 DNA polymerase has high processivity under the following reaction conditions: about 40 mM Tris-HCl, pH 7.5, about 15 mM _MgCl2 , about 25 mM NaCl, about 5 mM DTT, about 0.25 mM each dNTP, about 0.00025 mM to about 0.125 mM ddTTP, 50 μg/mL single-stranded gDNA, about 100 μM random primer (n=6), about 0.5 to about 1 unit of T7 DNA polymerase, reaction temperature greater than 37°C.

[00189] In another specific example, Taq polymerase has high processivity at temperatures of about 70°C when reacted with DNA samples in a 10-fold molar excess and randomly selected primers (n=6). The amplification reaction under these conditions may further include a buffer such as Tris-HCl at about 20 mM, pH about 7, about 1 mM to 2 mM _MgCl2 , about 0.2 mM of each dNTP, and about 0.0002 mM to about 0.1 mM ddTTP. In addition, a stabilizing agent such as glycerol, gelatin, BSA, or a nonionic surfactant may be added.

[00190] In another specific example, the Klenow fragment is highly processive under the following reaction conditions: about 10 mM Tris-HCl, pH 7.9, about 10 mM _MgCl2 , about 50 mM NaCl, about 1 mM DTT, and about 100 g/mol BSA.

[00191] In another specific example, Bst DNA polymerase has high processivity under the following reaction conditions: about 20 mM Tris-HCl, pH 8.8, about 10 mM (NH ₄ ) ₂ SO ₄ , about 10 mM KCl, about 2 mM MgSO ₄ , and about 0.1% nonionic surfactant (e.g., TRITON™ X-100 from The Dow Chemical Co.).

[00192] Both amplification processes described herein generate multiple gDNA fragments without the need for a fragmentation process. Single-stranded gDNA serves as a template for multiple genotyping target loci 54, and multiple amplicons of each genotyping target locus 54 are generated. When introduced into a flow cell 20 with genotyping templates 48 or 49, the amplified gDNA fragments (genotyping target loci 54) hybridize to the corresponding complementary regions of the genotyping templates 48 or 49.

[00193] Sets

[00194] Genotyping nucleotides 10 or 10' and flow cell 20 may be part of a genotyping kit.

[00195] In one example, the kit comprises: a flow cell 20 including a substrate 30, 30' having recesses 38 separated by intermediate regions 40, and first and second capture primers 22, 24 or 22, 24' attached within each of the recesses 38; and a genotyping probe fluid comprising a liquid carrier and a genotyping oligonucleotide 10 or 10' in the liquid carrier, wherein the genotyping oligonucleotide 10 or 10' comprises a first primer sequence 12, a probe sequence 14 representing a genotyping target locus 54, a restriction endonuclease site 16, 16' and a second primer sequence 18, 18' which is at least partially complementary to a second capture primer 24, 24'. [00196] The kit may also include genotyping library preparation components, such as a whole genome sample, a polymerase (which may be a low processivity polymerase as defined herein), a plurality of primers, and free nucleotides (in some examples including naturally occurring nucleotides, and in other examples including a mixture of naturally occurring nucleotides and dideoxythymidine triphosphate [ddTTP]).

[00197] Any example of genotyping oligonucleotide 10 or 10' may be used in the kit, and any example of flow cell 20 may be used in the kit.

[00198] In an alternative embodiment, the kit may include an unstructured flow cell with primers across the surface of the flow channel.

[00199] additional notes

[00200] It should be understood that all combinations of the above concepts and additional concepts described in more detail below (provided that such concepts are not mutually contradictory) are considered to be part of the inventive subject matter described herein. In particular, all combinations of claimed subject matter appearing at the end of this description are considered to be part of the inventive subject matter described herein. It should also be understood that terminology expressly used herein, which may also appear in any description incorporated herein by reference, is intended to have the meaning most consistent with the particular concepts described herein.

[00201] Although several examples are described in detail, it should be understood that the examples described may be changed. Therefore, the above description should be considered as not limiting.

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SEQUENCE LIST

<110> Illumina Cambridge Ltd.

Illumina, Inc.

<120> GENOTYPING KITS

<130> IP-1897-PCT

<150> 62/981866

<151> 02/26/2020

<160> 3

<170> PatentIn, version 3.5

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<212> DNA

<213> Artificial sequence

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<223> Synthesized

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aatgatacgg cgaccaccga 20

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<212> DNA

<213> Artificial sequence

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<223> Synthesized

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caagcagaag acggcatacg a 21

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<212> DNA

<213> Artificial sequence

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<223> Synthesized

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gttcgtcttc tgccgtatgc t 21

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Claims (60)

1. A kit for obtaining a monoclonal cluster of amplicons with a unique target locus for genotyping, containing: flow cell, including: substrate; and first and second capture primers attached to the substrate; and genotyping probe fluid medium comprising: liquid carrier; and a genotyping oligonucleotide in a liquid carrier, wherein the genotyping oligonucleotide comprises: first primer sequence; a region of the index sequence directly attached to the first primer sequence; a region of the priming site directly attached to a region of the index sequence; a probe sequence directly attached to the priming site region, wherein the probe sequence is a target genotyping locus having a nucleobase of interest, wherein the last base of the probe sequence is a nucleobase of the target genotyping locus that is located immediately adjacent to the nucleobase of interest; restriction endonuclease site; And a second primer sequence that is at least partially complementary to the second capture primer; moreover: a restriction endonuclease site is located between the probe sequence and the second primer sequence; or the probe sequence is directly attached to the second primer sequence, and the restriction endonuclease site is integrated into the second primer sequence at a cleavage distance from the last base of the probe sequence. 2. The kit of claim 1, wherein the genotyping oligonucleotide comprises a plurality of different genotyping oligonucleotides, and wherein each of the different genotyping oligonucleotides comprises a probe sequence that is different from the sequence of each other of the different genotyping oligonucleotides. 3. The kit according to one of claims 1 or 2, wherein the second capture primer includes a cleavage site. 4. The kit of claim 3, wherein the restriction endonuclease site is sensitive to a restriction endonuclease selected from the group consisting of a 4-base cleaving restriction endonuclease, a 5-base cleaving restriction endonuclease, and a 6-base cleaving restriction endonuclease. 5. A set according to one of items 1-4, in which: the substrate has recesses separated by intermediate regions; and The first and second capture primers are attached inside each of the recesses. 6. A method for obtaining a monoclonal cluster of amplicons with a unique target locus for genotyping, comprising: introducing a genotyping probe fluid into a flow cell comprising first and second capture primers, wherein the genotyping probe fluid comprises a plurality of different genotyping oligonucleotides, wherein each of the different genotyping oligonucleotides comprises: first primer sequence; a unique region of index sequence directly attached to the first primer sequence; a region of the priming site directly attached to a region of the index sequence; a unique probe sequence directly attached to the priming site region, wherein the unique probe sequence is a target genotyping locus having a nucleobase of interest, wherein the last base of the probe sequence is a nucleobase of the target genotyping locus that is located immediately adjacent to the nucleobase of interest; restriction endonuclease site; And a second primer sequence that is at least partially complementary to the second capture primer; moreover: a restriction endonuclease site is located between the probe sequence and the second primer sequence; or the probe sequence is directly attached to the second primer sequence, and the restriction endonuclease site is integrated into the second primer sequence at a cleavage distance from the last base of the probe sequence; as a result, each of the different genotyping oligonucleotides reacts to form a unique clonal population of amplicons from each of the different genotyping oligonucleotides; linearization of amplicons to obtain probe matrices; sequencing at least one probe-determining portion of the probe templates to determine each of the probe sequences; removing at least the nascent chains from the probe templates, thereby exposing the 3' OH group at the end of the probe templates; hybridization of different samples with probe matrices; and conducting genotyping reactions of samples on accessible 3' OH groups. 7. The method according to claim 6, wherein the linearization of the amplicons comprises: cleaving the amplicons attached to the second capture primers at the cleavage sites of the second capture primers; and denaturation of the cleaved regions of the amplicons attached to the second capture primers to yield probe templates. 8. The method according to claim 7, wherein sequencing at least one probe-determining section of the probe templates comprises performing a base extension reaction along the at least one probe-determining section using the second capture primer as a sequencing primer. 9. The method of claim 8, wherein removing at least nascent chains from the probe matrices comprises: cleavage of restriction endonuclease sites; and denaturation of the remaining nascent chain from the probe templates. 10. The method according to one of claims 6-9, further comprising comparing each determined probe sequence with one of the different clonal populations of amplicons. 11. The method according to one of paragraphs 6-10, further comprising, prior to hybridization of the various samples to the probe matrices, obtaining the various samples by: bringing a single-stranded genomic DNA template into contact with a low processivity polymerase, a plurality of primers and free nucleotides, thereby forming complementary fragments of the single-stranded genomic DNA template; and displacement of complementary fragments from a single-stranded genomic DNA template, thereby forming at least some of the different patterns. 12. The method of claim 11, further comprising denaturing the double-stranded genomic deoxyribonucleic acid (DNA) to thereby form a single-stranded genomic DNA template. 13. The method of claim 11, further comprising repeating a given number of cycles of both contacting and displacing to form additional complementary fragments in each of the given number of cycles. 14. The method of claim 11, wherein the low processivity polymerase is selected from the group consisting of T4 DNA polymerase, T7 DNA polymerase, Taq polymerase, Stoffel fragment, Klenow fragment, Bsu DNA polymerase, Bst DNA polymerase and engineered polymerase. 15. The method according to one of paragraphs 6-10, further comprising, prior to hybridization of the various samples to the probe matrices, obtaining the various samples by: bringing a single-stranded genomic DNA template into contact with a polymerase, a plurality of primers and a mixture of free nucleotides, including natural nucleotides and dideoxythymidine triphosphate, thereby forming truncated complementary fragments of the single-stranded genomic DNA template; and displacement of truncated complementary fragments from a single-stranded genomic DNA template, thereby forming at least some of the different patterns. 16. The method of claim 15, wherein the natural nucleotides comprise deoxyadenine triphosphate, deoxythymine triphosphate, deoxyguanine triphosphate and deoxycytosine triphosphate, and wherein the mixture of free nucleotides comprises deoxythymine triphosphate and dideoxythymidine triphosphate in a ratio in the range of about 10:5 to about 10:0.01. 17. The method of claim 15, further comprising denaturing the double-stranded genomic deoxyribonucleic acid (DNA) to thereby form a single-stranded genomic DNA template. 18. The method of claim 15, further comprising repeating a given number of cycles of both contacting and displacing to form additional truncated complementary fragments in each of the given number of cycles. 19. The method of claim 15, wherein the polymerase is selected from the group consisting of T4 DNA polymerase, T7 DNA polymerase, Taq polymerase, Stoffel fragment, Klenow fragment, Bsu DNA polymerase, Bst DNA polymerase and an engineered polymerase.

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