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WO2019023951A1 - 核酸测序方法 - Google Patents

核酸测序方法 Download PDF

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Publication number
WO2019023951A1
WO2019023951A1 PCT/CN2017/095520 CN2017095520W WO2019023951A1 WO 2019023951 A1 WO2019023951 A1 WO 2019023951A1 CN 2017095520 W CN2017095520 W CN 2017095520W WO 2019023951 A1 WO2019023951 A1 WO 2019023951A1
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WIPO (PCT)
Prior art keywords
solid support
sequencing
nucleic acid
reaction
labeled
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2017/095520
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English (en)
French (fr)
Inventor
杨晋
徐讯
王卉
谢槟
李卓坤
赵胜明
陈奥
徐崇钧
章文蔚
倪鸣
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MGI Tech Co Ltd
Complete Genomics Inc
Original Assignee
MGI Tech Co Ltd
Complete Genomics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to CN202410439381.7A priority Critical patent/CN118086478A/zh
Priority to EP17920285.8A priority patent/EP3663407B1/en
Priority to DK17920285.8T priority patent/DK3663407T3/da
Priority to CN202410439616.2A priority patent/CN118109572A/zh
Priority to EP23155059.1A priority patent/EP4209597B1/en
Priority to EP25154432.6A priority patent/EP4553171A3/en
Priority to CN202410439618.1A priority patent/CN118086480A/zh
Priority to CN202410439435.XA priority patent/CN118086479A/zh
Priority to FIEP17920285.8T priority patent/FI3663407T3/fi
Priority to CN201780093472.8A priority patent/CN111032883B/zh
Priority to US16/635,131 priority patent/US11692221B2/en
Priority to ES23155047T priority patent/ES3014460T3/es
Priority to EP23155047.6A priority patent/EP4212628B1/en
Priority to EP25154434.2A priority patent/EP4553172A3/en
Priority to PL17920285.8T priority patent/PL3663407T3/pl
Priority to RS20230242A priority patent/RS64086B1/sr
Priority to ES17920285T priority patent/ES2941083T3/es
Priority to PT179202858T priority patent/PT3663407T/pt
Priority to ES23155059T priority patent/ES3018947T3/es
Application filed by MGI Tech Co Ltd, Complete Genomics Inc filed Critical MGI Tech Co Ltd
Priority to PCT/CN2017/095520 priority patent/WO2019023951A1/zh
Priority to HUE17920285A priority patent/HUE061474T2/hu
Publication of WO2019023951A1 publication Critical patent/WO2019023951A1/zh
Anticipated expiration legal-status Critical
Priority to US18/144,124 priority patent/US20230323445A1/en
Priority to US18/144,121 priority patent/US20230272467A1/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept

Definitions

  • the invention relates to the field of nucleic acid sequencing.
  • the present invention relates to a method of sequencing a nucleic acid using a immersion reaction scheme comprising sequentially immersing a solid support having nucleic acid molecules immobilized thereon in a different reaction vessel to effect nucleic acid sequencing.
  • the second generation of sequencing technology is based on the development of the first generation of Sanger sequencing technology, with low cost, high throughput, automation and other features, greatly promoting the development of the gene sequencing industry.
  • Second-generation sequencing technology has been widely used in whole-genome sequencing, transcriptome sequencing, and metagenomic sequencing. It is a powerful tool for analyzing the evolution and classification of organisms, studying cancer-related genes such as autism, and conducting in vitro diagnostics. It has promoted people's understanding of life sciences and promoted the development of health industry.
  • the existing second-generation sequencing technologies mainly include Illumina's SBS sequencing technology (Sequencing-by-Synthesis), Life Technologies' semiconductor sequencing technology, and BGI's Joint Probe Anchor Polymerization Technology (cPAS).
  • SBS sequencing technique uses dNTPs (dATP, dCTP, dGTP, dTTP) with two modified portions (fluorescent labeling and blocking groups), wherein dATP, dCTP, dGTP, and dTTP carry different fluorescent labels, respectively.
  • the reagent excises the reversible blocking group and the fluorescent label (referred to as regeneration reaction) and proceeds to the next polymerization reaction (P085, genomics, 2016 first edition, Yang Huanming).
  • the semiconductor sequencing technology utilizes the pH change caused by pyrophosphate released by the dNTPs polymerized during the extension of the template strand, and the change in the bottom of the chip detects the change to obtain DNA strand sequence information (P083, Genomics, 2016, first edition, Yang Huanming).
  • SOLiD The second-generation sequencing technology solution SOLiD developed by ABI in the United States uses SBL sequencing (Sequencing-by-Ligation) for high-throughput sequencing.
  • SOLiD's SBL sequencing method uses a mixture of ligase and 8-base single-stranded fluorescent probes with different primers to achieve a read length of 35-70 nt, which has the advantage of high accuracy (P093, Genomics, 2016).
  • Combinatorial probe-anchor synthesis cPAS uses DNA molecular anchors and fluorescent probes to polymerize on DNA nanospheres (DNB), followed by high-resolution imaging systems to acquire optical signals. After the digital processing, the sequence to be tested can be obtained; wherein the DNB passes the line Amplification enhances the signal and reduces the error rate of a single copy (see BGISEQ-500 sequencing platform).
  • the biochemical reaction part of the existing second-generation sequencing technology is mainly carried out by a flow channel system.
  • a typical flow channel system can be found in WO2007123744, which uses a solid substrate comprising a flow cell comprising one or more fluid channels to which the polynucleotide is attached, and the fluorescent labeling reagent is controllably controlled by a flow guiding system Flow through the fluid channel to contact the polynucleotide.
  • the platform uses a flow cell to pump sequencing reagents from a refrigeration unit to a flow cell at a specific point in time, through a sequencing chip under the control of a complex set of fluid control devices, and Auxiliary with fine temperature control facilities to ensure that biochemical reactions are fully carried out in the shortest possible time ( 2500 System Guide, illumina, 2015).
  • the present invention provides a method for sequencing nucleic acids using a immersion reaction scheme, which comprises sequentially immersing solid supports on which nucleic acid molecules are immobilized on different ones.
  • the reaction vessel is used to effect nucleic acid sequencing.
  • immersion reaction protocol as used herein is referred to relative to the flow channel system used in the prior art, which means moving the nucleic acid to be sequenced to the reaction reagent during sequencing of the nucleic acid.
  • Contact rather than using a flow channel, moves the reagents into contact with the nucleic acid.
  • the immersion reaction scheme has at least the following advantages: 1. Recycling of biochemical reagents; 2. Good extensibility, flexible use, and greatly improved flux; 3. No complicated temperature and fluid control system, uniform reaction; 4 Significantly reduce the cost of gene sequencing.
  • nucleic acid sequencing can be accomplished using a variety of sequencing methods.
  • sequencing methods include, but are not limited to, electrophoresis, synthetic sequencing, ligation sequencing, combined probe anchor polymerization sequencing, hybridization sequencing, single molecule sequencing, and real-time sequencing methods.
  • the process of determining the nucleotide sequence of a target nucleic acid can be an automated process.
  • the invention provides a method of sequencing a nucleic acid using a immersion reaction protocol, the immersion reaction scheme comprising the steps of:
  • reaction solutions immersing the first batch of solid support in one or more reaction solutions to generate a signal representative of the nucleotide sequence of the nucleic acid molecule on the solid support, the one or more The reaction solutions are each placed in separate reaction vessels,
  • the first batch of the solid support is washed to remove the residual reaction solution on the solid support.
  • steps b through e may be repeated one or more times, such as repeating 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 Or more times.
  • the washing is performed by soaking the solid support in a reaction vessel containing the washing solution.
  • the soaking includes any means of contacting all nucleic acid molecules immobilized on a solid support with a reagent or solution in a reaction vessel.
  • the solid support can be partially or fully immersed in the reaction vessel, for example, in a vertical, oblique or horizontal manner, provided that all of the nucleic acid molecules immobilized on the solid support are in contact with the reagents in the reaction vessel.
  • nucleic acid molecule as used herein may be any type of nucleic acid, for example, the nucleic acid may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or an analog of DNA or RNA made from a nucleotide analog. It can be used interchangeably with “polynucleotide”. Nucleic acids can be single stranded, double stranded or contain both single stranded and double stranded sequences.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • Nucleic acids can be single stranded, double stranded or contain both single stranded and double stranded sequences.
  • the sequenced nucleic acid can be in the form of a single molecule (which can be a natural molecule, a modified molecule such as a labeled molecule, or a nucleic acid comprising a nucleotide analog), a concatamer of sequences (eg, a DNA nanosphere) ), etc., may be amplified (eg, amplified as a concatamer, amplified into multiple individual molecules having the same or similar sequences, etc.), and/or may be in any other form.
  • the exact sequence of the nucleic acid molecule can be known or unknown.
  • Nucleotide analogs comprising modified nucleobases can also be used in the methods described herein.
  • Exemplary modified nucleobases that may be included in a polynucleotide, whether having a native backbone or a similar structure include, for example, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, 2-amino Indole, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine , 2-thiouracil, 2-thiothymidine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6 - azouracil, 6-azocytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-haloadenine or guanine,
  • Nucleic acid molecules that are sequenced in particular embodiments of the invention can be of any length.
  • exemplary lengths of useful nucleic acids include, for example, at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 1,000. , 5,000 or 10,000, 100,000 nucleotides or longer. Alternatively or additionally, the length may be no longer than 1,000,000, 100,000, 10,000, 1,000, 100 nucleotides or less.
  • the length of the nucleic acid molecule can also include all integers between the above exemplary numbers.
  • nucleic acids that can be sequenced using the methods described herein can, for example, be within the scope of short polynucleotides, fragments, cDNA, genes, and genomic fragments.
  • the nucleic acid can be obtained from a particular biological source.
  • the nucleic acid is a human nucleic acid obtained from a human, for example, a sample of human tissue.
  • the nucleic acid is a human mitochondrial nucleic acid.
  • the nucleic acid can be obtained from a metagenomic sample.
  • the nucleic acid can be obtained from an environmental source that no longer contains living organisms.
  • the term "immobilized” means attached directly or indirectly to a solid support via a covalent bond or a non-covalent bond. In certain embodiments of the present disclosure, covalent attachment may be used, but typically only needed is
  • the nucleic acid remains immobilized or attached to the solid support under conditions where it is desired to use a solid support, such as in applications requiring nucleic acid amplification and/or sequencing.
  • an oligonucleotide to be used as a capture primer or amplification primer eg, a primer having a partially double-stranded structure of the invention
  • Immobilization can occur via hybridization to surface-attached oligonucleotides, in which case the immobilized oligonucleotide or polynucleotide can be in the 3'-5' direction.
  • Another way of non-covalent attachment may be to bind the nucleic acid binding protein to the solid support by amination modification and to capture the nucleic acid molecule by the nucleic acid binding protein.
  • immobilization can occur by other means than base pair hybridization, such as the covalent attachment described above.
  • Non-limiting examples of nucleic acid attachment to solid support include nucleic acid hybridization, biotin streptavidin binding, sulfhydryl binding, photoactivated binding, covalent binding, antibody-antigen, via hydrogel or other porous polymer. Physical limitations, etc.
  • Various exemplary methods for immobilizing nucleic acids on a solid support can be found, for example, in G. Steinberg-Tatman et al, Bioconjugate Chemistry 2006, 17, 841-848; Xu X. et al. Journal of the American Chemical Society 128 (2006).
  • the solid support can comprise an inert substrate or matrix that has been chemically functionalized, for example by applying a layer or coating of an intermediate material that allows covalent attachment to the multicore A reactive group of a glycoside.
  • the intermediate material can be attached directly or indirectly to the solid support via a covalent bond or a non-covalent bond.
  • a support can include a polyacrylamide hydrogel layer on an inert substrate such as glass.
  • the polynucleotide can be directly covalently attached to an intermediate layer (eg, a hydrogel), but the intermediate layer itself can be non-covalently attached to other substrates or substrates (eg, glass substrates) layers.
  • reaction vessel means any device capable of containing a reaction solution, including but not limited to tanks, channels, wells, test tubes, cups, dishes, and the like.
  • the reaction vessel may have any suitable shape, for example, the reaction vessel may be square, spherical, conical, cylindrical, irregular, or the like.
  • the reaction vessel may have any suitable size, for example, its size may be arbitrarily adjusted according to the volume of the reaction solution to be contained, for example, it may be sized to accommodate at least 1 ⁇ L, at least 5 ⁇ L, at least 10 ⁇ L, at least 20 ⁇ L, at least 50 ⁇ L, at least 100 ⁇ L, at least 1 mL, at least 5 mL, at least 10 mL, at least 20 mL, at least 50 mL, at least 100 mL, at least 200 mL, at least 500 mL, at least 1 L or more, or may be sized to accommodate up to 1 ⁇ L, up to 5 ⁇ L, up to 10 ⁇ L, Up to 20 ⁇ L, up to 50 ⁇ L, up to 100 ⁇ L, up to 1 mL, up to 5 mL, up to 10 mL, up to 20 mL, up to 50 mL, up to 100 mL, up to 200 mL, up to 500 mL, up to
  • reaction solution refers to an agent that is used in any sequencing method suitable for use in the soaking reaction schemes described herein.
  • sequencing methods can include, but are not limited to, electrophoretic sequencing, synthetic sequencing, ligation sequencing, combined probe anchor polymerization sequencing, hybrid sequencing, single molecule sequencing, and real-time sequencing methods. Therefore, the one or more reaction solutions used in step b in the immersion reaction scheme of the present invention and the regeneration solution used in step d depend on the sequencing method employed. It is determined by the sequencing method that one or more of the reaction solutions used in step b in the immersion reaction scheme of the present invention and the regeneration solution used in step d are within the capabilities of those skilled in the art.
  • Ligation sequencing as used herein is a variety of ligation sequencing methods well known in the art. Basically, ligation sequencing involves the hybridization and ligation of labeled (eg, fluorescently labeled) sequencing probes and anchor probes (also referred to as "primers" in SOLiD sequencing) to DNA strands.
  • the sequencing probe contains one or two fixed known sequencing bases (single base sequencing probe or double base sequencing probe) and a series of degenerate or universal bases that allow for sequencing probes and nucleic acid templates Perform complementary pairing.
  • the solid support to which the nucleic acid molecule is immobilized is immobilized to the anchor probe prior to step b (also in SOLiD sequencing)
  • the one or more reaction solutions of step b include a sequencing probe comprising a labeled (eg, fluorescent label) a solution of a needle, ligase or a mixture thereof, provided that the solid support is contacted with each of the labeled sequencing probe and ligase, and wherein the signal on the solid support is complementary to solid support
  • a labeled sequencing probe of the nucleic acid molecule on the object is produced, the labeled sequencing probe being linked via a ligase to an anchor probe that is complementary to the same nucleic acid molecule.
  • the specific composition of the one or more reaction solutions of step b will vary depending on the particular ligation sequencing method employed.
  • the one or more reaction solutions of step b can include only one reaction solution comprising a ligase and a labeled sequencing probe, optionally, further The anchor probe is included; or the one or more reaction solutions of step b may also include two reaction solutions, one of which contains an anchor probe and the other of which contains a labeled sequencing probe, and At least one of the two reaction solutions comprises a ligase, in which case step b comprises the steps of: immersing the solid support in a reaction solution containing the anchor probe, washing, and then immersing in the inclusion of labeled sequencing The reaction solution of the probe is washed.
  • the one or more reaction solutions of step b may further comprise three reaction solutions comprising an anchor probe, a labeled sequencing probe and a ligase, respectively, in which case step b comprises the following process: The solid support is immersed in a reaction solution containing an anchor probe, washed, then immersed in a reaction solution containing the labeled sequencing probe, washed, and then immersed in a reaction solution containing a ligase, and washed.
  • the one or more reaction solutions of step b may comprise only one comprising both a ligase, a first anchor probe, a second anchor probe, and a labeled sequencing probe.
  • a reaction solution, or a plurality of reaction solutions each containing a ligase, a first anchor probe, a second anchor probe, a labeled sequencing probe, or a mixture thereof.
  • ligase refers to a nucleic acid modifying enzyme that catalyzes the intramolecular formation and intermolecular formation of phosphodiester bonds between the 5'-phosphate and 3'-hydroxyl ends of a nucleic acid strand.
  • Ligase can be obtained from recombinant or natural sources.
  • One or more low temperature (eg, room temperature or lower) ligases eg, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, and/or E. coli DNA ligase
  • the ligase may also be a thermostable ligase.
  • a thermostable ligase from a thermophilic organism can be used.
  • thermostable DNA ligases include, but are not limited to, Tth DNA ligase (from Thermus thermophilus, available from, for example, Eurogentec and GeneCraft); Pfu DNA ligase (from intense Hyperthermophilic ligase of Pyrococcus furiosus; Taq ligase (from Thermus aquaticus), and any other suitable thermostable ligase, or any combination thereof.
  • Synthetic sequencing as used herein is a variety of synthetic sequencing methods well known in the art. Basically, synthetic sequencing involves first hybridizing the sequenced nucleic acid molecule to a sequencing primer, followed by polymerization of the labeled nucleic acid molecule as a template at the 3' end of the sequencing primer (eg, fluorescent labeling) in the presence of a polymerase. Nucleotide. After polymerization, the labeled nucleotide is identified by detecting the label. After removal of the label (eg, fluorophore) from the labeled nucleotide, the next polymerization sequencing cycle begins.
  • a sequencing primer e.g, fluorescent labeling
  • the sequenced core The acid molecule is immobilized on a solid support by hybridization to a sequencing primer immobilized on a solid support.
  • the solid support on which the nucleic acid molecule is immobilized is contacted with the sequencing primer prior to step b, such that the sequencing primer hybridizes to the solid support Nucleic acid molecule.
  • the one or more reaction solutions of step b comprise a nucleoside comprising a polymerase, labeled (eg, fluorescently labeled) a solution of an acid or a mixture thereof, provided that the solid support is contacted with each of the polymerase, the labeled nucleotide, and wherein the signal on the solid support is complementary bound to the solid support
  • the labeled nucleotide of the nucleic acid molecule is produced, and the labeled nucleotide is polymerized via a polymerase with a nucleic acid molecule on a solid support as a template to the 3' end of the sequencing primer.
  • the regeneration solution of step d includes reagents for removing the label from the labeled (eg, fluorescently labeled) nucleotide.
  • the solid support is immersed in a reaction solution comprising the polymerase and labeled (in step b) For example, fluorescently labeled nucleotides.
  • the labeled nucleotide may further comprise a 3' blocking group.
  • the 3' blocking group prevents the incorporation of other nucleotides when the labeled nucleotide is polymerized onto the growing nucleotide strand.
  • the 3' blocking group is removed along with the label in step d.
  • Suitable 3' blocking groups and reagents for removing 3' blocking groups from nucleotides are well known in the art, and non-limiting examples of such 3' blocking groups and reagents can be found, for example, in Greene & Wuts. , Protective Groups in Organic Synthesis, John Wiley & Sons. Guillier; Metzker et al.
  • the label on the labeled nucleotide itself can be used as a 3' blocking group.
  • a label may be of a size or structure sufficient to function to block the incorporation of other nucleotides into the polynucleotide strand. The blockage may be due to steric hindrance or may be due to a combination of size, charge and structure.
  • the labeling and blocking groups on the labeled nucleotide may be The same, but preferably the label and blocking groups are removed from the nucleotide in the same manner.
  • the regenerating reagent of step d described herein comprises an agent that removes both the label and the 3' blocking group from the labeled nucleotide.
  • step d described herein can include separately soaking the solid support to an agent that can remove the label from the labeled nucleotide and removing the 3' resistance from the labeled nucleotide. Broken group of reagents.
  • polymerase refers to an enzyme that synthesizes a nucleic acid strand or polymer, including DNA polymerases and RNA polymerases.
  • the polymerase used herein is a DNA polymerase. May be used is one polymerase Sequenase TM (7 DNA polymerase from bacteriophage enzyme, which sequence is modified to improve its properties - see Tabor and Richarson, Proc.Nat.Acad.Sci.USA, 84: 4767-4771 (1987), available from, for example, United States Biochemical Corporation, Cleveland, Ohio).
  • TM Sequenase
  • Other polymerases that can be used in place of Sequenase (TM) include, but are not limited to, the Klenow fragment of DNA polymerase I, AMV reverse transcriptase, and Taq polymerase. Further description of polymerases that can be used in the methods of the invention can also be found in WO05024010 and WO06120433, the entire contents of which are hereby incorporated by reference.
  • the polymerization conditions generally used are the polymerization conditions of these enzymes known in the art.
  • the polymerization conditions include a temperature in the range of from about room temperature to about 45 °C; and pH 7 buffer to 8, preferably pH 7.3 to 7.7; an enzyme concentration of from about 0.01 units / [mu] l to about 1 unit / The microliter is for a period of from about 1 to about 20 minutes, preferably from 1 to 5 minutes.
  • Typical for Sequenase TM buffer consisting of: 0.040M Tris HCl (pH7.5) 0.050M sodium chloride, 0.010M magnesium chloride, 0.010M dithiothreitol.
  • these typical conditions include a temperature in the range of from about 10 ° C to about 45 ° C, preferably from about 15 ° C to about 40 ° C; a buffer of pH 6.8 to 7.4, preferably pH 7.0 To 7.4; the enzyme concentration is from about 0.01 unit/microliter to about 1 unit/microliter, preferably from about 0.02 to about 0.15 unit/microliter, and the reaction time is from about 1 to about 40 minutes.
  • a typical buffer for the Klenow fragment of DNA polymerase I consists of 0.05 M trishydroxymethylammonium chloride, pH 7.5 0.05 M magnesium chloride, 0.05 M sodium chloride, 0.010 M dithiothreitol.
  • the term “complementary” or “substantially complementary” refers to a hybrid or base between nucleotides or nucleic acids. Pairing or duplex formation. If a nucleotide of one nucleic acid at a given position is capable of forming a hydrogen bond with a nucleotide of another nucleic acid, then the two nucleic acids are considered to be complementary to each other at that position.
  • Complementary nucleotides are typically A and T (or A and U) or C and G.
  • RNA or DNA molecules are said to be substantially complementary.
  • hybridization refers to a sufficient hydrogen bonding between complementary nucleotide or nucleotide bases, which may be, for example, Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonding, Stable and specific binding occurs between nucleic acid strands.
  • Hybridization capacity is determined according to stringent conditions, including appropriate buffer concentrations and temperatures, which allow for specific hybridization to a target nucleic acid having a fully or partially complementary region. Therefore, not all nucleotides of a nucleic acid need to be complementary.
  • a nucleic acid strand is "substantially complementary" when the nucleic acid strand hybridizes to all, part or overlapping regions of the target nucleic acid.
  • T m generally refers to a double stranded nucleic acid molecule so that half dissociated into single strands.
  • a suitable temperature control device can be used to adjust the temperature in each reaction vessel to the temperature required when the reaction solution therein reacts or is washed with the washing solution.
  • the temperature required for each reaction vessel can be different.
  • each counter The temperature required for the container may be partially the same.
  • the temperatures required for each reaction vessel may be the same.
  • the temperatures of the reaction vessels respectively containing the one or more reaction solutions in step b described herein may be the same or different from each other and may be the same or different than the temperature of the reaction vessel in step d.
  • a temperature control device can be employed to control the temperature of all of the reaction vessels.
  • different temperature control devices can be employed to separately control the temperature of each reaction vessel.
  • Suitable temperature control devices that can be used in the method of the present invention are readily available, for example, from a variety of temperature control devices available on the market, such as water baths.
  • the temperature of each reaction vessel may be determined conventionally depending on the sequencing method employed, and the temperature of each reaction vessel may be, for example, but not limited to, at least about 0 ° C, at least about 5 ° C, at least about 10 ° C, at least about 15 ° C, at least about 20 ° C, at least about 25 ° C, at least about 30 ° C, at least about 35 ° C, at least about 40 ° C, at least about 45 ° C, at least about 50 ° C, at least about 55 ° C, at least about 60 ° C or higher. In some cases, temperatures below 0 °C may be required.
  • the term “signal” includes any signal that can be detected, including but not limited to optical signals, electrical signals, electromagnetic signals, radiated signals, and the like.
  • the term “signal representing a nucleotide sequence of a nucleic acid molecule” means that the signal carries information of the nucleotide sequence and can be decoded into the nucleotide sequence of the nucleic acid molecule being sequenced. For example, where a labeled (eg, fluorescently labeled) sequencing probe is used, the signal generated by the label carries the nucleotide sequence information of the sequencing probe, and the signal is used to know the sequenced nucleic acid molecule and the sequencing The nucleotide sequence of the position where the probe is complementary to the pair.
  • a labeled eg, fluorescently labeled
  • the signal generated by the label carries the identity information of the nucleotide, and the signal is used to know the nucleotide molecule being sequenced and the nucleotide The nucleotide sequence of the position of the complementary pair.
  • the signal is a fluorescent signal.
  • the label is a fluorescent label.
  • the manner in which fluorescent labels or signals are detected is well known in the art. For example, it can be realized by a device that detects the wavelength of fluorescence. Such devices are well known in the art.
  • such a device can be a confocal scanning microscope that scans the surface of the solid support with a laser to image the fluorophore directly on the nucleic acid molecule being sequenced.
  • each of the generated signals can be observed, for example, with a sensitive 2-D detector, such as a charge coupled detector (CCD).
  • CCD charge coupled detector
  • Other techniques such as scanning near-field optical microscopy (SNOM) can also be used, for example.
  • the solid support can be used to protect the nucleic acid molecule, the labeled probe, and/or the labeled nucleotide at the time of detection prior to detecting the signal on the solid support in step c.
  • the protective reagent is contacted, for example, by soaking the solid support in a reaction vessel containing the protective reagent. Protection as used herein
  • the reagent may be, for example, an agent that prevents dehydration or degradation of the nucleic acid, a reagent that prevents fluorescence quenching, and the like.
  • Such protective agents include, but are not limited to, p-phenylenediamine (PPD), propyl gallate (NPG), 1,4-diazobicyclo-octane (DABCO), ascorbic acid (vitamin C), Vectashield, SlowFade, SlowFade Light and more.
  • PPD p-phenylenediamine
  • NPG propyl gallate
  • DABCO 1,4-diazobicyclo-octane
  • vitamin C ascorbic acid
  • Vectashield SlowFade
  • SlowFade Light and more.
  • a wash solution as used herein is any solution that is capable of washing away a substance on its solid support that is non-specifically bound thereto and does not adversely affect subsequent reactions.
  • the wash reagent contains a buffer, such as an organic salt, to maintain a stable pH of from about pH 6 to pH 9, and may also contain monovalent or divalent cations to remove non-specifically bound molecules from the solid support.
  • Exemplary washing reagents can include, for example, 100 mM Tris-HCl buffer at pH 6.5, TE buffer (Tris-HCl pH 8, 10 mM and EDTA, 1 mM), and the like.
  • the wash solutions used in the various steps in the immersion reaction schemes described herein may be the same or different, as desired, and may be adapted and selected depending on the sequencing method employed, such adjustments and selections It is within the abilities of those skilled in the art.
  • the reagents for nucleic acid sequencing may or may not be reused.
  • the one or more reaction solutions in step b and/or the regeneration solution in step d can be reused at least once, for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10 are reused. , 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 100 One or more times.
  • the method further comprises replacing or not replacing the one of step b at each iteration during the optional repeating of steps b through e of step f One or more reaction solutions and/or the regeneration solution of step d.
  • the number of times the reaction solution is repeatedly used is related to the degree of attenuation of the sequencing signal, that is, if the sequencing signal is significantly weakened after repeated use of the reaction solution to make the sequencing result inaccurate, such repeated use is inappropriate and should be replaced with a new one.
  • Reaction solution In a preferred embodiment, the one or more reaction solutions in step b can be reused at least 10 times without significantly affecting the intensity of the sequencing signal. In a preferred embodiment, the regeneration solution in step d can be reused at least 20 times without significantly affecting the intensity of the sequencing signal.
  • the time for changing the reaction solution can be determined based on the reaction time of the reaction solution with the solid support during each reaction.
  • the reaction time of the reaction solution with the solid support during each sequencing reaction depends on the reaction occurring and the reaction temperature.
  • the reaction temperature may be a temperature suitable for the function of the sequencing enzyme (eg, ligase or polymerase) used, for example, may be at least about 5 ° C, at least about 10 ° C, at least about 15 ° C, at least about 20 ° C, at least about 25 ° C, at least about 30 ° C, at least about 35 ° C, at least about 40 ° C, at least about 45 ° C, at least about 50 ° C, at least about 55 ° C, to Less than about 60 ° C or higher, such as from about 10 ° C to about 45 ° C, preferably from about 15 ° C to about 40 ° C, such temperatures can be readily determined by one skilled
  • the time of each sequencing reaction may be the time required for the sequencing enzyme to be used to incorporate the labeled probe or nucleotide into the template nucleic acid strand, such time being readily determinable by those skilled in the art, for example, may be at least 1 s, at least 5 s, at least 10 s, at least 15 s, at least 20 s, at least 25 s, at least 30 s, at least 40 s, at least 50 s, at least 1 min, at least 1.5 min, at least 2 min, at least 2.5 min, at least 3 min, at least 3.5 min, at least 4 min, At least 5 min, at least 10 min, at least 15 min, at least 20 min, at least 30 min or longer.
  • the reaction temperature may be a temperature suitable for the reaction to occur, for example, At least about 5 ° C, at least about 10 ° C, at least about 15 ° C, at least about 20 ° C, at least about 25 ° C, at least about 30 ° C, at least about 35 ° C, at least about 40 ° C, at least about 45 ° C, at least about 50 ° C, At least about 55 ° C, at least about 60 ° C or higher, such temperature can be readily determined by one skilled in the art based on the method or reagent used to remove the label from the labeled probe or nucleotide.
  • the time of such a reaction can be readily determined by one skilled in the art, for example, can be at least 1 s, at least 5 s, at least 10 s, at least 15 s, at least 20 s, at least 25 s, at least 30 s, at least 40 s, at least 50 s, at least 1 min, at least 1.5 min. At least 2 min, at least 2.5 min, at least 3 min, at least 3.5 min, at least 4 min, at least 5 min, at least 10 min, at least 15 min, at least 20 min, at least 30 min or longer.
  • the sequencing reaction in step b is at least about 5 ° C, at least about 10 ° C, at least about 15 ° C, at least about 20 ° C, at least about 25 ° C, at least about 30 ° C, at least about 35 ° C, at least about 40 ° C, In one embodiment carried out at a temperature of at least about 45 ° C, at least about 50 ° C, at least about 55 ° C, at least about 60 ° C or higher, it may be less than or equal to about 1 hour, less than or equal to about 2 hours, less than or Equal to about 3 hours, less than or equal to about 4 hours, less than or equal to about 5 hours, less than or equal to about 6 hours, less than or equal to about 7 hours, less than or equal to about 8 hours, less than or equal to about 9 hours, less than or equal to About 10 hours, less than or equal to about 12 hours, less than or equal to about 15 hours, less than or equal to about 18 hours, less than or equal to about 20 hours, less than or equal to about 22 hours
  • the time interval for replacing one or more of the reaction solutions of step b is less than about 8 hours, preferably About 4 hours. In a preferred embodiment, when the reaction temperature of the one or more reaction solutions of step b is below about 45 ° C, the time interval for replacing the one or more reaction solutions of step b is less than about 24 The hour is preferably about 10 hours.
  • the immersion reaction scheme of the present invention differs at least in comparison to the flow path type reaction scheme employed by the existing second generation sequencing technology in that the method of the present invention allows simultaneous soaking of more than one solid support, thereby greatly improving Sequencing flux.
  • the method of the present invention allows for simultaneous soaking of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more Solid support.
  • Such more than one solid support that is simultaneously soaked is referred to herein as a batch of solid support.
  • a batch of solid support may also consist of only one solid support.
  • the immersion reaction scheme of the present invention enables nested batch processing of solid supports.
  • the nested batch processing includes, during the nucleic acid sequencing of the first batch of solid supports using the immersion protocol of the present invention, starting another batch of solid support at the end of one sequencing cycle Nucleic acid sequencing, and after the second batch of solid support begins nucleic acid sequencing, nucleic acid sequencing can also be initiated on the third batch of solid support; similarly, nucleic acid is started in the N-1 batch of solid support After sequencing, nucleic acid sequencing can be initiated on the Nth batch of solid support, with the condition that 1 ⁇ N ⁇ t cycles / t rate limit , and N is an integer value, where t cycle is the total time from step b to step e ( The total time referred to herein as a sequencing cycle, therefore each "sequencing cycle" as used herein begins with soaking a solid support to one or more reaction solutions of step b in the immersion reaction scheme of the present invention and Ending the washing of the solid support in step e), the rate
  • N 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 may be performed at most in one sequencing cycle. Nested batch processing of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more batch solid supports.
  • the time of other programs in the sequencing cycle can be adjusted based on the time required for the rate limiting program such that the total time of the sequencing cycle is exactly an integral multiple of the time required for the rate limiting program.
  • the immersion reaction scheme as described herein further comprises: after soaking the first batch of solid support in one or more reaction solutions of step b and before the end of step e Soaking a second batch of solid support on which the nucleic acid molecule is immobilized in one or more reaction solutions of step b, and subsequently operating the second batch of solid support according to steps b through f;
  • the process is iteratively repeated N-1 times, where 1 ⁇ N ⁇ t cycles / t rate limit , and N is an integer value, where t cycle is the total time of step b to step e, and t speed limit is at step b
  • the time required for each soaking, washing, detection of step c, soaking of step d, and washing of step e takes the longest time.
  • step b "iteratively iteratively” means that a nucleic acid is optionally immobilized thereon after immersing the first batch of solid support in one or more reaction solutions of step b and before the end of step e Soaking a second batch of solid support of the molecule in one or more reaction solutions of step b, followed by immersing the second batch of solid support in one or more reaction solutions of step b and Before the end of step e, the third batch of solid support on which the nucleic acid molecule is immobilized is optionally soaked in one or more reaction solutions of step b, followed by soaking the third batch of solid support After the one or more reaction solutions of step b are followed and before the end of step e, the fourth batch of solid support on which the nucleic acid molecules are immobilized is optionally soaked in one or more reaction solutions of step b And so on, until the process is iteratively repeated for the solid support of the N+1th batch, where 1 ⁇ N ⁇ t cycles / t rate limit
  • N 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more batches of solid support.
  • humectants include, but are not limited to, polyols such as glycerin, propylene glycol, 1,3-butylene glycol, sorbitol, polyethylene glycol, and the like. It should be understood that the addition of such a humectant should not result in the reaction solution and/or the wash solution, the solid support, and the nucleic acid molecules on the solid support. Any adverse effects are not and do not significantly increase the amount of reagent carried on the solid support. In a preferred embodiment, an appropriate amount of glycerin is added to the one or more reaction solutions of step b, the regeneration solution of step d, and/or the wash solution.
  • the amount of humectant added can be any effective amount that will keep the surface of the solid support moist. It should be understood that such amounts should not adversely affect the reaction solution and/or wash solution, solid support, and nucleic acid molecules on the solid support, and will not significantly increase the amount of reagent carried on the solid support. .
  • the amount of humectant added may be, for example, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, based on the volume of the reaction solution and/or wash solution, At least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, An amount of at least about 14%, at least about 15%, at least about 20% or more.
  • about 5% to 10% glycerol is added to the one or more reaction solutions of step b, the regeneration solution of step d, and/or the wash solution.
  • the immersion reaction scheme provided by the present invention further comprises adding an appropriate amount of the solid support in the one or more reaction solutions of step b, the regeneration solution of step d, and/or the washing solution.
  • the addition of such reagents may help to avoid the reaction solution and/or wash solution remaining on the solid support when the solid support is removed from the reaction solution and/or wash solution.
  • the drying caused by gravity acting away from the upper end of the solid support.
  • the upper end of the solid support means the end that is first exposed to the air when the solid support is removed from the solution.
  • such an agent can be, for example, a surfactant.
  • surfactants may include, but are not limited to, Tween-20, stearic acid, lauric acid, sodium lauryl sulfate, sodium dioctyl succinate (Aloso-OT), dodecylbenzenesulfonate Sodium, sodium glycocholate, benzalkonium chloride, benzalkonium bromide, lecithin, fatty acid glycerides, and the like. It should be understood that the addition of such agents, such as surfactants, should not have any adverse effect on the reaction solution and/or wash solution, solid support, and nucleic acid molecules on the solid support, and will not significantly increase the solid support.
  • the amount of reagent carried on is added to one or more of the reaction solutions of step b, the regeneration solution of step d, and/or the wash solution.
  • the amount of such an agent added, such as a surfactant may be such that it can retain the reaction solution and/or the washing solution remaining on the solid support when the solid support is taken out from the reaction solution and/or the washing solution. Any effective amount. It should be understood that such amounts should not adversely affect the reaction solution and/or wash solution, solid support, and nucleic acid molecules on the solid support, and will not significantly increase the amount of reagent carried on the solid support. .
  • the amount of agent can be, for example, at least about 0.001%, at least about 0.005%, at least about 0.01%, at least about 0.02%, at least about 0.03%, at least about 0.04%, at least about 0.05, based on the volume of the reaction solution and/or wash solution. %, at least about 0.06%, at least about 0.07%, at least about 0.08%, at least about 0.09%, at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6.
  • from about 0.05% to about 1% Tween-20 is added to one or more of the reaction solutions of step b, the regeneration solution of step d, and/or the wash solution.
  • the immersion reaction scheme provided by the present invention further includes optimizing the rate of movement of the solid support between the various reaction vessels to minimize the time during which the solid support is exposed to the air. It should be understood that such rate optimization should not cause any adverse effects on the reaction solution, the solid support, and the nucleic acid molecules on the solid support.
  • optimizing the rate of movement of the solid support between the various reaction vessels includes optimizing the rate at which the solid support is removed from the solution or reagent in the reaction vessel such that the solid support is from solution or reagent
  • the removal is as fast as possible without any adverse effects on the reaction solution, the solid support, and the nucleic acid molecules on the solid support due to faster withdrawal.
  • faster removal of the solid support from the solution or reagent helps to avoid drying of its surface due to evaporation and hydrophobicity, while also helping to reduce the solution or reagent carried on its surface.
  • Amount thereby reducing contamination of subsequent procedures, however, too fast a rate of extraction may cause damage to the solid support and the nucleic acid molecules immobilized thereon, thereby affecting the quality of the sequencing, thus requiring a solid support depending on the particular use,
  • the nucleic acid molecule immobilized thereon and the reaction solution or reagent optimize the extraction rate.
  • the rate at which the solid support is removed from the solution or reagent in the reaction vessel can be, for example, but not limited to, at least 1 mm/s, at least 5 mm/s, at least 10 mm/s, at least 15 mm/s, at least 20 mm/ s, at least 25 mm/s, at least 30 mm/s, at least 40 mm/s, at least 50 mm/s or higher.
  • the rate at which the solid support is removed from the solution or reagent in the reaction vessel can be, for example, but not limited to, no greater than 50 mm/s, no greater than 40 mm/s, no greater than 30 mm/s, no Above 20mm/s, not above 10mm/s or lower.
  • the rate at which the solid support is removed from the solution or reagent in the reaction vessel can be, for example, about 20 mm/s.
  • optimizing the rate of movement of the solid support between the various reaction vessels includes optimizing the rate of movement of the solid support after removal from the solution or reagent in the reaction vessel to expose the solid support to the air The time in is minimized. Not subject to theory, it is considered to be in the solution or reagent from the reaction vessel After removal, moving the solid support as quickly as possible helps to avoid drying of its surface due to evaporation and hydrophobicity.
  • the rate of movement of the solid support after removal from the solution or reagent in the reaction vessel is optimized such that the solid support is exposed to air for less than or equal to 100 ms, less than or equal to 200 ms, less than or equal to 500ms, less than or equal to 1s, less than or equal to 1.5s, less than or equal to 2s, less than or equal to 2.5s, less than or equal to 3s, less than or equal to 3.5s, less than or equal to 4s, less than or equal to 4.5s, less than or equal to 5s Less than or equal to 5.5s, less than or equal to 6s, less than or equal to 6.5s, less than or equal to 7s, less than or equal to 7.5s, less than or equal to 8s, less than or equal to 8.5s, less than or equal to 9s, less than or equal to 9.5s , less than or equal to 10 s, less than or equal to 15 s, less than or equal to 20 s, less than or equal to 30 s, less than or equal
  • the present invention provides a method of sequencing a nucleic acid using a immersion reaction protocol comprising the steps of:
  • reaction solutions immersing the first batch of solid support in one or more reaction solutions to generate a signal representative of the nucleotide sequence of the nucleic acid molecule on the solid support, the one or more The reaction solutions are each placed in separate reaction vessels,
  • the first batch of the solid support is washed to remove the residual reaction solution on the solid support.
  • a humectant such as glycerin, and/or a surfactant, such as Tween-20, is optionally added to the one or more reaction solutions of step b, the regeneration solution of step d and/or the wash solution, and optionally
  • a surfactant such as Tween-20
  • the rate of movement of the solid support between the reaction vessels is optimized to minimize the time during which the solid support is exposed to the air,
  • the method further comprises iteratively iteratively processing the solid support of the second batch to the Nth batch (ie, nested batch processing) as described herein, wherein 1 ⁇ N ⁇ t cycles / t limits Speed , and N is an integer value.
  • a reaction solution referred to herein as a first reaction solution
  • the first reaction solution comprising a ligase and a label (eg, fluorescent) a labeled probe, optionally comprising an anchor probe
  • the nucleic acid molecule is used as a template for performing a nucleic acid ligation reaction between the anchor probe and the sequencing probe
  • the first batch of solid support is removed from the first reaction solution, and the first batch of solid support is washed to remove the first reaction solution remaining on the solid support.
  • a humectant such as glycerin, and/or a surfactant, such as Tween-20, is added to the first reaction solution, the regeneration solution and/or the wash solution, and optionally a solid support is provided in each reaction vessel.
  • a humectant such as glycerin
  • a surfactant such as Tween-20
  • the method further comprises iteratively iteratively processing the solid support of the second batch to the Nth batch (ie, nested batch processing) as described herein, wherein 1 ⁇ N ⁇ t cycles / t limits Speed , and N is an integer value.
  • the invention provides a method of sequencing a nucleic acid using a immersion reaction protocol, the immersion reaction scheme comprising the steps of:
  • the first batch of solid support is removed from the first reaction solution, and the first batch of solid support is washed to remove the first reaction solution remaining on the solid support.
  • a humectant such as glycerin, and/or a surfactant, such as Tween-20, is added to the first reaction solution, the regeneration solution and/or the wash solution, and optionally a solid support is provided in each reaction vessel.
  • a humectant such as glycerin
  • a surfactant such as Tween-20
  • the method further comprises iteratively iteratively processing the solid support of the second batch to the Nth batch (ie, nested batch processing) as described herein, wherein 1 ⁇ N ⁇ t cycles / t limits Speed , and N is an integer value.
  • the invention also provides an apparatus for use in a immersed reaction scheme as described herein to sequence a nucleic acid molecule, the apparatus comprising:
  • reaction vessels each comprising a reaction solution for contacting a nucleic acid molecule to produce a signal representative of the nucleotide sequence of the nucleic acid molecule
  • reaction vessel comprising a reaction solution for eliminating said signal from a solid support
  • a temperature control device for example a water bath for controlling the temperature of the reaction vessels in steps a to c.
  • FIG. 1 shows a flow diagram (showing one sequencing cycle) of two exemplary embodiments of the immersion reaction scheme of the present invention, wherein an exemplary embodiment uses a protective reagent that protects nucleic acids and labels upon detection.
  • Figure 2 shows a comparison of the soaking reaction protocol of the present invention with the sequencing data of BGISEQ-500 SE50.
  • Figure 3 shows the sequencing results of the improved immersion reaction protocol of the present invention.
  • Figure 4 shows that the addition of a certain amount of surfactant to the polymerization reagent can improve the drying of the upper end of the chip.
  • the left side of the figure shows the lower end of the chip, and the right side of the figure shows the top of the chip.
  • Condition 1 5%-10% glycerol + fast moving chip was added to the first reaction solution;
  • Condition 2 10% glycerol + fast moving chip was added to the first reaction solution + 0.05%-1 was added to the first reaction solution %Tween-20.
  • Example 1 Nucleic acid sequencing using a immersion reaction protocol.
  • MGIEasy TM DNA library preparation kit (Shenzhen Hua Taizhi Ltd. made) to a standard strain of E. coli DNA extraction was prepared starting library for the sequencing, loaded onto a sequencing chip.
  • the reagents in the BGISEQ-500 high-throughput kit (SE50 V3.0, Shenzhen Huada Zhizhi Technology Co., Ltd., item number PF-UM-PEV30) were used and according to the flow chart of Figure 1A (described herein).
  • An exemplary embodiment of the immersion reaction protocol performs 10 cycles of sequencing of the obtained nucleic acid molecules on the chip, wherein three chips are simultaneously immersed.
  • MGIEasy TM DNA library prepared using the kit standard strain of E. coli DNA extraction was prepared starting library for the sequencing, loaded onto sequencing chips with high throughput BGISEQ-500 kit (SE50 V3.0) of the sequencing reagents The sequencing reaction was performed on a BGISEQ-500 sequencer as a control.
  • Q30 refers to an error in sequencing 1000 bases.
  • the ESR is the sequencing index of the BGISEQ-500 sequencer, which means the ratio of effective DNA nanospheres.
  • MappingRate refers to the ratio of sequencing data to the reference genome.
  • BarcodeSplitRate refers to the barcode resolution rate. The larger the number, the better the sequencing quality.
  • the quality of the sequencing data obtained by the immersion reaction scheme of the Ming is basically similar to the quality of the sequencing data obtained by the BGISEQ-500 sequencer (the difference is within 5%, and the barcode resolution is better than that of the BGISEQ-500 sequencer).
  • Example 2 Using the experimental procedure similar to that of Example 1, the chip was subjected to 15 sequencing cycles using different immersion reaction schemes.
  • Immersion scheme 1 The same experimental procedure as in Example 1, 15 sequencing cycles were performed on the chip;
  • Immersion scheme 2 On the basis of the immersion scheme 1, 5-10% glycerol was added to the polymerization reagent and the chip was rapidly moved at 20 mm/s during the experiment;
  • the chip was sequenced using a BGISEQ-500 sequencer as a control.
  • A represents the fluorescence of detecting the A base.
  • C indicates a fluorescent signal detecting a C base
  • G indicates a fluorescent signal detecting a G base
  • T indicates a fluorescent signal detecting a T base
  • AVG indicates an average value of detecting a fluorescent signal of four bases.
  • the signal value at the upper end of the chip is significantly reduced; after adding a certain amount of Tween-20, the signal value at the upper end of the chip is not significantly different from other regions, and the uniformity is good) (as shown in Fig. 4).
  • Example 3 Nested batch processing of the immersion reaction scheme of the present invention.
  • the nested batch processing of the chip is applied in the experimental procedure of Example 1, and when the first group of three chips is subjected to polymerization reaction and fluorescent labeling is detected, The other three chips of the two groups were immersed in the sequencing reaction reagent (ie, the first reaction solution) to start the sequencing reaction.
  • the sequencing reaction reagent ie, the first reaction solution
  • the polymerization reagent was changed every 10 sequencing cycles, and the regeneration reagent was replaced every 20 sequencing cycles.
  • the cost is 25% of the same type of sequencing of the BGISEQ-500 sequencer. A total of 60 complete sequencing cycles of all two sets of chips were completed in 2 hours.
  • the sequencing throughput of the present invention is more than five times that of the BGISEQ-500 sequencer or the HiSeq-2500 platform during the same sequencing time, and the throughput can be further increased by increasing the number of batches processed by nested batches. cut costs.
  • Example 1 The sequencing reagents that have been used in Example 1 were reused for the sequencing procedure described in Example 1, and the sequencing data was analyzed as described in Example 1 and compared to the unused reagents. The results of the comparison are shown in Table 1 below, showing that the used reagents can be sequenced normally.
  • Example 5 Stability study of reagents used in the immersion reaction scheme of the present invention.
  • sequencing reagent (sequencing reagent reference example 1, including polymerization reagent, washing reagent 1, washing reagent 2, regeneration reagent) was placed in a water bath at 45 ° C and 55 ° C for different times, including 4 hours, 8 hours, 24 hours.
  • the analysis results of the quality of the sequencing data are shown in Table 2. It can be seen that after treatment at 55 ° C for 4 hours or after 45 ° C for 8 hours, the sequencing reagent is still stable, and the sequencing quality is the same as that of the unheated sequencing reagent.
  • the photo group is basically similar. Therefore, based on this result, the replacement cycle of the sequencing reagent in the immersion reaction scheme can be determined to be 4 hours at 55 ° C or 10 hours at 45 ° C.

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Abstract

本发明提供一种使用浸泡式反应方案对核酸进行测序的方法，所述浸泡式反应方案包括使其上固定有核酸分子的固体支持物依次浸泡在不同的反应容器中以实现核酸测序。

Description

核酸测序方法 技术领域
本发明涉及核酸测序领域。具体地，本发明涉及使用浸泡式反应方案对核酸进行测序的方法，所述浸泡式反应方案包括使其上固定有核酸分子的固体支持物依次浸泡在不同的反应容器中以实现核酸测序。
背景技术
第二代测序技术基于第一代Sanger测序技术发展而来，具有低成本，高通量，自动化等特征，极大地推进了基因测序产业的发展。二代测序技术目前已经广泛应用于全基因组测序，转录组测序，宏基因组测序等，是分析生物的进化与分类，研究癌症，自闭症等疾病相关基因，以及进行体外诊断等的有力工具，促进了人们对于生命科学的进一步了解，也推动了健康产业的发展。
现有的第二代测序技术主要包括Illumina的SBS测序技术(Sequencing-by-Synthesis，合成法测序)，Life Technologies公司的半导体测序技术，和BGI的联合探针锚定聚合技术(cPAS)。SBS测序技术使用同时具有两个修饰部分(荧光标记和阻断基团)的dNTP(dATP,dCTP,dGTP,dTTP)，其中dATP、dCTP、dGTP、dTTP分别携带不同的荧光标记。由于阻断基团的存在，在发生聚合反应时，在每个核酸模板上只会加上一个互补的dNTP，检测该dNTP所携带的荧光标记即可确定该添加的互补dNTP的身份，随后用试剂对可逆阻断基团和荧光标记进行切除(被称为再生反应)，进行下一步聚合反应(P085，基因组学，2016年第一版，杨焕明著)。半导体测序技术利用在模板链延伸时聚合上去的dNTP释放的焦磷酸导致的pH值变化，通过芯片底部的传感器检测这一变化从而获得DNA链序列信息(P083，基因组学，2016年第一版，杨焕明著)。美国ABI公司开发的第二代测序技术方案SOLiD，则是采用SBL测序法(Sequencing-by-Ligation，连接法测序)来进行高通量测序。SOLiD的SBL测序法使用连接酶和8碱基单链荧光探针混合物，配合不同引物的循环连接，能够实现35-70nt之间的读长，其优势是准确度高(P093，基因组学，2016年第一版，杨焕明著)。联合探针锚定聚合技术(combinatorial probe–anchor synthesis，cPAS)使用DNA分子锚和荧光探针在DNA纳米球(DNB)上进行聚合，随后高分辨率成像系统对光信号进行采集，光信号经过数字化处理后即可获得待测序列；其中，DNB通过线 性扩增增强信号，降低单拷贝的错误率(参见BGISEQ-500测序平台)。
现有二代测序技术的生化反应部分主要采用流道式系统来进行。典型的流道式系统可参见WO2007123744，其使用包括流通池的固体衬底，流通池包括多核苷酸附接于其中的一个或多个流体通道，并通过导流系统将荧光标记试剂可控地流动通过流体通道从而与多核苷酸接触。以illumina的HiSeq 2500平台为例，该平台使用流动槽，将测序反应试剂在特定时间点的特定位置从制冷设备泵入流动槽，在一套复杂的流体控制设备的控制下流经测序芯片，并且辅助以精细的温控设施，确保在尽可能短的时间内让生化反应得以充分进行(
Figure PCTCN2017095520-appb-000001
2500 System Guide，illumina，2015)。
现有二代测序方法需要精确的温度控制和流体控制，成本较高，且容易出现反应不均匀现象。试剂都是一次性使用，利用率较低，进一步提高了测序成本。采用流道式方案的现有二代测序平台如HiSeq 2500等还存在通量较低的情况，每一台的通量小于1WGS/天，参考：https://www.illumina.com.cn/systems/hiseq_2500_1500/performance_specifications.html。
因此，本领域迫切需要新的高通量的、低成本的二代测序方法。
发明内容
针对现有二代测序方法存在的问题，本发明提供了使用浸泡式反应方案对核酸进行测序的方法，所述浸泡式反应方案包括使其上固定有核酸分子的固体支持物依次浸泡在不同的反应容器中以实现核酸测序。如本文所用的术语“浸泡式反应方案”是相对于现有技术使用的流道式系统而提及的，其意指在对核酸进行测序的过程中，使待测序的核酸移动至与反应试剂接触，而非使用流动通道使反应试剂移动至与核酸接触。所述浸泡式反应方案至少具有以下优势：1.生化试剂重复利用；2.扩展性好，使用灵活，能够大幅提高通量；3.不需要复杂的温度和流体控制系统，反应较均匀；4.显著降低基因测序成本。
在本文所述的方法中，核酸测序可以利用多种测序方法实现。测序方法的实例包括但不限于电泳、合成法测序、连接法测序、联合探针锚定聚合测序、杂交测序、单分子测序和实时测序方法。在一些实施方案中，确定靶核酸的核苷酸序列的过程可以是自动化过程。
应当理解，任何测序过程都可以结合到本文所述的方法中。此外，应当理解，可以容易地实施其它已知的测序过程，用于本文所述的方法。
因此，在一个实施方案中，本发明提供了使用浸泡式反应方案对核酸进行测序的方法，所述浸泡式反应方案包括以下步骤：
a.提供其上固定有核酸分子的第一批次的固体支持物，
b.将所述第一批次的固体支持物浸泡在一种或多种反应溶液中，以在固体支持物上产生代表所述核酸分子的核苷酸序列的信号，所述一种或多种反应溶液各自置于分开的反应容器中，
其中在将所述第一批次的固体支持物从每一种反应溶液中移出后，洗涤所述第一批次的固体支持物，以除去固体支持物上残留的反应溶液，
c.检测所述第一批次的固体支持物上的所述信号，
d.将所述第一批次的固体支持物浸泡在置于另外的反应容器中的再生溶液中，以消除固体支持物上的所述信号，
e.从再生溶液中移出所述第一批次的固体支持物，洗涤所述第一批次的固体支持物，以除去固体支持物上残留的再生溶液，
f.任选地，重复步骤b至e或b至c。
在一个实施方案中，步骤b至e可重复一次或多次，例如重复1、2、3、4、5、10、15、20、30、40、50、60、70、80、90、100或更多次。
在某些实施方案中，洗涤通过将固体支持物浸泡在包含洗涤溶液的反应容器中进行。
如本文所用，所述浸泡包括使得固体支持物上固定的所有核酸分子与反应容器中的试剂或溶液接触的任何方式。固体支持物可以例如以垂直、倾斜或水平的方式部分或全部浸泡在反应容器中，条件是固体支持物上固定的所有核酸分子与反应容器中的试剂接触。
如本文所用的被测序的“核酸分子”可以是任何类型的核酸，例如核酸可以是脱氧核糖核酸(DNA)、核糖核酸(RNA)或由核苷酸类似物制成的DNA或RNA的类似物，其可以与“多核苷酸”互换使用。核酸可以是单链的、双链的或含有单链和双链序列两者。核酸分子可以来源于双链DNA(dsDNA)形式(例如，基因组DNA、PCR和扩增产物等)，或者可以来源于如DNA(ssDNA)或RNA的单链形式并且其可以转化为dsDNA形式， 并且反之亦然。在一些实施方案中，被测序的核酸可以是单分子的形式(可以是天然分子、修饰分子如标记的分子，或包括核苷酸类似物的核酸)、序列的多联体(例如DNA纳米球)，等等)，可以扩增(例如扩增为多联体、扩增为多个具有相同或相似序列的个体分子，等等)，和/或可以为任何其它形式。核酸分子的准确序列可以是已知的或未知的。以下是核酸的示例性实例：基因或基因片段(例如，探针、引物、EST或SAGE标签)、基因组DNA、基因组DNA片段、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、核糖酶、cDNA、核酸文库、重组多核苷酸、合成多核苷酸、分枝多核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、任何上述序列的核酸探针、引物或扩增拷贝。
核酸可以包括核苷酸或核苷酸类似物。核苷酸通常含有糖、核碱基和至少一个磷酸基。核苷酸可以是无碱基的(即，缺少核碱基)。核苷酸包括脱氧核糖核苷酸、修饰的脱氧核糖核苷酸、核糖核苷酸、修饰的核糖核苷酸、肽核苷酸、修饰的肽核苷酸、修饰磷酸盐糖主链核苷酸及其混合物。核苷酸的实例包括(例如)腺苷一磷酸(AMP)、腺苷二磷酸(ADP)、腺苷三磷酸(ATP)、胸苷一磷酸(TMP)、胸苷二磷酸(TDP)、胸苷三磷酸(TTP)、胞苷酸(CMP)、胞苷二磷酸(CDP)、胞苷三磷酸(CTP)、鸟苷一磷酸(GMP)、鸟苷二磷酸(GDP)、鸟苷三磷酸(GTP)、尿苷一磷酸(UMP)、尿苷二磷酸(UDP)、尿苷三磷酸(UTP)、脱氧腺苷酸(dAMP)、脱氧腺苷二磷酸(dADP)、脱氧腺苷三磷酸(dATP)、脱氧胸腺嘧啶核苷一磷酸(dTMP)、脱氧胸腺嘧啶核苷二磷酸(dTDP)、脱氧胸苷三磷酸(dTTP)、去氧胞二磷(dCDP)、脱氧胞苷三磷酸(dCTP)、脱氧鸟苷一磷酸(dGMP)、脱氧鸟苷二磷酸(dGDP)、脱氧鸟苷三磷酸(dGTP)、脱氧尿苷一磷酸(dUMP)、脱氧尿苷二磷酸(dUDP)和脱氧尿苷三磷酸(dUTP)。还可以在本文所述的方法中使用包含修饰的核碱基的核苷酸类似物。无论是具有天然主链还是类似结构，可以包含在多核苷酸中的示例性修饰的核碱基包括(例如)肌苷、黄嘌呤、次黄嘌呤、异胞嘧啶、异鸟嘌呤、2-氨基嘌呤、5-甲基胞嘧啶、5-羟甲基胞嘧啶、2-氨基腺嘌呤、6-甲基腺嘌呤、6-甲基鸟嘌呤、2-丙基鸟嘌呤、2-丙基腺嘌呤、2-硫脲嘧啶、2-硫胸腺嘧啶、2-硫胞嘧啶、15-卤代脲嘧啶、15-卤代胞嘧啶、5-丙炔基尿嘧啶、5-丙炔基胞嘧啶、6-偶氮尿嘧啶、6-偶氮胞嘧啶、6-偶氮胸腺嘧啶、5-尿嘧啶、4-硫尿嘧啶、8-卤代腺嘌呤或鸟嘌呤、8-氨基腺嘌呤或鸟嘌呤、8-硫腺嘌呤或鸟嘌呤、8-硫烷基腺嘌呤或鸟嘌呤、8-羟基腺嘌呤或鸟嘌呤、5-卤素取代的尿嘧啶或胞嘧啶、7-甲基鸟嘌呤、7-甲基腺嘌呤、8-氮杂鸟嘌呤、 8-氮杂腺嘌呤、7-去氮鸟嘌呤、7-去氮腺嘌呤、3-去氮鸟嘌呤、3-去氮腺嘌呤等。如本领域中已知的，某些核苷酸类似物不能引入多核苷酸，例如，核苷酸类似物，如腺苷5′-磷酰硫酸。
在本发明的具体实施方案中被测序的核酸分子可以具有任意长度。一般说来，有用的核酸的示例性长度包括(例如)至少约5、10、20、30、40、50、60、70、80、90、100、150、200、300、400、500、1,000、5,000或10,000、100,000个核苷酸或更长。作为另外一种选择或者另外，所述长度可以不长于1,000,000、100,000、10,000、1,000、100个核苷酸或更少。核酸分子的长度还可以包括以上示例性数目之间的所有整数。因此，可以使用本发明所述的方法测序的核酸可以(例如)在短多核苷酸、片段、cDNA、基因和基因组片段的范围内。
在本发明的具体实施方案中进行测序的核酸分子可以为任意数量，例如可以是2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90或100个或者更多个相同或不同的核酸分子。进行测序的核酸分子的数量还可以是例如200、300、400、500、1000、5000、10000、50000、1x105、2x105、3x105、4x105、5x105、6x105、7x105、8x105、9x105、1x106、2x106、3x106、4x106、5x106、6x106、7x106、8x106、9x106或1x107个或更多个相同或不同的核酸分子。进行测序的核酸分子的数量还可以包括以上示例性数目之间的所有整数。
核酸可以从任何来源获得。例如，核酸可以由从一种生物获得的核酸分子制备，或者由从包括一种或多种生物的天然来源获得的核酸分子群制备。核酸分子的来源包括但不限于细胞器、细胞、组织、器官或生物体。可以用作核酸分子来源的细胞可以是原核的(例如细菌)；或真核的，如真菌(例如酵母)、植物、原生动物和其它寄生虫，和动物(包括昆虫、线虫)，和哺乳动物(例如，大鼠、小鼠、猴、非人类灵长类动物和人类)；或者核酸分子可以来源于病毒。
在一些实施方案中，核酸可以从特定生物来源获得。在一个优选的实施方案中，核酸是从人获得的人核酸，例如，人组织的样品。在另一个优选的实施方案中，核酸是人线粒体核酸。在另一个优选的实施方案中，核酸可以从宏基因组样品获得。在其它实施方案中，核酸可以从不再包含活生物的环境来源获得。
如本文所用的，术语“固定”意指经由共价键或非共价键直接或间接附接至固体支持物。在本公开内容的某些实施方案中，可以使用共价附接，但是通常仅需要的是 在期望使用固体支持物的条件下(例如在需要核酸扩增和/或测序的应用中)核酸保持固定或附接至固体支持物。通常，待用作捕获引物或扩增引物的寡核苷酸(例如本发明的具有部分双链结构的引物)被固定，使得3'末端对于酶促延伸是可利用的并且该引物序列的至少一部分能够杂交至互补核酸序列。固定可以经由杂交至表面附接的寡核苷酸发生，在这种情况下固定的寡核苷酸或多核苷酸可以为3'-5'方向。另一种非共价附接的方式可以是通过氨基化修饰将核酸结合蛋白质结合在固体支持物上，并通过核酸结合蛋白质捕获核酸分子。可选地，固定可以通过除碱基配对杂交之外的其他方式发生，例如上文描述的共价附接。核酸与固体支持物附接方式的非限制性示例包括核酸杂交、生物素链霉亲和素结合、巯基结合、光活化结合、共价结合、抗体-抗原、经由水凝胶或其他多孔聚合物的物理限制等。用于将核酸固定在固体支持物上的各种示例性方法可参见例如G.Steinberg-Tatman等人,Bioconjugate Chemistry 2006,17,841-848；Xu X.等人Journal of the American Chemical Society 128(2006)9286-9287；美国专利申请US 5639603、US 5641658、US2010248991；国际专利申请WO 2001062982、WO 2001012862、WO 2007111937、WO0006770，为了所有目的，特别是为了与制备形成其上固定有核酸的固体支持物有关的全部教导，以上文献均通过引用全文并入本文。
如本文所使用的，术语“固体支持物”意指核酸可以与其附接的任何不可溶的基底或基质，诸如例如，乳胶珠、葡聚糖珠、聚苯乙烯表面、聚丙烯表面、聚丙烯酰胺凝胶、金表面、玻璃表面、芯片、传感器、电极和硅晶片。固体支持物的表面可以是任何所需形状，所述形状包括，例如，适合用于特定应用的平面的、球形的或多孔的。例如，固体支持物可以是平面的玻璃表面。固体支持物还可以被安装在流动池的内部，以允许与多个试剂的溶液相互作用。
在某些实施方案中，固体支持物可以包括惰性基底或基质，该惰性基底或基质已经例如通过应用中间材料的层或涂层被化学官能化，所述中间材料具有允许共价附接至多核苷酸的反应性基团。中间材料可以经由共价键或非共价键直接或间接附接至固体支持物。作为用于非共价附接至固体支持物的非限制性实例，这类支持物可以包括在惰性基底例如玻璃上的聚丙烯酰胺水凝胶层。在这类实施方案中，多核苷酸可以直接共价附接至中间层(例如，水凝胶)，但是中间层本身可以非共价附接至其他基底或基质(例如玻璃基底)层。
如本文所用，术语“反应容器”意指能够容纳反应溶液的任何装置，包括但不限于槽、渠、孔、试管、杯、皿等。反应容器可以具有任何合适的形状，例如反应容器可以是方形、球形、锥形、圆柱体形、不规则形状等。反应容器可以具有任何合适的尺寸，例如其尺寸可以根据所需要容纳的反应溶液的体积而进行任意的调整，例如其尺寸可以允许容纳至少1μL、至少5μL、至少10μL、至少20μL、至少50μL、至少100μL、至少1mL、至少5mL、至少10mL、至少20mL、至少50mL、至少100mL、至少200mL、至少500mL、至少1L或更多的反应溶液，或者其尺寸可以允许容纳至多1μL、至多5μL、至多10μL、至多20μL、至多50μL、至多100μL、至多1mL、至多5mL、至多10mL、至多20mL、至多50mL、至多100mL、至多200mL、至多500mL、至多1L或更多的反应溶液。反应容器可由任何合适的材料制成，例如可由玻璃、金属诸如不锈钢、聚合材料如塑料等制成。应理解，反应容器的材料应不会不利地影响反应溶液的反应活性。
如本文所用，术语“反应溶液”是指适合用于本文所述的浸泡式反应方案的任何测序方法中所使用的试剂。如本文所述，这样的测序方法可以包括但不限于电泳测序、合成法测序、连接法测序、联合探针锚定聚合测序、杂交测序、单分子测序和实时测序方法。因此，在本发明的浸泡式反应方案中步骤b中所使用的一种或多种反应溶液以及步骤d中所使用的再生溶液取决于所采用的测序方法。根据测序方法确定在本发明的浸泡式反应方案中步骤b中所使用的一种或多种反应溶液以及步骤d中所使用的再生溶液在本领域技术人员的能力范围内。
因此，在一个具体的实施方案中，本发明涉及使用本文所述的浸泡式反应方案对核酸分子进行连接法测序的方法。在这样的实施方案中，步骤b的一种或多种反应溶液包含用于在连接法测序中在被测序的核酸分子上产生连接产物并生成代表所述核酸分子的核苷酸序列的信号的试剂，并且步骤d的再生溶液包含用于从被测序的核酸分子上消除所述信号并使得能够起始下一个连接测序反应的试剂。
如本文所用的连接法测序是本领域熟知的各种连接法测序方法。基本地，连接法测序涉及经标记(例如荧光标记)的测序探针和锚定探针(在SOLiD测序中也被称为“引物”)与DNA链的杂交和连接。测序探针包含一或两个固定已知的测序碱基(单碱基测序探针或双碱基测序探针)和一系列简并或通用碱基，其使得测序探针与核酸模板之间进行互补配对。锚定探针序列与核酸模板上的衔接子序列(衔接子序列意指 核酸模板中的序列已知的寡核苷酸)互补并提供起始连接的位点的已知序列。连接之后，对模板成像并鉴定测序探针中的所述一或两个已知碱基。在锚定探针-测序探针复合物的完全移除或者经过切割除去标记(例如荧光团)之后并再生连接位点之后，开始下一个连接测序循环。
因此，在使用本文所述的浸泡式反应方案对核酸分子进行连接法测序的一个实施方案中，步骤b的一种或多种反应溶液包括包含锚定探针、经标记(例如荧光标记)的测序探针、连接酶或其混合物的溶液，前提是固体支持物与锚定探针、经标记的测序探针、连接酶中的每一种均发生接触，并且其中固体支持物上的所述信号由互补结合至固体支持物上的核酸分子的经标记的测序探针产生,所述经标记的测序探针经由连接酶连接至互补结合至同一核酸分子的锚定探针。在使用本文所述的浸泡式反应方案对核酸分子进行连接法测序的另一个实施方案中，在步骤b之前将其上固定有核酸分子的固体支持物与锚定探针(在SOLiD测序中也被称为“引物”)接触，以使得锚定探针杂交至所述固体支持物上的核酸分子，并且步骤b的一种或多种反应溶液包括包含经标记(例如荧光标记)的测序探针、连接酶或其混合物的溶液，前提是固体支持物与经标记的测序探针和连接酶中的每一种均发生接触，并且其中固体支持物上的所述信号由互补结合至固体支持物上的核酸分子的经标记的测序探针产生,所述经标记的测序探针经由连接酶连接至互补结合至同一核酸分子的锚定探针。
所述步骤b的一种或多种反应溶液的具体组成取决于所采用的具体连接法测序方法而不同。例如，在采用单一锚定探针的情况下，步骤b的一种或多种反应溶液可以包括仅一种反应溶液，该反应溶液包含连接酶和经标记的测序探针，任选地，还包含锚定探针；或者步骤b的一种或多种反应溶液也可以包括两种反应溶液，其中一种反应溶液包含锚定探针，另一种反应溶液包含经标记的测序探针，并且这两种反应溶液至少之一包含连接酶，在这样的情况下，步骤b包括如下过程：将固体支持物浸泡在包含锚定探针的反应溶液中、洗涤，随后浸泡在包含经标记的测序探针的反应溶液中、洗涤。或者，步骤b的一种或多种反应溶液还可以包括三种反应溶液，其分别包含锚定探针、经标记的测序探针和连接酶，在这样的情况下，步骤b包括如下过程：将固体支持物浸泡在包含锚定探针的反应溶液中、洗涤，随后浸泡在包含经标记的测序探针的反应溶液中、洗涤，随后浸泡在包含连接酶的反应溶液中、洗涤。类似地，在采 用双重锚定探针的情况下，步骤b的一种或多种反应溶液可以包括同时包含连接酶、第一锚定探针、第二锚定探针和经标记的测序探针的仅一种反应溶液，或分别包含连接酶、第一锚定探针、第二锚定探针、经标记的测序探针或其混合物的多种反应溶液。
关于连接法测序，特别是关于锚定探针、测序探针的描述，可参见例如WO2013066975、美国专利No.60/992,485、61/026,337、61/035,914和61/061,134中的记载。关于连接法测序的详细描述还可参见例如Landegren,U.,Kaiser,R.,Sanders,J.&Hood,L.A ligase-mediated gene detection technique.Science 241,1077–1080(1988)，美国专利No.6969488、美国专利No.6172218和美国专利No.6306597(其公开内容在此全部引入作为参考)。在优选的实施方案中，连接法测序包括联合探针锚定连接(cPAL)测序(参见WO2013066975的记载)。
如本文所用，术语“连接酶”是指催化核酸链的5'-磷酸和3'-羟基末端之间磷酸二酯键的分子内形成和分子间形成的核酸修饰酶。连接酶可以获自重组或天然来源。可以使用一种或多种低温(例如，室温或更低)连接酶(例如，T3 DNA连接酶、T4 DNA连接酶、T7 DNA连接酶和/或大肠杆菌DNA连接酶)。连接酶也可以是热稳定的连接酶。可以使用来自嗜热生物的热稳定连接酶。热稳定DNA连接酶的例子包括但不限于：Tth DNA连接酶(来自嗜热栖热菌(Thermus thermophilus)，可购自例如欧基公司(Eurogentec)和GeneCraft公司)；Pfu DNA连接酶(来自激烈火球菌(Pyrococcusfuriosus)的超嗜热连接酶)；Taq连接酶(来自水生栖热菌(Thermus aquaticus))，以及任何其他合适的热稳定连接酶，或其任意组合。
在另一个具体的实施方案中，本发明涉及使用本文所述的浸泡式反应方案对核酸分子进行合成法测序的方法。在这样的实施方案中，步骤b的一种或多种反应溶液包含用于在合成法测序中以被测序的核酸分子为模板进行聚合反应并生成代表所述核酸分子的核苷酸序列的信号的试剂，并且步骤d的再生溶液包含用于从被测序的核酸分子上消除所述信号并使得能够起始下一个聚合测序反应的试剂。
如本文所用的合成法测序是本领域熟知的各种合成法测序方法。基本地，合成法测序涉及首先将被测序的核酸分子与测序引物杂交，随后在聚合酶的存在下，以被测序的核酸分子为模板在测序引物的3’端聚合经标记(例如荧光标记)的核苷酸。聚合之后，通过检测所述标记来鉴定该经标记的核苷酸。在从经标记的核苷酸上除去标记(例如荧光团)之后，开始下一个聚合测序循环。在一个实施方案中，被测序的核 酸分子通过与固定在固体支持物上的测序引物杂交而固定在固体支持物上。在另一个实施方案中，在本发明的浸泡式反应方案中，在步骤b之前将其上固定有核酸分子的固体支持物与测序引物接触，以使得测序引物杂交至所述固体支持物上的核酸分子。
因此，在使用本文所述的浸泡式反应方案对核酸分子进行合成法测序的一个实施方案中，步骤b的一种或多种反应溶液包括包含聚合酶、经标记(例如荧光标记)的核苷酸或其混合物的溶液，前提是固体支持物与聚合酶、经标记的核苷酸中的每一种均发生接触，并且其中固体支持物上的所述信号由互补结合至固体支持物上的核酸分子的经标记的核苷酸产生，所述经标记的核苷酸经由聚合酶以固体支持物上的核酸分子为模板聚合至测序引物的3’端。在这样的实施方案中，步骤d的再生溶液包括用于从经标记(例如荧光标记)的核苷酸除去标记的试剂。在使用本文所述的浸泡式反应方案对核酸分子进行合成法测序的优选实施方案中，在步骤b中将固体支持物浸泡在一种反应溶液中，所述反应溶液包含聚合酶和经标记(例如荧光标记)的核苷酸。
适合用于合成法测序的经标记(例如荧光标记)的核苷酸以及用于从经标记(例如荧光标记)的核苷酸除去标记的试剂是本领域熟知的，这样的核苷酸和试剂的非限制性实例可参见例如WO04018497、WO04018493、美国专利No.7427673和美国专利No.7057026(其公开内容在此全部引入作为参考)中公开的经标记的核苷酸和用于从经标记的核苷酸除去标记的试剂。
在某些实施方案中，经标记的核苷酸还可包含3’阻断基团。所述3’阻断基团在该经标记的核苷酸聚合到生长的核苷酸链上时阻止其他核苷酸的掺入。优选地，3’阻断基团在步骤d中与标记一起除去。合适的3’阻断基团以及用于从核苷酸除去3’阻断基团的试剂是本领域熟知的，这样的3’阻断基团和试剂的非限制性实例可参见例如：Greene&Wuts，Protective Groups in Organic Synthesis，John Wiley&Sons。Guillier；Metzker等人(Nucleic Acids Research,22(20):4259-4267,1994)；WO91/06678、WO2002/029003、WO2014139596、WO2004/018497，上述参考文献均通过引用整体并入本文。
在优选的实施方案中，经标记的核苷酸上的标记本身可用作3’阻断基团。不受理论约束，例如这样的标记可以具有足以发挥阻断其他核苷酸掺入到多核苷酸链上的大小或结构。所述阻断可能是由于空间位阻造成的，或者可能是由于大小、电荷和结构的组合造成的。在某些实施方案中，经标记的核苷酸上的标记和阻断基团可以是不 同的，但优选可采用相同的方式从核苷酸上除去所述标记和阻断基团。在这样的情况下，本文所述的步骤d的再生试剂包含可从经标记的核苷酸上同时除去标记和3’阻断基团的试剂。在另外的一些实施方案中，本文所述的步骤d可以包括将固体支持物分别浸泡到可从经标记的核苷酸上除去标记的试剂和可从经标记的核苷酸上除去3’阻断基团的试剂中。
如本文所用，术语“聚合酶”是指合成核酸链或聚合物的酶，包括DNA聚合酶和RNA聚合酶。优选地，本文所用的聚合酶是DNA聚合酶。可以使用的一种聚合酶是SequenaseTM(来源于噬菌体7 DNA聚合酶的酶，其被修饰以改善其测序性质-参见Tabor and Richarson,Proc.Nat.Acad.Sci.USA,84:4767-4771(1987)，可购自例如United States Biochemical Corporation,Cleveland,Ohio)。可用于代替SequenaseTM的其它聚合酶包括但不限于DNA聚合酶I的Klenow片段，AMV逆转录酶以及Taq聚合酶。关于可用于本发明的方法的聚合酶的其他描述还可参见WO05024010和WO06120433，其全部内容通过引用并入本文。
通常使用的聚合条件是本领域已知的这些酶的聚合条件。在SequenaseTM的情况下，聚合条件包括在约室温至约45℃范围内的温度；pH 7至8的缓冲液，优选pH 7.3至7.7；酶浓度为约0.01单位/微升至约1单位/微升，反应时间为约1至约20分钟，优选1至5分钟。用于SequenaseTM的典型缓冲液由以下组成：0.040M Tris HCl(pH7.5)0.050M氯化钠，0.010M氯化镁，0.010M二硫苏糖醇。在DNA聚合酶I的Klenow片段的情况下，这些典型的条件包括在约10℃至约45℃范围内的温度，优选约15℃至约40℃；pH 6.8至7.4的缓冲液，优选pH 7.0至7.4；酶浓度为约0.01单位/微升至约1单位/微升，优选约0.02至约0.15单位/微升，反应时间为约1至约40分钟。用于DNA聚合酶I的Klenow片段的典型缓冲液由以下组成：0.05M三羟甲基氨基甲烷氯化物，pH 7.5 0.05M氯化镁，0.05M氯化钠，0.010M二硫苏糖醇。
应当理解，这些条件仅是示例性的。当使用其它聚合酶时，应该使用最适合它们的条件，因为通常希望尽可能快地进行聚合反应。为此，通常对于逆转录酶需要使用42℃的温度；对于Klenow聚合酶为24℃；对于SequenaseTM为37℃；和对于Taq聚合酶为72℃。此外，为了增强反应，特别是在使用经修饰的dNTP的情况下，使用显著过量的dNTP(超过化学计量)或修饰其他条件如盐浓度可以是有利的。
如本文所用，术语“互补”或“基本互补”是指核苷酸或核酸之间的杂交或碱基 配对或者双链体形成。如果在一个给定位置上一个核酸的核苷酸能够与另一个核酸的核苷酸形成氢键，则这两个核酸被认为在该位置上是彼此互补的。互补核苷酸一般是A和T(或A和U)或者C和G。当在最佳地比对和比较并适当地进行了核苷酸插入或缺失的情况下，一条链的核苷酸与另一条链的至少大约80％、通常至少大约90％到约95％，甚至大约98％到100％配对时，这两个单链RNA或DNA分子被称为基本互补。
如本文所用，术语“杂交”是指互补的核苷酸或核苷酸碱基之间足够的氢键结合，其可以是，例如，Watson-Crick，Hoogsteen或反向的Hoogsteen氢键结合，以使得核酸链之间发生稳定和特异性结合。杂交能力根据严格条件(包括合适的缓冲液浓度和温度，其允许特异性杂交至具有完全或部分互补性区域的靶核酸)确定。因此，不是核酸的所有核苷酸都需要是互补的。此外，当核酸链与靶核酸的所有、部分或重叠区域杂交时，核酸链是“基本互补”的。建立用于设计本发明的寡核苷酸或引物的严格杂交条件的定性和定量考虑事项是本领域已知的，参见，例如，Ausubel等人，Short Protocols in Molecular Biology(4th ed.，John Wiley & Sons 1999)；Sambrook等人，Molecular Cloning：A Laboratory Manual(3d ed.，Cold Spring Harbor Laboratory Press 2001)：Nucleic Acid Hybridisation：A Practical Approach(B.D.Hames&S.J.Higgins eds.，IRL Press 1985)。
如本文所用，术语“Tm”用于本文通常是指让半数的双链核酸分子解离成单链的温度。计算核酸的Tm的公式是本领域公知的。正如标准参考文献指出的，当核酸处于阳离子浓度为0.5M或更低的水溶液中时，可以通过公式Tm＝81.5+16.6(log10[Na+])0.41(％[G+C])-675/n-1.0m简单估计Tm值，(G+C)含量在30％和70％之间，n是碱基数，m是错配碱基对百分比(参见例如，Sambrook J等人(2001),Molecular Cloning,A Laboratory Manual,(3rd Ed.,Cold Spring Harbor Laboratory Press)。其他参考文献包含更复杂的计算方法，这些方法在计算Tm时考虑到结构和序列特性(还可以参见，Anderson and Young(1985),Quantitative Filter Hybridization,Nucleic Acid Hybridization,和Allawi and Santa Lucia(1997),Biochemistry 36:10581-94)。
因此，在本发明的具体实施方案中，可以使用合适的温度控制设备来将各个反应容器内的温度调节至其中的反应溶液发生反应或用洗涤溶液洗涤时所需要的温度。在一个实施方案中，各反应容器所需的温度可以是不同的。在另一个实施方案中，各反 应容器所需的温度可以是部分相同的。在另一个实施方案中，各反应容器所需的温度可以是相同的。例如，在本文所述的步骤b中分别包含所述一种或多种反应溶液的反应容器的温度可以彼此相同或不同，并且可以与步骤d中的反应容器的温度相同或不同。在一个实施方案中，可以采用一个温度控制设备来控制所有反应容器的温度。在另一个实施方案中，尤其是各反应容器所需的温度不相同的情况下，可以采用不同的温度控制设备来分别控制各反应容器的温度。可用于本发明的方法的合适的温度控制设备是容易获得的，例如可购自市面上可获得的各种温度控制设备，例如水浴锅。在进行核酸测序时，各反应容器的温度可以取决于所采用的测序方法，根据反应容器内进行的反应来常规地确定，各反应容器的温度可以是例如但不限于至少约0℃、至少约5℃、至少约10℃、至少约15℃、至少约20℃、至少约25℃、至少约30℃、至少约35℃、至少约40℃、至少约45℃、至少约50℃、至少约55℃、至少约60℃或更高。在某些情况下，低于0℃的温度可能是需要的。
如本文所用，术语“信号”包括能够被检测到的任何信号，包括但不限于光信号、电信号、电磁信号、放射信号等。术语“代表核酸分子的核苷酸序列的信号”意指该信号携带核苷酸序列的信息并能够被解码为被测序的核酸分子的核苷酸序列。例如，在使用经标记(例如荧光标记)的测序探针的情况下，由标记产生的信号携带测序探针的核苷酸序列信息，并且通过该信号可获知被测序的核酸分子上与该测序探针互补配对的位置的核苷酸序列。又例如，在使用经标记(例如荧光标记)的核苷酸的情况下，由标记产生的信号携带核苷酸的身份信息，并且通过该信号可获知被测序的核酸分子上与该核苷酸互补配对的位置的核苷酸序列。在优选的实施方案中，所述信号是荧光信号。在优选的实施方案中，所述标记是荧光标记。检测荧光标记或信号的方式是本领域熟知的。例如，可以通过检测荧光的波长的装置来实现。这样的装置是本领域熟知的。例如，这样的装置可以是共焦扫描显微镜，其用激光扫描固体支持物的表面，以便使直接结合被测序的核酸分子上的荧光团成像。另外，可以例如用灵敏的2-D探测器，如电荷偶连的探测器(CCD)观察所产生的每一种信号。还可以例如使用诸如扫描近场光学显微方法(SNOM)的其他技术。
在一个实施方案中，在步骤c的检测固体支持物上的信号之前，可以将该固体支持物与用于在检测时保护核酸分子、经标记的探针和/或经标记的核苷酸的保护试剂接触，例如将该固体支持物浸泡在包含所述保护试剂的反应容器中。如本文所用的保护 试剂可以是例如防止核酸脱水或降解的试剂，防止荧光淬灭的试剂等。这样的保护试剂包括但不限于对苯二胺(PPD)、没食子酸丙酯(NPG)、1,4-重氮基二环-辛烷(DABCO)、抗坏血酸(维生素C)、Vectashield、SlowFade、SlowFade Light等。
如本文所用的洗涤溶液是能够将固体支持物上与其非特异性结合的物质洗掉并且不会不利地影响后续反应的任何溶液。适当地，洗涤试剂含有缓冲剂，例如有机盐，以保持约pH 6至pH 9的稳定pH，并且可能还包含一价或二价阳离子，从而从固体支持物除去非特异性结合的分子。示例性的洗涤试剂可以包括例如pH 6.5的100mM Tris-HCl缓冲液、TE缓冲液(Tris-HCl pH 8,10mM和EDTA,1mM)等。根据需要，在本文所述的浸泡式反应方案中的各步骤中使用的洗涤溶液可以是相同或不同的，并且可以取决于所采用的测序方法而进行适应性调整和选择，这样的调整和选择在本领域技术人员的能力范围内。
在本发明的方法中，用于核酸测序的试剂可以或可以不重复使用。例如，步骤b中的一种或多种反应溶液和/或步骤d中的再生溶液可重复使用至少1次，例如重复使用至少2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、19、20、21、22、23、24、25、26、27、28、29、30、40、50、60、70、80、100次或更多次。因此，在本文所述的浸泡式反应方案的一个实施方案中，方法还包括在步骤f的任选重复步骤b至e的过程中，在每次重复时更换或不更换步骤b的所述一种或多种反应溶液和/或步骤d的所述再生溶液。
通常，反应溶液重复使用的次数与测序信号的减弱程度相关，也就是说，如果重复使用反应溶液后测序信号显著减弱以使得测序结果不准确时，这样的重复使用是不合适的，应更换新的反应溶液。在优选实施方案中，步骤b中的一种或多种反应溶液可重复使用至少10次而不显著影响测序信号的强度。在优选实施方案中，步骤d中的再生溶液可重复使用至少20次而不显著影响测序信号的强度。
或者，可以根据每次反应过程中反应溶液与固体支持物的反应时间来确定更换反应溶液的时间。每次测序反应过程中反应溶液与固体支持物的反应时间取决于所发生的反应和反应温度。例如，在测序反应的情况下(例如在步骤b的反应容器中)，反应温度可以是适合于所使用的测序酶(例如连接酶或聚合酶)发挥功能所需要的温度，例如可以是至少约5℃、至少约10℃、至少约15℃、至少约20℃、至少约25℃、至少约30℃、至少约35℃、至少约40℃、至少约45℃、至少约50℃、至少约55℃、至 少约60℃或更高，例如约10℃至约45℃范围内的温度，优选约15℃至约40℃，这样的温度可以由本领域技术人员根据所使用的测序酶容易地确定。每次测序反应的时间可以是所用的测序酶将经标记的探针或核苷酸掺入到模板核酸链上所需的时间，这样的时间可以由本领域技术人员容易地确定，例如可以是至少1s、至少5s、至少10s、至少15s、至少20s、至少25s、至少30s、至少40s、至少50s、至少1min、至少1.5min、至少2min、至少2.5min、至少3min、至少3.5min、至少4min、至少5min、至少10min、至少15min、至少20min、至少30min或更长时间。又例如，在从经标记的探针或核苷酸除去标记的反应的情况下(例如在步骤d的反应容器中)，反应温度可以是适合于发生所述反应所需要的温度，例如可以是至少约5℃、至少约10℃、至少约15℃、至少约20℃、至少约25℃、至少约30℃、至少约35℃、至少约40℃、至少约45℃、至少约50℃、至少约55℃、至少约60℃或更高，这样的温度可以由本领域技术人员根据所使用的用于从经标记的探针或核苷酸除去标记的方法或试剂容易地确定。这样的反应的时间可以由本领域技术人员容易地确定，例如可以是至少1s、至少5s、至少10s、至少15s、至少20s、至少25s、至少30s、至少40s、至少50s、至少1min、至少1.5min、至少2min、至少2.5min、至少3min、至少3.5min、至少4min、至少5min、至少10min、至少15min、至少20min、至少30min或更长时间。
因此，在其中步骤b的测序反应在至少约5℃、至少约10℃、至少约15℃、至少约20℃、至少约25℃、至少约30℃、至少约35℃、至少约40℃、至少约45℃、至少约50℃、至少约55℃、至少约60℃或更高的温度下进行的一个实施方案中，可以以小于或等于约1小时、小于或等于约2小时、小于或等于约3小时、小于或等于约4小时、小于或等于约5小时、小于或等于约6小时、小于或等于约7小时、小于或等于约8小时、小于或等于约9小时、小于或等于约10小时、小于或等于约12小时、小于或等于约15小时、小于或等于约18小时、小于或等于约20小时、小于或等于约22小时、小于或等于约24或更多小时，优选小于或等于约4小时或10小时的时间间隔更换测序反应试剂。在优选的实施方案中，当步骤b的一种或多种反应溶液的反应温度在约55℃以下时，更换步骤b的一种或多种反应溶液的时间间隔为小于约8小时，优选为约4小时。在优选的实施方案中，当步骤b的所述一种或多种反应溶液的反应温度在约45℃以下时，更换步骤b的所述一种或多种反应溶液的时间间隔为小于约24小时，优选为约10小时。
与现有二代测序技术采用的流道式反应方案相比，本发明的浸泡式反应方案的不同之处至少还在于：本发明的方法允许同时浸泡多于一个固体支持物，从而极大地提高测序通量。例如，本发明的方法允许同时浸泡2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20个或更多个固体支持物。这样的同时浸泡的多于一个固体支持物在本文中被称为一个批次的固体支持物。然而，应理解，这样的一个批次的固体支持物也可以仅由一个固体支持物组成。
更特别地，本发明的浸泡式反应方案使得固体支持物的嵌套批次处理成为可能。所述嵌套批次处理包括在使用本发明的浸泡式方案对第一批次固体支持物进行核酸测序的过程中，在一个测序循环还未结束时即开始对另一批次固体支持物进行核酸测序，并且在该第二批次固体支持物开始进行核酸测序后，还可以对第三批次固体支持物开始进行核酸测序；类似地，在第N-1批次固体支持物开始进行核酸测序后，可以对第N批次固体支持物开始进行核酸测序，其条件是1≤N≤t循环/t限速，且N为整数值，其中t循环是步骤b至步骤e的总时间(在本文称为一个测序循环的总时间，因此如本文所用的每个“测序循环”开始于在本发明的浸泡式反应方案中将固体支持物浸泡至步骤b的一种或多种反应溶液且结束于步骤e的洗涤该固体支持物)，t限速是在步骤b的每次浸泡、洗涤、步骤c的检测、步骤d的浸泡以及步骤e的洗涤中耗时最长程序的时间(在本文称为限速步骤的时间)。因此，例如，如果一个测序循环的总时间为120秒，并且在该测序循环中的限速程序为步骤c的检测程序，其需要进行40秒，则基于上述等式，N≤120秒/40秒＝3，因而在一个测序循环中，可以最多进行N＝3个批次固体支持物的嵌套批次处理。又例如，如果一个测序循环的总时间为120秒，并且在该测序循环中的限速程序为步骤c的检测程序，其需要进行35秒，则基于上述等式，N≤120秒/35秒＝3.4，因而在一个测序循环中，可以最多进行N＝3个批次固体支持物的嵌套批次处理。在本发明的实施方案中，在一个测序循环中，可以最多进行N＝1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30或更多个批次固体支持物的嵌套批次处理。在优选的实施方案中，可以基于限速程序所需要的时间，调整测序循环中其它程序的时间，以使得测序循环的总时间刚好为限速程序所需时间的整倍数。
因此，在一个实施方案中，如本文所述的浸泡式反应方案还包括：在将第一批次的固体支持物浸泡在步骤b的一种或多种反应溶液中之后且在步骤e结束之前，将其 上固定有核酸分子的第二批次的固体支持物浸泡在步骤b的一种或多种反应溶液中，随后按照步骤b至f操作所述第二批次的固体支持物；任选迭代地重复此过程N-1次，其中1≤N≤t循环/t限速，且N为整数值，其中t循环是步骤b至步骤e的总时间，t限速是在步骤b的每次浸泡、洗涤、步骤c的检测、步骤d的浸泡以及步骤e的洗涤中耗时最长程序的时间。如本文所用，“迭代地重复”意指在将第一批次的固体支持物浸泡在步骤b的一种或多种反应溶液中之后且在步骤e结束之前，任选将其上固定有核酸分子的第二批次的固体支持物浸泡在步骤b的一种或多种反应溶液中，随后在将第二批次的固体支持物浸泡在步骤b的一种或多种反应溶液中之后且在步骤e结束之前，任选将其上固定有核酸分子的第三批次的固体支持物浸泡在步骤b的一种或多种反应溶液中，随后在将第三批次的固体支持物浸泡在步骤b的一种或多种反应溶液中之后且在步骤e结束之前，任选将其上固定有核酸分子的第四批次的固体支持物浸泡在步骤b的一种或多种反应溶液中，以此类推，直到对第N+1批次的固体支持物迭代地重复此过程，其中1≤N≤t循环/t限速，且N为整数值。
在实践中，将第N批次的固体支持物浸泡在步骤b的一种或多种反应溶液中与将第N+1批次的固体支持物浸泡在步骤b的一种或多种反应溶液中之间的时间间隔tN满足以下条件：
tN≥t限速，且
Figure PCTCN2017095520-appb-000002
在一个实施方案中，本发明的方法允许一个测序循环使用N＝1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30或更多个批次的固体支持物。因此，相比现有的第二代测序技术，本发明的浸泡式反应方案极大地提高了测序通量，并且能够在单位时间内对更多的核酸进行测序。
在一个实施方案中，本发明提供的浸泡式反应方案还包括在步骤b的一种或多种反应溶液、步骤d的再生溶液和/或洗涤溶液中添加适当量的保湿剂。在优选的实施方案中，在第一反应溶液中添加适当量的保湿剂。不受理论约束，据认为这样的保湿剂的添加可能有助于在将固体支持物从反应溶液和/或洗涤溶液中取出来而暴露于空气时，避免其表面可能因为蒸发和疏水性而导致的干燥。这样的保湿剂包括但不限于多元醇例如甘油、丙二醇、1,3-丁二醇、山梨醇、聚乙二醇等。应理解，这样的保湿剂的添加应不会对反应溶液和/或洗涤溶液、固体支持物以及固体支持物上的核酸分子造 成任何不利的影响，并且还不会显著增加固体支持物上携带的试剂的量。在优选的实施方案中，在步骤b的一种或多种反应溶液、步骤d的再生溶液和/或洗涤溶液中添加适当量的甘油。添加的保湿剂的量可以是能够使固体支持物表面保持湿润的任何有效量。应理解，这样的量应不会对反应溶液和/或洗涤溶液、固体支持物以及固体支持物上的核酸分子造成任何不利的影响，并且还不会显著增加固体支持物上携带的试剂的量。添加的保湿剂的量可以是例如基于反应溶液和/或洗涤溶液的体积，至少约0.05％、至少约0.1％、至少约0.5％、至少约1％、至少约2％、至少约3％、至少约4％、至少约5％、至少约6％、至少约7％、至少约8％、至少约9％、至少约10％、至少约11％、至少约12％、至少约13％、至少约14％、至少约15％、至少约20％或更多的量。在优选的实施方案中，在步骤b的一种或多种反应溶液、步骤d的再生溶液和/或洗涤溶液中添加约5％-10％的甘油。
在一个实施方案中，本发明提供的浸泡式反应方案还包括在步骤b的一种或多种反应溶液、步骤d的再生溶液和/或洗涤溶液中添加适当量的用于在将固体支持物从反应溶液和/或洗涤溶液中取出时将固体支持物上残留的反应溶液和/或洗涤溶液中保留在其上的试剂。不受理论约束，据认为这样的试剂的添加可能有助于在将固体支持物从反应溶液和/或洗涤溶液中取出来时，避免固体支持物上残留的反应溶液和/或洗涤溶液可能因重力作用脱离固体支持物上端而导致的干燥。如本文所用，固体支持物的上端意指将固体支持物从溶液中取出时最先暴露于空气中的端部。在优选的实施方案中，这样的试剂可以是例如表面活性剂。这样的表面活性剂可以包括但不限于Tween-20、硬脂酸、月桂酸、十二烷基硫酸钠、二辛基琥珀酸磺酸钠(阿洛索-OT)、十二烷基苯磺酸钠、甘胆酸钠、苯扎氯铵、苯扎溴铵、卵磷脂、脂肪酸甘油酯等。应理解，这样的试剂例如表面活性剂的添加应不会对反应溶液和/或洗涤溶液、固体支持物以及固体支持物上的核酸分子造成任何不利的影响，并且还不会显著增加固体支持物上携带的试剂的量。在优选的实施方案中，在步骤b的一种或多种反应溶液、步骤d的再生溶液和/或洗涤溶液中添加适当量的Tween-20。添加的这样的试剂例如表面活性剂的量可以是能够在将固体支持物从反应溶液和/或洗涤溶液中取出时将固体支持物上残留的反应溶液和/或洗涤溶液中保留在其上的任何有效量。应理解，这样的量应不会对反应溶液和/或洗涤溶液、固体支持物以及固体支持物上的核酸分子造成任何不利的影响，并且还不会显著增加固体支持物上携带的试剂的量。添加的这样的试剂例如表面活性 剂的量可以是例如基于反应溶液和/或洗涤溶液的体积，至少约0.001％、至少约0.005％、至少约0.01％、至少约0.02％、至少约0.03％、至少约0.04％、至少约0.05％、至少约0.06％、至少约0.07％、至少约0.08％、至少约0.09％、至少约0.1％、至少约0.2％、至少约0.3％、至少约0.4％、至少约0.5％、至少约0.6％、至少约0.7％、至少约0.8％、至少约0.9％、至少约1％、至少约2％、至少约3％、至少约4％、至少约5％或更多的量。在优选的实施方案中，在步骤b的一种或多种反应溶液、步骤d的再生溶液和/或洗涤溶液中添加约0.05％-1％的Tween-20。
在一个实施方案中，本发明提供的浸泡式反应方案还包括优化固体支持物在各反应容器之间的移动速率，以使得固体支持物暴露于空气中的时间最小化。应理解，这样的速率优化应不会对反应溶液、固体支持物以及固体支持物上的核酸分子造成任何不利的影响。
因此，在一个具体的实施方案中，优化固体支持物在各反应容器之间的移动速率包括优化固体支持物从反应容器中的溶液或试剂中取出的速率，以使得固体支持物从溶液或试剂中尽可能快地取出而同时不会因较快的取出而对反应溶液、固体支持物以及固体支持物上的核酸分子造成任何不利的影响。不受理论约束，据认为将固体支持物从溶液或试剂中更快地取出有助于避免其表面可能因为蒸发和疏水性而导致的干燥，同时还有助于减少其表面携带的溶液或试剂的量，从而降低对后续程序的污染，然而，过快的取出速率可能会对固体支持物及其上固定的核酸分子带来损害，从而影响测序质量，因而需要根据具体使用的固体支持物、其上固定的核酸分子以及反应溶液或试剂对取出速率进行优化。在一个实施方案中，固体支持物从反应容器中的溶液或试剂中取出的速率可以是例如但不限于至少1mm/s、至少5mm/s、至少10mm/s、至少15mm/s、至少20mm/s、至少25mm/s、至少30mm/s、至少40mm/s、至少50mm/s或更高。在另一个实施方案中，固体支持物从反应容器中的溶液或试剂中取出的速率可以是例如但不限于不高于50mm/s、不高于40mm/s、不高于30mm/s、不高于20mm/s、不高于10mm/s或更低。在优选的实施方案中，固体支持物从反应容器中的溶液或试剂中取出的速率可以是例如约20mm/s。
在一个具体的实施方案中，优化固体支持物在各反应容器之间的移动速率包括优化固体支持物在从反应容器中的溶液或试剂中取出后的移动速率，以使得固体支持物暴露于空气中的时间最小化。不受理论约束，据认为在从反应容器中的溶液或试剂中 取出后，尽可能快地移动固体支持物有助于避免其表面可能因为蒸发和疏水性而导致的干燥。在一个实施方案中，优化固体支持物在从反应容器中的溶液或试剂中取出后的移动速率，以使得固体支持物暴露于空气中的时间小于或等于100ms、小于或等于200ms、小于或等于500ms、小于或等于1s、小于或等于1.5s、小于或等于2s、小于或等于2.5s、小于或等于3s、小于或等于3.5s、小于或等于4s、小于或等于4.5s、小于或等于5s、小于或等于5.5s、小于或等于6s、小于或等于6.5s、小于或等于7s、小于或等于7.5s、小于或等于8s、小于或等于8.5s、小于或等于9s、小于或等于9.5s、小于或等于10s、小于或等于15s、小于或等于20s、小于或等于30s、小于或等于1min、小于或等于2min、小于或等于5min或小于或等于10min。
因此，在优选的实施方案中，本发明提供了使用浸泡式反应方案对核酸进行测序的方法，所述浸泡式反应方案包括以下步骤：
a.提供其上固定有核酸分子的第一批次的固体支持物，
b.将所述第一批次的固体支持物浸泡在一种或多种反应溶液中，以在固体支持物上产生代表所述核酸分子的核苷酸序列的信号，所述一种或多种反应溶液各自置于分开的反应容器中，
其中在将所述第一批次的固体支持物从每一种反应溶液中移出后，洗涤所述第一批次的固体支持物，以除去固体支持物上残留的反应溶液，
c.检测所述第一批次的固体支持物上的所述信号，
d.将所述第一批次的固体支持物浸泡在置于另外的反应容器中的再生溶液中，以消除固体支持物上的所述信号，
e.从再生溶液中移出所述第一批次的固体支持物，洗涤所述第一批次的固体支持物，以除去固体支持物上残留的再生溶液，
f.任选地，重复步骤b至e或b-c，
其中任选地在步骤b的一种或多种反应溶液、步骤d的再生溶液和/或洗涤溶液中添加了保湿剂，例如甘油，和/或表面活性剂，例如Tween-20，并且任选地固体支持物在各反应容器之间的移动速率被优化以使得固体支持物暴露于空气中的时间最小化，
任选地，所述方法还包括如本文所述的迭代地重复处理第二批次至第N批次(即嵌套批次处理)的固体支持物，其中1≤N≤t循环/t限速，且N为整数值。
在另一个优选的实施方案中，本发明提供了使用浸泡式反应方案对核酸进行连接 法测序的方法，所述浸泡式反应方案包括以下步骤：
a.提供其上固定有核酸分子的第一批次的固体支持物，
b.将所述第一批次的固体支持物浸泡在置于反应容器中的反应溶液(在本文称为第一反应溶液)中，所述第一反应溶液包含连接酶和经标记(例如荧光标记)的测序探针，任选地还包含锚定探针，其中在反应容器中，以核酸分子为模板在锚定探针和测序探针之间进行核酸连接反应，和
将所述第一批次的固体支持物从第一反应溶液中移出，并洗涤所述第一批次的固体支持物，以除去固体支持物上残留的第一反应溶液，
c.检测所述第一批次的固体支持物上的标记，
d.将所述第一批次的固体支持物浸泡在置于另外的反应容器中的再生溶液中，所述再生溶液包含可从经标记(例如荧光标记)的测序探针上除去标记的试剂或可将经标记(例如荧光标记)的测序探针从核酸分子上移除的试剂，
e.从再生溶液中移出所述第一批次的固体支持物，洗涤所述第一批次的固体支持物，以除去固体支持物上残留的再生溶液，
f.任选地，重复步骤b至e或b至c，
其中任选地在第一反应溶液、再生溶液和/或洗涤溶液中添加了保湿剂，例如甘油，和/或表面活性剂，例如Tween-20，并且任选地固体支持物在各反应容器之间的移动速率被优化以使得固体支持物暴露于空气中的时间最小化，
任选地，所述方法还包括如本文所述的迭代地重复处理第二批次至第N批次(即嵌套批次处理)的固体支持物，其中1≤N≤t循环/t限速，且N为整数值。
在另一个优选的实施方案中，本发明提供了使用浸泡式反应方案对核酸进行合成法测序的方法，所述浸泡式反应方案包括以下步骤：
a.提供其上固定有核酸分子的第一批次的固体支持物，
b.将所述第一批次的固体支持物浸泡在置于反应容器中的反应溶液(在本文称为第一反应溶液)中，所述第一反应溶液包含聚合酶和经标记(例如荧光标记)的核苷酸，任选地，所述经标记的核苷酸还可包含3’阻断基团，其中在反应容器中，以核酸分子为模板进行核酸聚合反应，和
将所述第一批次的固体支持物从第一反应溶液中移出，并洗涤所述第一批次的固体支持物，以除去固体支持物上残留的第一反应溶液，
c.检测所述第一批次的固体支持物上的标记，
d.将所述第一批次的固体支持物浸泡在置于另外的反应容器中的再生溶液中，所述再生溶液包含可从经标记(例如荧光标记)的核苷酸上除去标记的试剂，
e.从再生溶液中移出所述第一批次的固体支持物，洗涤所述第一批次的固体支持物，以除去固体支持物上残留的再生溶液，
f.任选地，重复步骤b至e或b至c，
其中任选地在第一反应溶液、再生溶液和/或洗涤溶液中添加了保湿剂，例如甘油，和/或表面活性剂，例如Tween-20，并且任选地固体支持物在各反应容器之间的移动速率被优化以使得固体支持物暴露于空气中的时间最小化，
任选地，所述方法还包括如本文所述的迭代地重复处理第二批次至第N批次(即嵌套批次处理)的固体支持物，其中1≤N≤t循环/t限速，且N为整数值。
在其它实施方案中，本发明还提供了用于本文所述的浸泡式反应方案以对核酸分子进行测序的设备，所述设备包括：
a)一个或多个反应容器，其各自包含用于与核酸分子接触以产生代表所述核酸分子的核苷酸序列的信号的反应溶液；
b)包含洗涤试剂的一个或多个反应容器；
c)包含用于从固体支持物上消除所述信号的反应溶液的反应容器；
d)检测所述信号的装置；和
e)温度控制设备(例如水浴锅)，其用于控制步骤a至c中的反应容器的温度。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是，本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了本发明的浸泡式反应方案的两个示例性实施方案的流程图(显示一个测序循环)，其中一个示例性实施方案使用了在检测时保护核酸和标记的保护试剂。
图2显示本发明的浸泡式反应方案与BGISEQ-500 SE50的测序数据对比。
图3显示本发明的改进的浸泡式反应方案的测序结果。
图4显示在聚合反应试剂里添加一定量的表面活性剂能够改善芯片上端的干燥。图左边表示芯片下端，图右边表示芯片上端。条件1：在第一反应溶液中添加5％-10％甘油+快速移动芯片；条件2：在第一反应溶液中添加10％甘油+快速移动芯片+在第一反应溶液中添加0.05％-1％Tween-20。
实施例
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1-利用浸泡式反应方案进行核酸测序。
根据制造商的说明书，采用MGIEasyTMDNA文库制备试剂盒(深圳华大智造科技有限公司)以大肠杆菌标准菌株为原料提取DNA制备用于测序的文库，加载到测序芯片上。根据制造商的说明，采用BGISEQ-500高通量试剂盒(SE50 V3.0，深圳华大智造科技有限公司，货号PF-UM-PEV30)中的试剂并按照图1A的流程图(本文所述的浸泡式反应方案的一个示例性实施方案)对获得的芯片上的核酸分子进行10个循环的测序，其中使用3张芯片同时浸泡。
将获得的测序数据上传至BGI Online(参见https://www.bgionline.cn/)进行分析或者使用BGISEQ-500测序仪自带的软件分析以对测序数据的质量进行分析并输出可视化报告，如图2所示。
采用MGIEasyTMDNA文库制备试剂盒以大肠杆菌标准菌株为原料提取DNA制备用于测序的文库，加载到测序芯片上，并采用BGISEQ-500高通量试剂盒(SE50 V3.0)中的测序试剂，在BGISEQ-500测序仪上进行测序反应作为对照。
在图2中，Q30指的是测序1000个碱基出现一个错误，数字越大表示测序质量越好；ESR是BGISEQ-500测序仪的测序指标，指有效的DNA纳米球比例，数字越大表示测序质量越好；MappingRate指的是测序数据相对于参考基因组的比对率，数字越大表示测序质量越好；BarcodeSplitRate指的是条形码拆分率，数字越大表示测序质量越好。
通过与使用BGISEQ-500测序仪对相同芯片进行测序的对照组进行比较显示，本发 明的浸泡式反应方案获得的测序数据的质量与BGISEQ-500测序仪获得的测序数据的质量基本相似(差距在5％以内，条形码拆分率比BGISEQ-500测序仪更好)。
实施例2-不同浸泡式反应方案的比较
采用与实施例1相似的实验程序，利用不同的浸泡式反应方案对芯片进行15个测序循环。
首先，采用以下浸泡式方案对芯片进行测序：
浸泡式方案1：与实施例1相同的实验程序，对芯片进行15个测序循环；
浸泡式方案2：在浸泡式方案1的基础上，在聚合反应试剂中添加5-10％甘油并在实验过程中以20mm/s快速移动芯片；
使用BGISEQ-500测序仪对芯片进行测序作为对照。
针对这三种测序，分别计算第15个测序循环检测的荧光信号强度相对于第5个测序循环检测的荧光信号强度的下降比率，结果如图3所示，其中A表示检测A碱基的荧光信号；C表示检测C碱基的荧光信号；G表示检测G碱基的荧光信号；T表示检测T碱基的荧光信号；AVG表示检测四种碱基荧光信号的平均值。
我们发现在添加甘油、并且提高芯片抬升速度到20mm/s后，第15个循环相对于第5个循环的荧光信号下降程度减少到10％左右，相比浸泡式方案1的超过20％的下降，有了明显的改善(如图3所示)。
此外，在5％-10％甘油+快速移动芯片(条件1)的基础上，进一步在测序试剂里添加了0.05％-1％的Tween-20(条件2)。采用与实施例1相同的实验程序，对芯片进行多个测序循环后，利用BGISEQ-500测序仪自带的分析软件获得芯片上的测序信号值的热图，结果如图4所示。发现与条件1测序的芯片相比，条件2测序的芯片上端的干燥现象得到了明显的缓解，并且芯片上端的数据质量和芯片的其他区域差别不显著(如果不添加表面活性剂，在30个循环后，芯片上端的信号值明显降低；在添加一定量Tween-20以后，芯片上端的信号值和其他区域没有显著差别，均匀性较好)(如图4所示)。
实施例3-本发明的浸泡式反应方案的嵌套批次处理。
根据本发明的浸泡式反应方案的一个实施方案，在实施例1的实验程序中应用芯片的嵌套批次处理，在第一组的3张芯片完成聚合反应后进行荧光标记检测时，将第二组的另外3张芯片浸泡到测序反应试剂(即第一反应溶液)中开始测序反应。在实 验中，每10个测序循环更换一次聚合反应试剂，每20个测序循环更换一次再生试剂。经比较，成本是BGISEQ-500测序仪同类型测序的25％。最终于2小时内完成了所有两组芯片的总共60次完整测序循环。而相比之下，在目前的BGISEQ-500测序仪或者illumina HiSeq-2500平台上，完成60次完整测序循环需要10小时以上。因此，在相同的测序时间内，本发明的测序通量是BGISEQ-500测序仪或HiSeq-2500平台的5倍以上，并且通过增加嵌套批次处理的批次数量，可以进一步增加通量和降低成本。
实施例4-测序试剂的重复使用不影响测序质量
将已在实施例1中使用过的测序试剂再次用于进行实施例1所述的测序程序，并如实施例1所述分析测序数据，并与未使用过的试剂进行比较。比较结果如下表1所示，显示使用过的试剂可以正常测序。
表1：使用过与未使用过的试剂的比较：
Figure PCTCN2017095520-appb-000003
实施例5-在本发明的浸泡式反应方案中使用的试剂的稳定性研究。
将测序试剂(测序试剂参考实施例1，包括聚合反应试剂，洗涤试剂1，洗涤试剂2，再生试剂)分别放在45℃和55℃水浴锅里处理不同的时间，包括4小时，8小时，24小时。
然后将热处理过的试剂用于在BGISEQ-500测序仪上进行核酸测序，通过BGISEQ-500测序仪自带的软件比较测序数据质量，确定试剂稳定性。
测序数据质量的分析结果如表2所示，可以看出，在55℃处理4小时，或者45℃处理8小时以后，测序试剂依然稳定，测序质量与使用未经过热处理的测序试剂的对 照组基本相似。因此，根据此结果，可以将浸泡式反应方案中的测序试剂的更换周期确定为55℃下4小时，或45℃下10小时。
表2：浸泡式反应方案中，测序试剂的稳定性分析：
Figure PCTCN2017095520-appb-000004

Claims (22)

  1. 一种使用浸泡式反应方案对核酸分子进行测序的方法，所述浸泡式反应方案包括以下步骤：
    a.提供其上固定有核酸分子的第一批次的固体支持物，
    b.将所述第一批次的固体支持物浸泡在一种或多种反应溶液中，以在固体支持物上产生代表所述核酸分子的核苷酸序列的信号，所述一种或多种反应溶液各自置于分开的反应容器中，
    其中在将所述第一批次的固体支持物从每一种反应溶液中移出后，洗涤所述第一批次的固体支持物，以除去固体支持物上残留的反应溶液，
    c.检测所述第一批次的固体支持物上的所述信号，
    d.将所述第一批次的固体支持物浸泡在置于另外的反应容器中的再生溶液中，以消除固体支持物上的所述信号，
    e.从再生溶液中移出所述第一批次的固体支持物，洗涤所述第一批次的固体支持物，以除去固体支持物上残留的再生溶液，
    f.任选地，重复步骤b至e或b至c。
  2. 权利要求1的方法，其还包括：在将所述第一批次的固体支持物浸泡在步骤b的一种或多种反应溶液中之后且在步骤e结束之前，将其上固定有核酸分子的第二批次的固体支持物浸泡在步骤b的一种或多种反应溶液中，随后按照步骤b至f操作所述第二批次的固体支持物；
    任选迭代地重复此过程N-1次，
    其中1≤N≤t循环/t限速，且N为整数值，
    其中t循环是步骤b至步骤e的总时间，t限速是在步骤b的每次浸泡、洗涤、步骤c的检测、步骤d的浸泡以及步骤e的洗涤中耗时最长程序的时间。
  3. 权利要求2的方法，其中将第N批次的固体支持物浸泡在步骤b的一种或多种反应溶液中与将第N+1批次的固体支持物浸泡在步骤b的一种或多种反应溶液中之间的时间间隔tN满足以下条件：
    tN≥t限速，且
    Figure PCTCN2017095520-appb-100001
  4. 权利要求1至3中任一项的方法，其中每个批次的固体支持物由一个或多于一个固体支持物组成。
  5. 权利要求1-4中任一项的方法，其中所述信号包括荧光信号。
  6. 权利要求1-5中任一项的方法，其中所述测序包括连接法测序。
  7. 权利要求6的方法，其中步骤b的一种或多种反应溶液包括包含锚定探针、经标记的测序探针、连接酶或其混合物的溶液，前提是固体支持物与锚定探针、经标记(例如荧光标记)的测序探针、连接酶中的每一种均发生接触，并且其中固体支持物上的所述信号由互补结合至固体支持物上的核酸分子的经标记的测序探针产生,所述经标记的测序探针经由连接酶连接至互补结合至同一核酸分子的锚定探针，
    优选地，步骤d的再生溶液包含可从经标记(例如荧光标记)的测序探针上除去标记的试剂或可将经标记(例如荧光标记)的测序探针从核酸分子上移除的试剂。
  8. 权利要求6的方法，其还包括：在步骤b之前将其上固定有核酸分子的固体支持物与锚定探针接触，以使得锚定探针杂交至所述固体支持物上的核酸分子。
  9. 权利要求8的方法，其中步骤b的一种或多种反应溶液包括包含经标记(例如荧光标记)的测序探针、连接酶或其混合物的溶液，前提是固体支持物与经标记的测序探针和连接酶中的每一种均发生接触，并且其中固体支持物上的所述信号由互补结合至固体支持物上的核酸分子的经标记的测序探针产生,所述经标记的测序探针经由连接酶连接至互补结合至同一核酸分子的锚定探针，
    优选地，步骤d的再生溶液包含可从经标记(例如荧光标记)的测序探针上除去标记的试剂或可将经标记(例如荧光标记)的测序探针从核酸分子上移除的试剂。
  10. 权利要求1-5中任一项的方法，其中所述测序包括合成法测序。
  11. 权利要求10的方法，其中步骤b的一种或多种反应溶液包括包含聚合酶、测序引物、经标记(例如荧光标记)的核苷酸或其混合物的溶液，前提是固体支持物与聚合酶、测序引物和经标记的核苷酸中的每一种均发生接触，并且其中固体支持物上的所述信号由互补结合至固体支持物上的核酸分子的经标记的核苷酸产生，所述经标记的核苷酸经由聚合酶以固体支持物上的核酸分子为模板聚合至测序引物的3’端，
    优选地，步骤d的再生溶液包含可从经标记(例如荧光标记)的核苷酸上除去标记的试剂，
    例如，所述经标记的核苷酸还可包含3’阻断基团。
  12. 权利要求10的方法，其中所述核酸分子通过与固定在固体支持物上的测序引物杂交而固定在固体支持物上。
  13. 权利要求10的方法，其还包括：在步骤b之前将其上固定有核酸分子的固体支持物与测序引物接触，以使得测序引物杂交至所述固体支持物上的核酸分子。
  14. 权利要求12或13的方法，其中步骤b的一种或多种反应溶液包括包含聚合酶、经标记(例如荧光标记)的核苷酸或其混合物的溶液，前提是固体支持物与聚合酶和经标记的核苷酸中的每一种均发生接触，并且其中固体支持物上的所述信号由互补结合至固体支持物上的核酸分子的经标记的核苷酸产生，所述经标记的核苷酸经由聚合酶以固体支持物上的核酸分子为模板聚合至测序引物的3’端，
    优选地，步骤d的再生溶液包含可从经标记(例如荧光标记)的核苷酸上除去标记的试剂，
    例如，所述经标记的核苷酸还可包含3’阻断基团。
  15. 权利要求1-5中任一项的方法，其中所述测序包括联合探针锚定聚合(cPAS)测序。
  16. 前述权利要求中任一项的方法，其中所述洗涤通过将固体支持物浸泡在包含洗涤溶液的反应容器中进行。
  17. 前述权利要求中任一项的方法，其还包括：在步骤b的一种或多种反应溶液、步骤d的再生溶液和/或洗涤溶液中添加保湿剂，例如甘油。
  18. 前述权利要求中任一项的方法，其还包括：优化固体支持物在各反应容器之间的移动速率，以使得固体支持物暴露于空气中的时间最小化。
  19. 前述权利要求中任一项的方法，其还包括：在步骤b的一种或多种反应溶液、步骤d的再生溶液和/或洗涤溶液添加用于在将固体支持物从反应溶液和/或洗涤溶液中取出时将固体支持物上残留的反应溶液和/或洗涤溶液中保留在其上的试剂，例如表面活性剂，例如Tween-20。
  20. 前述权利要求中任一项的方法，其还包括：使用温度控制设备(例如水浴锅)调节各个反应容器中的温度。
  21. 前述权利要求中任一项的方法，其中在步骤f的任选重复步骤b至e的过程中，在每次重复时更换或不更换步骤b的所述一种或多种反应溶液和/或步骤d的所述再生溶液，
    例如，当步骤b的所述一种或多种反应溶液的反应温度在约55℃以下时，更换步骤b的所述一种或多种反应溶液的时间间隔为小于约8小时，
    例如，当步骤b的所述一种或多种反应溶液的反应温度在约45℃以下时，更换步骤b的所述一种或多种反应溶液的时间间隔为小于约24小时。
  22. 一种用于权利要求1-21中任一项的浸泡式反应方案以对核酸分子进行测序的设备，所述设备包括：
    a)一个或多个反应容器，其各自包含用于与核酸分子接触以产生代表所述核酸分子的核苷酸序列的信号的反应溶液；
    b)包含洗涤试剂的一个或多个反应容器；
    c)包含用于从固体支持物上消除所述信号的反应溶液的反应容器；
    d)检测所述信号的装置；和
    e)温度控制设备(例如水浴锅)，其用于控制步骤a至c中的反应容器的温度。
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