CN116287167B

CN116287167B - Method for sequencing nucleic acid molecules

Info

Publication number: CN116287167B
Application number: CN202310136389.1A
Authority: CN
Inventors: 李汉东; 成昌梅; 肖育劲; 刘金枝; 邹艳; 吴高椿; 丁利杰; 黄岩; 张朝文; 高群
Original assignee: Shenzhen Dadao Sequencing Biotechnology Co ltd
Current assignee: Shenzhen Dadao Sequencing Biotechnology Co ltd
Priority date: 2023-02-20
Filing date: 2023-02-20
Publication date: 2024-06-25
Anticipated expiration: 2043-02-20
Also published as: CN116287167A; WO2024174850A1

Abstract

The present invention provides a method for sequencing a nucleic acid molecule. In the sequencing process, based on the specific identifiers of two groups of nucleotides, aiming at the same position to be tested of a sequencing template, the nucleotides with preset identifiers in a first group of nucleotides are introduced into the position to be tested in the form of forming a five-membered complex to obtain first identifier information, and then the nucleotides with preset identifiers in a second group of nucleotides are introduced into the same position in the form of covalent extension to obtain second identifier information, so that the first identifier information and the second identifier information are doubly confirmed by adopting binary system, and the accuracy of base identification is improved. The sequencing method provided by the invention is based on the identification of the preset identifiers of two groups of nucleotides, can be realized by a simple single-excitation light source and a monochromatic light path system hardware sequencing instrument, and is low in cost.

Description

Method for sequencing nucleic acid molecules

Technical Field

The invention relates to the technical field of gene sequencing, in particular to a sequencing method of nucleic acid molecules.

Background

As one of key technologies for genomics research, gene sequencing technology, has rapidly progressed over the past 40 years. Twice in 1977, the nobel prize was given to the Sanger invention dideoxy chain termination method (Sanger sequencing, i.e., the first generation sequencing technique), followed by the appearance of the first generation gene sequencer ABI3700, and in 2003, sequencing was completed for a human genome project sequencing length of up to 30 hundred million base pairs. However, the first generation sequencing technology fails to meet the increasing demands of whole genome sequencing in terms of throughput, cost, read length, sequencing speed, and data analysis systems, and thus the next generation sequencing (next generation sequencing, NGS) technology has emerged.

NGS techniques are also known as large-scale parallel sequencing or deep sequencing, including second, third and fourth generation sequencing techniques. Currently, representative second generation sequencing platforms are 454 from Roche, switzerland, genome sequencer (GA), hiSeq 2000 and MiSeq from Illumina, oligo ligation detection sequencing (sequencing by oligo ligation detection, SOLiD) 5500XL from ABI, ion Torrent personalized operations genome sequencer (personal genome machine, PGM) from Life Technologies; third generation sequencing platforms have HeliScope genetic analysis system from Helicos Biosciences and single molecule real-time (single molecule real time, SMRT) sequencing technology from PacificBiosciences; the fourth generation of sequencing technology is nanopore sequencing technology available from Oxford Nanopore Technologies, uk. The following are the basic principles and application features of these sequencing techniques.

Second generation sequencing technology the first generation sequencing technology developed on the basis of Sanger sequencing method is a gold standard for sequencing, the sequencing length can reach 1000bp, the accuracy is almost 100%, but the defects of low flux, high cost and long time consumption exist, and the large-scale application of the technology is seriously affected. For this purpose, second generation sequencing techniques were developed. The core principle of the second generation sequencing technology is sequencing-by-synthesis, the basic steps of which include library preparation, generation of monoclonal DNA clusters, and sequencing reactions. Compared with the first generation sequencing technology, the second generation sequencing technology has the following characteristics: (1) high throughput. The second generation sequencing technology does not depend on traditional capillary electrophoresis, the sequencing reaction is carried out on a chip, and hundreds of millions of points on the chip can be sequenced simultaneously; (2) cost reduction. The cost per Mb base of the second generation sequencing technology is reduced by 96.0-99.9% compared with that of Sanger sequencing method; (3) The sensitivity is high, for example, the design of a Roche 454 sequencing platform of '1 fragment=1 magnetic bead=1 read length' can ensure the detection of low-abundance DNA information; (4) the read length is shorter and subsequent splicing is necessary; (5) The polymerase chain reaction (polymerase chain reaction, PCR) process may introduce bias and mismatch.

Roche454 sequencing platform Roche454 (Genome sequence 20 System) was the first NGS sequencing platform, offered by LIFE SCIENCES in the United states in 2005 and purchased by Roche, switzerland in 2007. The Roche company developed Roche GS Titanium, roche GS FLX+, roche GS Junior, and Roche GS Junior+ on this basis. Roche454 is a sequencing platform based on microemulsion PCR and pyrosequencing techniques. The principle is that a single-stranded DNA template is limited on emulsified magnetic beads and microemulsion PCR is carried out in emulsion droplets to generate thousands of template DNA clusters to be detected. The beads were then placed in microtiter plate wells with pyrophosphate sequencing substrates for enzyme-linked chemiluminescent reactions and multiple copies of a single DNA molecule were sequenced in parallel on a large scale. The Roche454 sequencing platform has the advantages of long sequencing read length and short time consumption, such as the Roche GS FLX+ read length of 1kb, and one round of sequencing time of only 23 hours. The accuracy of Roche454 sequencing is up to 99.9%, the length and accuracy equivalent to those of Sanger sequencing are achieved, and the sequencing cost is reduced.

The main application fields of Roche 454 sequencing include microbial community diversity analysis (such as 16s, 18s and ITS amplicon sequencing), metagenomic research of complex environment samples, de novo sequencing of microbial genomes, transcriptome sequencing, exon sequencing, target region capture sequencing, pathogen detection and the like. However, the Roche 454 platform also suffers from disadvantages such as relatively high cost of pyrosequencing reagents, relatively complex sample preparation, difficulty in handling duplicate and homologous base polymerization regions, and accumulation of errors due to reagent flushing. With the update of the sequencing instrument, the Roche 454 platform is difficult to upgrade and improve again from the aspects of expansibility and cost, so Roche company announced the production of the pyrosequencing instrument to be phased out from 2016.

Illumina sequencing platform-Solexa in the United states, the company Solexa in 2006, purchased by Illumina in 2007, after which Illumina developed GA II X, hiSeq 2500, hiSeq 3000, hiSeq 4000, hiSeq X Ten, hiSeq X Five, nextSeq500, miSeq series and MiniSeq systems. The Illumina sequencing platform is sequencing-by-synthesis based on bridge PCR and fluorescent reversible terminators. The single-stranded DNA is fixed on the surface of a chip of 8 channels to form an oligonucleotide bridge, the chip is placed in a flow cell, and different monoclonal DNA clusters are generated in each channel through PCR amplification. Adding DNA polymerase and 4 fluorescence marked dNTP reversible terminators, then carrying out synthesis reaction, only adding a single base each time, detecting fluorescence signals to determine the base type while synthesizing, then cutting off the 3' -end extension termination group of dNTP, and continuing adding bases to carry out sequencing reaction. The Illumina has the highest flux in the NGS platform, kampmann et al completed the whole genome sequencing work of 9 influenza a virus samples in one sequencing cycle in one channel using GA ii X. The newly proposed NextSeq500 combines sample preparation and sequencing functions, the push button operation of which can be switched between many common sequencing applications. The system is capable of sequencing the entire human genome and up to 16 exomes in one day, and the NextSeq500 takes only 12 hours to run 75 sequencing cycles. In addition, illumina sequencing costs are lowest and therefore is most widely used, almost covering all aspects of sequencing applications such as genome-wide from-head sequencing, resequencing, exon and target region capture sequencing; transcriptome sequencing, digital gene expression profiling, microrna sequencing, and degradation group sequencing; methylation sequencing of apparent histology, simplified methylation sequencing, and methylation DNA co-immunoprecipitation sequencing, among others. However, the Illumina platform may result in increased costs for later data pruning and analysis due to shorter read lengths.

SOLiD sequencing platform SOLiD sequencing technology was developed by Agencourt, inc. of America and purchased from ABI, inc. in 2006. ABI pushes out the first sequencing platform of SOLiD in 2007, and SOLiD 5500XL in 2010. As with the Roche 454 sequencing platform, SOLiD also employs microemulsion PCR, except that it employs oligonucleotide ligation sequencing. After the microemulsion PCR, the template strand was attached to a SOLiD slide via magnetic beads for sequencing reactions. First, the universal primer is complementarily paired with the linker on the template strand, and 16 8 base probes and ligase are added to compete for the site of ligation with the primer. After ligation, the last 3 bases of the probe are cleared, the fluorescent signal is released, the substrate is added multiple times until it extends to the end of the strand to be detected, and then a new primer is used to perform a new round of sequencing reaction. The new primer differs from the first primer by the same length and by one base from the position of the adaptor pairing, and 5 such primers are required to complete the sequencing of the strand to be detected. The double-base coding strategy is adopted for connection reaction, namely, each base of every two adjacent sites corresponds to one of 4 fluorescent signals in 16 arbitrarily combined 8-base probes, and when a nucleotide chain is extended, each site is scanned 2 times, the accuracy reaches 99.94%, but the double-base coding strategy can also cause linkage decoding errors. SOLiD can produce 75-110 bp read length in one cycle, and a sequencing sequence of 300 Gb. As with Illumina, SOLiD can be used for determining single nucleotide polymorphism, deletion, insertion and other genomic structural variations in whole genome resequencing, and can also be used for target region capture sequencing, chromatin co-precipitation sequencing and RNA sequencing, but has limited application due to the defects of long reading length, high cost and difficult analysis of data results.

Ion Torrent PGM and Proton semiconductor sequencing the Ion Torrent PGM and Proton (2012) were subsequently pushed out from Life Technologies purchased in 2010 by the american Ion Torrent company, which are sequencing platforms between the second generation and the third generation, the core technology of which is semiconductor sequencing developed by Ion Torrent company. Semiconductor sequencing techniques also employ microemulsion PCR, except that it detects a change in pH, rather than a fluorescent signal, caused by release of hydrogen ions when a single nucleotide is paired with a template strand immobilized on a chip. Ion Torrent PGM has 3 chips, 314 chips are suitable for minigenome sequencing, 316 and 318 chips are used for whole transcriptome sequencing and chromatin co-immunoprecipitation sequencing. The Ion Torrent PGM flux is low, but the speed is high, the cost is low, and the instrument scale is small, so the method is widely applied, and is suitable for 16s RNA sequencing, de novo sequencing and resequencing of microorganisms and viruses, target region capture sequencing, single nucleotide polymorphism detection, short tandem repeat sequencing, mixed infection identification, mitochondrial DNA sequencing and the like. However, ion Torrent PGM also has drawbacks such as accumulation of errors due to multiple elution processes, high error rate when reading highly repetitive sequences and homomultimeric sequences, and the like. The existing Roche GS Junior, ion TorrentPGM and MiSeq 3 small sequencers for clinical and small laboratory.

Third generation sequencing technology the third generation sequencing technology is characterized by single molecule sequencing, i.e., sequencing while synthesis is directly performed without PCR. This not only simplifies the sample handling process, avoids mismatches that may be introduced by amplification, but is also not affected by guanine and cytosine or adenine and thymine levels, so third generation sequencing techniques can directly sequence RNA and methylated DNA sequences. The existing third generation sequencing platforms include HeliScope genetic analysis system from Helicos Bioscience and PacBio RS single molecule real-time sequencing system from Pacific Biosciences.

HeliScope genetic analysis System HeliScope genetic analysis System was the first single molecule sequencing System, introduced by the company Helicos Bioscience in the United states in 2008. The basic process is that a single-stranded DNA template modified by 3' -end poly adenine is captured by a primer modified by poly thymine on a chip, under the action of DNA polymerase, fluorescent marked dNTPs are paired with a template chain, and base information can be obtained by collecting fluorescent signals. The system can generate 21-25 Gb per cycle, and the average read length of the sequence is 35 bp. The HeliScope genetic analysis system requires less sample size and has low sample quality, and can be used for detecting paleobiological information. HeliScope genetic analysis systems are most commonly used for gene expression analysis, such as gene expression capping analysis with Heli-Scope in the mammalian gene function annotation program.

PacBIO RS single molecule real-time sequencing System was developed by PacificBiosciences in 2010, and single DNA molecules were sequenced using four-color fluorescent-labeled dNTPs and zero-order waveguides (zeromode wave guides, ZMW) on a single molecule real-time chip. ZWM is a pore-like nano photoelectric structure with a diameter of 50-100 nm and a depth of 100nm, which decays exponentially when light enters, and only the part near the substrate is illuminated. DNA polymerase is immobilized at the bottom of the ZMW, DNA synthesis is performed after adding templates, primers and four-color fluorescent labeled dNTPs, and only the dNTPs participating in the reaction can stay at the bottom of the ZMW, so that fluorescent signals of the dNTPs are recognized. The PacBIO RS single-molecule real-time sequencing system has the advantage of long reading time, and the latest PacBIO RS II reading time can reach 20kb. PacBIO RS generates 400Mb sequence per cycle, with cost of $2-17/Mb, which has low flux, high cost and single base recognition error rate up to 14% compared with other NGS platforms, but can be improved by increasing cycle times, and can also be used in combination with second generation sequencing technology to reduce cost and increase accuracy. Rhoads et al have shown that PacBIO RS is suitable for de novo sequencing, mutation detection of gene structure, sequencing of complex repeats, detection of gene subtypes, 4-methylcytosine and 6-methyladenine.

The fourth generation sequencing technology is a single-molecule sequencing technology, but is different from a HeliScope genetic analysis system and a PacBIO RS single-molecule real-time sequencing platform, and does not need to carry out synthesis reaction, fluorescent marking, elution and CCD (charge couple device, CCD) camera shooting, so that double spanning from optical detection to electronic conduction detection and short-reading long-sequencing is realized, and the method is a real single-molecule sequencing technology, so the method is classified as the fourth generation sequencing technology. British Oxfold

Nanopore Technologies company has now proposed high throughput GridION (2012) and U disk size MinION (2013) sequencers, both in the trial phase. The companies Roche, illumina and Life Technologies, switzerland, etc., are also investing in nanopore sequencing, such as Roche invests Genia Technologies and Stratos Genomics. Compared with other NGS platforms, the nanopore sequencing technology has the advantages of long reading length, high throughput, low cost, short time consumption and relatively simple data analysis, and the future nanopore sequencing technology is expected to complete whole genome sequencing within several hours and hundreds of dollars after being put into the market. Since the size and shape of each base of the DNA molecule are different, the DNA molecule can cause characteristic current change when passing through the nanopore to form a circuit under the drive of electrophoresis, and thus the base type and arrangement sequence of the DNA molecule can be determined. The nanopore has two kinds of biological nanopores and solid-state nanopores, wherein the biological nanopores have alpha-hemolysin nanopores and MspA protein nanopores, and the solid-state nanopores are graphene nanopores. Compared with biological nanopores, solid-state nanopores are more stable. The cost of Minion is lower, the U disk size can reach 10kb, but DNA molecules pass through rapidly, it is difficult to distinguish base information and noise floor, the error rate is very high, therefore still need to improve. In application, nanopore sequencing can be used for single nucleotide, ssDNA, dsDNA, and RNA sequencing, while nanopore technology is also used for qualitative and quantitative characterization of DNA, micrornas, proteins, anions, cations, and organic molecules.

The update of the NGS platform realizes the high-throughput, low-cost and rapid detection of the whole genome, particularly the gradual maturation of the second generation sequencing technology, and promotes the application of NGS in the clinical field. More and more researchers select gene panel detection of NGS (a method of capturing target DNA and performing gene sequencing with several gene-corresponding probes on a chip) to study tumors, or identify potential drug targets by exon sequencing. Traditional diagnostic methods are also gradually being pursued in diagnosing childhood genetic disease, and whole exome sequencing is being used instead. NGS detection can help doctors determine treatment schemes, and can evaluate the risk of the same diseases of children in the future for parents of children, so that development of molecular diagnosis innovation and accurate medical treatment is certainly promoted. Overall, illumina sequencing platform is most commonly applied, sequencing cost is also remarkably reduced, and the goal of completing human genome sequencing for the first time is achieved at $1,000; roche 454 is mainly used for de novo sequencing and metagenomic research due to its long sequencing read length; ABI SOLiD has fewer applications than Illumina; ion Torrent PGM, which is intermediate between the second and third generations, is widely used because of its simplicity, rapidity, low cost and small-scale advantages. In clinical applications, the results of second generation sequencing still require verification of the sequencing gold standard, the first generation sequencing technology. Third generation and fourth generation sequencing technologies are currently in development, and although the read length is ten times longer than that of Sanger sequencing, the PacBIO RS single-molecule real-time sequencing system and the MinION sequencer have high error rates and still need to be improved. In addition, bioinformatics analysis of NGS data also presents a significant challenge. In summary, with a deeper understanding of genomic information, advances in sequencing technology, and innovations in bioinformatics analysis software, sequencing technology will play a greater role in biological and biomedical research.

The application of second generation sequencing to carry out gene detection is one of the most hot professional categories in the current biology field, and has been rapidly developed at home and abroad in recent years, so that the method can not only track the infectious disease path, but also predict the individual disease risk, effectively predict various diseases such as cancers, diabetes, down syndrome and the like, thereby providing effective help for later-stage defense and treatment.

From the above description, it can be known that the second generation sequencer, especially the hilleq X Ten of Illumina company, has the advantages of high throughput, low cost and accuracy. The technology of the bericukang and the technology of Huada gene company belong to the second generation technology. Third and fourth generation gene sequencing techniques, while promising, are not yet mainstream due to their low accuracy and low throughput. Regardless of the generation of technology, the performance sought after is mainly the following: (1) high accuracy, (2) high speed, (3) low cost, (4) portability (miniaturization).

Disclosure of Invention

Based on the above, the application aims to provide a sequencing method of nucleic acid molecules, which can be realized based on a simple single-excitation light source and a monochromatic light path system hardware sequencing instrument, and has high accuracy and low cost.

The application provides a sequencing method of a nucleic acid molecule, which comprises the following steps:

Providing a first set of nucleotides, wherein the nucleotides in the first set of nucleotides, which are paired with a site to be detected of the sequencing template, a sequencing primer, a first nucleic acid polymerase and non-catalytic competing metal ions react to form a non-covalent five-membered complex; the first set of nucleotides comprises a first dATP or analogue thereof, a first dTTP or analogue thereof, a first dCTP or analogue thereof, and a first dGTP or analogue thereof, each nucleotide or analogue thereof independently having a preset identity; detecting a preset mark correspondingly contained in the non-covalent five-membered complex, and obtaining first mark information;

scattering the non-covalent five-membered complex, retaining the sequencing template and the sequencing primer for covalent extension with a second set of nucleotides, a second nucleic acid polymerase, and a catalytic competing metal ion; the second set of nucleotides comprises a second dATP or analogue thereof, a second dTTP or analogue thereof, a second dCTP or analogue thereof, and a second dGTP or analogue thereof, each nucleotide or analogue thereof having a reversible terminator structure and each independently having a predetermined identity; detecting a preset mark correspondingly contained in a product obtained by covalent extension, and obtaining second mark information;

Combining the first identification information and the second identification information in binary system, and determining the type of the paired nucleotide according to the obtained combination information, so as to determine the type of the nucleotide of the site to be detected;

Wherein,

The condition satisfied by the first set of nucleotides and the second set of nucleotides includes that combination tag 1, combination tag 2, combination tag 3 and combination tag 4 are different from each other as defined below,

The combined identifier 1 is a preset identifier of a first dATP or analogue thereof in the first group of nucleotides and a preset identifier of a second dATP or analogue thereof in the second group of nucleotides;

the combined mark 2 is a preset mark of a first dTTP or an analogue thereof in the first group of nucleotides and a preset mark of a second dTTP or an analogue thereof in the second group of nucleotides;

The combined identifier 3 is a preset identifier of a first dCTP or an analogue thereof in the first group of nucleotides and a preset identifier of a second dCTP or an analogue thereof in the second group of nucleotides;

The combined signature 4 is a preset signature of a first dGTP or analogue thereof in the first set of nucleotides and a preset signature of a second dGTP or analogue thereof in the second set of nucleotides.

In some embodiments, the preset identity comprises a mark and a non-mark;

Optionally, the label comprises one or more of a fluorescent label, a chemiluminescent label, a bioluminescent label, an electrochemiluminescent label, an electrical signal label, and a magnetic signal;

further alternatively, the fluorescent label is selected from one or more of AF532, ATTO532, cy3B, cy, cy5, ATTO647N, ATTO647, and AF 647.

In some embodiments, the preset mark is marked, the identification information is marked as 1, the preset mark is unmarked, the identification information is marked as 0, and binary combination is performed by 1 and 0.

In some embodiments, the predetermined identity of the second dATP or analogue thereof or/and the second dGTP or analogue thereof in the second set of nucleosides is a label.

In some embodiments, the non-catalytic competing metal ions are selected from one or more of calcium ions, strontium ions, and barium ions.

In some embodiments, the catalytic competing metal ion is magnesium ion.

In some of these embodiments, the reversible terminator structure is selected from one of 3' -benzyl azide, 3' -R-SS-and 3' -NH ₂ -O-.

In some of these embodiments, the first nucleic acid polymerase and the second nucleic acid polymerase are each independently selected from one of Taq DNA polymerase, KOD DNA polymerase, 9°n DNA polymerase, and Klenow enzyme.

In some of these embodiments, the agent that breaks up the non-covalent five-membered complex comprises a ligand that complexes the non-catalytic competing metal ion;

optionally, the ligand is selected from one or more of EDTA, EGTA, NTA and HPTA;

Further alternatively, the reagent that breaks up the non-covalent five-membered complex comprises guanidine thiocyanate, sodium citrate, and EDTA;

Still further alternatively, the reagent for breaking up the non-covalent five-membered complex comprises water and 3.5M-4.5M guanidine thiocyanate, 40mM-45mM sodium citrate and 8mM-12mM EDTA, pH 7.5-8.5.

In some of these embodiments, the conditions under which the non-covalent five-membered complex is formed include one or more of the following items (1) and (2):

(1) The reaction temperature is 25.7-65.6 ℃ and the reaction time is 10-180 s; and, a step of, in the first embodiment,

(2) The initial reaction system comprises water, the first group of nucleotides, a sequencing template, a sequencing primer, 0.05U/mu L-0.2U/mu L of first nucleic acid polymerase, non-catalytic competitive metal ions, naCl, (NH ₄)₂SO₄, tween-20, EDTA, betaine and Tris;

Alternatively, the reaction initiation system comprises water and 0.2. Mu.M-1. Mu.M of the first set of nucleotides, 0.1pM-10pM sequencing template, 0.5. Mu.M-1.5. Mu.M sequencing primer, 0.05U/. Mu.L-0.2U/. Mu.L of the first nucleic acid polymerase, 0.45mM-0.55mM non-catalytic competing metal ion 、45mM-55mM NaCl、55mM-65mM(NH₄)₂SO₄、0.045％-0.055％ Tween-20、0.08mM-1mM EDTA、0.8M-1.2M betaine, 0mM-55mM LiCl, 0% -0.12% hydroxylamine, 45mM-55mM Tris and 0% -5.5% DMSO, and the pH is 7.5-9.

In some of these embodiments, the conditions for covalent extension include one or more of the following (1) and (2):

(1) The reaction temperature is 60-65 ℃ and the reaction time is 55-65 s; and, a step of, in the first embodiment,

(2) The initial reaction system comprises water, the first group of nucleotides, a sequencing template, a sequencing primer, 0.05U/mu L-0.2U/mu L of second nucleic acid polymerase, catalytic competitive metal ions, naCl, (NH ₄)₂SO₄, tween-20, EDTA, DMSO and Tris;

Alternatively, the reaction initiation system comprises water and 0.2. Mu.M-1. Mu.M of the second set of nucleotides, 0.1pM-10pM sequencing template, 0.5. Mu.M-1.5. Mu.M sequencing primer, 0.05U/. Mu.L-0.2U/. Mu.L of the second nucleic acid polymerase, 2mM-4mM catalytic competing metal ion 、45mM-55mM NaCl、55mM-65mM(NH₄)₂SO₄、0.045％-0.055％ Tween-20、0.08mM-1mM EDTA、4.5％-5.5％DMSO and 45mM-55mM Tris, pH 7.5-9.

In some of these embodiments, the sequencing method further comprises, after determining the identity of the nucleotide of the test site, cleaving the reversible terminator structure to determine the identity of the nucleotide of the next site to the test site;

alternatively, the method of determining the kind of the nucleotide of the next site is the same as the method of determining the kind of the nucleotide of the site to be detected.

Compared with the prior art, the application has the following beneficial effects:

In the sequencing process, based on the specific identifiers of two groups of nucleotides, aiming at the same position to be tested of a sequencing template, the nucleotides with preset identifiers in a first group of nucleotides are introduced into the position to be tested in the form of forming a five-membered complex to obtain first identifier information, and then the nucleotides with preset identifiers in a second group of nucleotides are introduced into the same position in the form of covalent extension to obtain second identifier information, so that the first identifier information and the second identifier information are doubly confirmed by adopting binary system, and the accuracy of base identification is improved. The sequencing method provided by the application is based on the identification of the preset identifiers of two groups of nucleotides, can be realized by a simple single-excitation light source and a monochromatic light path system hardware sequencing instrument, and is low in cost.

Drawings

In order to more clearly illustrate the technical solution in the embodiments of the present application and to more fully understand the present application and its advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments. It is evident that the figures in the following description are only some embodiments of the application, from which other figures can be obtained without inventive effort for a person skilled in the art.

FIG. 1 is a flow chart of the formation of a non-covalent five-membered complex and the extension of a covalent reversible terminator;

FIG. 2 is a schematic diagram of a non-covalent five-membered complex: template-sequencing primer-non-catalytic competing metal ion-polymerase-nucleoside;

FIG. 3 is a diagram of the test mentioned in example 1;

FIG. 4 is a diagram of the test mentioned in example 2;

FIG. 5 is a diagram of one embodiment mentioned in example 3;

FIG. 6 is a diagram of one embodiment mentioned in example 3;

FIG. 7 is a diagram of one embodiment mentioned in example 3;

FIG. 8 is a diagram of one embodiment mentioned in example 3;

FIG. 9 is a diagram of the test mentioned in example 4;

FIG. 10 is a diagram of the test mentioned in example 5;

FIG. 11 is a diagram of the test mentioned in example 6;

FIG. 12 is a test chart mentioned in example 7.

Detailed Description

The present application will be described in further detail with reference to the drawings, embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the application and are not intended to limit the scope of the application in order that the present disclosure may be more thorough and complete. It will also be appreciated that the present application may be embodied in many different forms and is not limited to the embodiments and examples described herein, but may be modified or altered by persons skilled in the art without departing from the spirit of the application, and equivalents thereof are also intended to fall within the scope of the application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the application, it being understood that the application may be practiced without one or more of these details.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing the embodiments and examples only and is not intended to be limiting of the invention.

Terminology

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

The term "and/or", "and/or" as used herein includes a selection of any one of two or more of the listed items and also includes any and all combinations of the listed items, including any two or more of the listed items, or all combinations of the listed items. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it should be understood that, in the present application, the technical solutions include technical solutions that all use "logical and" connection, and also include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical schemes of all "logical or" connections), also include any and all combinations of A, B, C, D, i.e., the combinations of any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical schemes of all "logical and" connections).

The terms "plurality", "plural", "multiple", and the like in the present invention refer to, unless otherwise specified, an index of 2 or more in number. For example, "one or more" means one kind or two or more kinds.

As used in this disclosure, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.

In the present invention, "suitable" in "suitable combination manner", "suitable manner", "any suitable manner", etc., are used to implement the technical scheme of the present invention, solve the technical problem of the present invention, and achieve the technical effects expected by the present invention.

In the present invention, "preferred", "better", "preferred" are merely embodiments or examples which are better described, and it should be understood that they do not limit the scope of the present invention.

In the present invention, "further", "still further", "particularly" and the like are used for descriptive purposes to indicate differences in content but should not be construed as limiting the scope of the invention.

In the present invention, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, a numerical range (i.e., a numerical range) is referred to, and optional numerical distributions are considered to be continuous within the numerical range and include two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range and each numerical value between the two numerical endpoints unless otherwise specified. Where a numerical range merely refers to integers within the numerical range, including both end integers of the numerical range, and each integer between the two ends, unless otherwise indicated, each integer is recited herein as directly, such as where t is an integer selected from 1 to 10, and where t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8,9, and 10. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or may vary within a predetermined temperature range. It should be appreciated that the constant temperature process described allows the temperature to fluctuate within the accuracy of the instrument control. Allows for fluctuations within a range such as + -5 ℃, + -4 ℃, + -3 ℃, + -2 ℃, + -1 ℃.

In the present invention,% (w/w) and wt% each represent weight percent,% (v/v) represents volume percent, and% (w/v) represents mass volume percent.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Unless otherwise indicated to the contrary by the intent and/or technical aspects of the present application, all references to which this application pertains are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are also incorporated into the present application by reference, but are not limited to being able to implement the present application. It should be understood that when a reference is made to the description of the application in conflict with the description, the application is modified in light of or adaptive to the description of the application.

Wherein,

The nucleotide refers to natural or non-natural, labeled or non-labeled nucleotide, the labeling site comprises phosphoric acid, pentose and base, the preferential labeling site is 7 th position of purine base and 5 th position of pyrimidine base, and can contain a reversible terminator structure or not, and the labeled or non-labeled nucleotide containing the reversible terminator structure is preferentially selected to ensure that sequencing is synchronous and avoid advance. The class of natural nucleotides or analogs thereof includes, but is not limited to, nucleotide Triphosphates (NTPs), such as ribonucleotide triphosphates (rtps), deoxyribonucleotide triphosphates (dntps), or non-natural analogs thereof, such as dideoxyribonucleotide triphosphates (ddntps) or a reversible terminating nucleotide triphosphate (rtNTP). Nucleotide analogs that participate in the formation of stable non-covalent five-membered complexes may include a reversible terminator structure at the 3' end that may prevent any possible covalent extension. U.S. patent application 7,057,026 (Illumina) describes the structure of reversible terminators linked at pentoses, another type of reversible terminator linked to a nitrogen-containing base of a nucleotide. Other reversible terminators that may similarly be used in connection with the methods described in the patents include those described in U.S. patent application 7,713,698 (university of Columbia).

Nucleotide analogs are polyphosphates containing modifications including, but not limited to, triphosphates, tetraphosphates, pentaphosphates, hexaphosphates, heptaphosphates, octaphosphates, and nonaphosphates. The linkage of Alpha and Beta phosphates may be a hydrolyzable oxygen atom, a non-hydrolyzable sulfur atom, a selenium atom, a methine group, and the like.

Nucleotide analogs that contain reversible terminators that prevent covalent extension of nucleotides to the 3' end of the primer from which they have been covalently extended. The irreversible-containing nucleotide analogs include 2',3' -dideoxynucleotides (ddNTPs, e.g., ddGTP, ddATP, ddTTP, ddCTP). Dideoxynucleotides lack the 3' -OH group of dNTPs, which would otherwise be involved in polymerase mediated primer extension. Irreversibly terminated nucleotides are particularly useful for genotyping applications. Alternatively, the reversible terminator structure includes, but is not limited to, 3' -benzyl azide, 3' -R-SS-and 3' -NH ₂ -O-, and the like.

Nucleic acid polymerases covalently add nucleotides one by one to an increasing nucleic acid strand, the covalently added nucleotides being complementary to the nucleotides of the template. Nucleic acid polymerases, including but not limited to DNA polymerases, RNA polymerases, reverse transcriptases, primer enzymes, and transferases. Typically, a nucleic acid polymerase has one or more active sites at which nucleotide binding and/or catalysis of nucleotide polymerization can occur. The nucleic acid polymerase can catalyze the polymerization of nucleotides to the 3' end of the first strand of the double-stranded nucleic acid molecule. For example, a nucleic acid polymerase catalyzes the addition of the next correct nucleotide to the 3' hydroxyl group of the first strand of a double-stranded nucleic acid molecule via a phosphodiester bond, thereby covalently binding the nucleotide to the first strand molecule of the double-stranded nucleic acid. Optionally, the nucleic acid polymerase need not be capable of undergoing covalent extension of nucleotides under one or more of the conditions used in the proprietary methods. For example, a mutant polymerase may be capable of forming a non-covalent five-membered complex but not catalyzing covalent extension of a nucleotide. Nucleic acid polymerases include, but are not limited to Taq polymerases,9degree N,Thermococcus kodakaraensis(KOD)DNA polymerase,Klenow,Moloney Murine Leukemia Virus(M-MLV)Reverse Transcriptase and Avian Myeloblastosis Virus REVERSE TRANSCRIPTASE (AMV RT).

The nucleotides involved in forming the non-covalent five-membered complex may include exogenous labels including, but not limited to, labeled affinity streptomycin and biotin, the exogenous labeled nucleotides may include a reversible or irreversible terminator moiety, the exogenous labeled nucleotides may be non-extendible or extendible, and the exogenous labeled nucleotides may be a mixture of both non-extendible or extendible groups.

The nucleic acid polymerase involved in the formation of the non-covalent five-membered complex may be exogenously labeled and may provide a paired donor or acceptor in the pair. Nucleotides involved in the formation of non-covalent five-membered complexes may be exogenously labeled and may provide a receptor or donor in a Fluorescence Resonance Energy Transfer (FRET) pair. Thus, FRET detects a Fluorescence Resonance Energy Transfer (FRET) pair (acceptor or donor) in a non-covalent five-membered complex, thereby effecting nucleotide base recognition, wherein the unlabeled nucleotide may include a reversible or irreversible terminator moiety, and the unlabeled nucleotide may be extendable or non-extendable. The labeled nucleotides may include a reversible or irreversible terminator moiety, and the unlabeled nucleotides may be extendable or non-extendable.

During the formation of the stable non-covalent five-membered complex, nucleotides (e.g., natural nucleotides or nucleotide analogs, labeled or unlabeled nucleotides) may be present in the mixture in various combinations. For example, at least 1, 2,3, 4 or more nucleotide types may be present. Or up to 4, 3, 2 or 1 nucleotide types may be present. Similarly, the one or more nucleotide types present may be complementary to at least 1, 2,3 or 4 base types in the template nucleic acid. Or the one or more nucleotide types present may be complementary to up to 4, 3, 2 or 1 base types in the template nucleic acid.

A primer refers to a nucleic acid that has binding (e.g., complementarity) to a nucleic acid at or near the template. Typically, the primers are bound in a configuration that allows replication of the template, e.g., by nucleic acid polymerase extension of the primers (e.g., the primers may need to be deblocked prior to replication). In some embodiments, the primer is capable of binding to a nucleic acid polymerase upon hybridization of the primer to the template, and the primer may be covalently extended in the presence of cofactor metal ions and nucleotides, or form a non-covalent five-membered complex in the presence of cofactor non-catalytic competing metal ions. The primers may have any suitable length. The primer may be a hairpin primer, a first portion of the template molecule that binds to a second portion of the template molecule, the first portion being a primer sequence and the second portion being a primer binding sequence. The primer may consist of DNA、RNA,PNA(peptide nucleic acid oligonucleotide),LNA(locked nucleic acid,also known as bridged nucleic acid(BNA) or an analogue thereof. Peptide nucleic acid mimics oligonucleotides (PNAs) because the ability to base pair is still present, whereas the sugar phosphate backbone has been replaced with a charge neutral polyamide. Locked Nucleic Acid (LNA) a modified RNA nucleotide in which the ribose moiety is modified as an additional bridge linking the 2 'oxygen and 4' carbon. The primer need not be extended by the formation of new phosphodiester bonds nor shortened by nucleic acid degradation during the step of forming a stable non-covalent five-membered complex or during the step of checking for a stable non-covalent five-membered complex.

The template is typically genomic DNA (gDNA), complementary DNA (cDNA) derived from reverse transcription of RNA or a plasmid containing cloned DNA fragments, or exosome DNA or RNA, or episomal DNA or RNA (cfDNA, cfrna), and can be obtained from preparation methods such as genomic isolation, genomic fragmentation, gene cloning and/or amplification. The template includes a genomic fragment of the same sequence as a portion of the genome. A set of genome fragments may comprise a percentage of from 0.1% to 100% genome. The template can be obtained by single molecule or single molecule concatemer or monoclonal multi-molecule cluster through amplification technology such as Polymerase Chain Reaction (PCR), rolling Circle Amplification (RCA), recombinase polymerase chain Reaction (RPA), multiple Displacement Amplification (MDA) and the like in a test tube solution or generated in situ on the surface of a chip. Exemplary methods for isolating, amplifying, and fragmenting nucleic acids to produce templates for analysis on an array are set forth in the following patents and articles. U.S. patent application 6,355,431(Illumina),"Molecular Cloning:A Laboratory Manual",3rd edition,Cold Spring Harbor Laboratory,New York(2001,Sambrook, et al), and "Current Protocols in Molecular Biology", john Wiley and Sons, baltimore, md. (1998, ausubel, et al).

The "polymerase" requires a metal ion as a cofactor for its active function, and typically, the polymerase has two binding sites for the metal ion, each of which functions to complex with and form a covalent phosphodiester. Alternatively, the first nucleic acid polymerase and the second nucleic acid polymerase are each independently selected from one of Taq DNA polymerase, KOD DNA polymerase, 9°n DNA polymerase, and Klenow enzyme. Wherein, the "non-catalytic competing metal ion" does not promote phosphodiester bond formation, but only non-covalent five-membered complexes, and thus does not allow covalent extension of the primer, in a process that can generate non-covalent five-membered complexes including primers, templates, polymerase non-catalytic competing metal ions, and labeled or unlabeled nucleotides with reversible terminator structures. Non-catalytic competing metal ions can interact with the polymerase, for example, by competitive binding compared to catalytic metal ions. The species of divalent non-catalytic competing metal ions include, but are not limited to, ca ²⁺、Zn²⁺、Co²⁺、Ni²⁺ and Sr ² ⁺. The species of trivalent non-catalytic competing metal ions include, but are not limited to, fe ³⁺、Co³⁺、Eu³⁺、Ni³⁺, and Tb ³⁺ ions. The addition of organic cationic compounds, the types of which include but are not limited to betaines, quaternary ammonium salts, and quaternary phosphonium salts, may further enhance the function of non-catalytic competing metal ions. Optionally, the non-catalytic competing metal ions are selected from one or more of calcium ions, strontium ions and barium ions. Optionally, the catalytic competing metal ion is magnesium ion.

Optionally, the preset identifier includes a mark and a non-mark. Optionally, the label comprises one or more of a fluorescent label, a chemiluminescent label, a bioluminescent label, an electrochemiluminescent label, an electrical signal label, and a magnetic signal; further alternatively, the fluorescent label is selected from one or more of AF532, ATTO532, cy3B, cy, cy5, ATTO647N, ATTO647, and AF 647. Optionally, the preset mark is marked, the identification information is marked as 1, the preset mark is unmarked, the identification information is marked as 0, and binary combination is performed by 1 and 0.

Optionally, the predetermined identity of the second dATP or analogue thereof or/and the second dGTP or analogue thereof in the second set of nucleosides is a label.

Optionally, the reagent that breaks up the non-covalent five-membered complex comprises a ligand that complexes the non-catalytic competing metal ion;

Still further alternatively, the reagent for breaking up the non-covalent five-membered complex comprises water and 3.5M-4.5M guanidine thiocyanate, 40mM-45mM sodium citrate and 8mM-12mM EDTA, pH 7.5-8.5. In some examples, the guanidine thiocyanate concentration is, for example, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5M, the sodium citrate concentration is, for example, 40, 41, 42, 43, 44, 45mm, the edta concentration is, for example, 8, 9, 10, 11, 12mm, and the ph is, for example, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5.

Optionally, the conditions for reacting to form the non-covalent five-membered complex include one or more of the following (1) and (2):

(1) The reaction temperature is 25.7-65.6deg.C (e.g., 25.7, 26, 30, 35, 40, 45, 50, 55, 60, 65, 65.5deg.C; alternatively 35-50deg.C; further alternatively 36.2-45.8deg.C) and the reaction time is 10s-180s (e.g., 10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180s; alternatively 30s-180s; further alternatively 60s-180 s); and, a step of, in the first embodiment,

Alternatively, the reaction initiation system comprises water and 0.2. Mu.M-1. Mu.M of the first set of nucleotides, 0.1pM-10pM sequencing template, 0.5. Mu.M-1.5. Mu.M sequencing primer, 0.05U/. Mu.L-0.2U/. Mu.L first nucleic acid polymerase, 0.45mM-0.55mM non-catalytic competing metal ion 、45mM-55mM NaCl、55mM-65mM(NH₄)₂SO₄、0.045％-0.055％ Tween-20、0.08mM-1mM EDTA、0.8M-1.2M betaine, 0mM-55mM LiCl, 0% -0.12% hydroxylamine, 45mM-55mM Tris and 0% -5.5% DMSO, The pH value is 7.5-9. in some examples, the concentration of each nucleotide in the first set of nucleotides is, for example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1. Mu.M, the concentration of the sequencing template is, for example, 0.1, 0.5, 1, 2, 3, 4,5, 6, 7, 8, 9, 10pM, the concentration of the sequencing primer is, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5. Mu.M, the concentration of the first nucleic acid polymerase is, for example, 0.05, 0.07, 0.09, 0.12, 0.14, 0.16, 0.18, 0.2U/. Mu.L, the concentration of non-catalytic competing metal ions being, for example, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55mM, the concentration of NaCl being, for example, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55mM, (the concentration of NH ₄)₂SO₄ being, for example, 55, 56, 57, 58, 59, 60, 61), 62. 63, 64, 65mM, tween-20, for example 0.045%, 0.046%, 0.047%, 0.048%, 0.049%, 0.05%, 0.051%, 0.052%, 0.053%, 0.054%, 0.055%, EDTA, for example 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1mM, betaine, for example 0.8, 0.9, 1, 1.1, 1.2M, liCl may or may not be added, the concentration of the hydroxyl amine may or may not be added, for example, 0, 5, 10, 20, 30, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55mM, the concentration of the hydroxyl amine may or may not be added, for example, 0%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, the concentration of Tris may be, for example, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55mM, DMSO, The concentration may be, for example, 0%, 1%, 2%, 3%, 4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, and the pH may be, for example, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.

In some of these examples, the conditions for covalent extension include one or more of the following items (1) and (2):

(1) The reaction temperature is 60-65 ℃ (for example, 60, 61, 62, 63, 64, 65 ℃), and the reaction time is 55s-65s (for example, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 s); and, a step of, in the first embodiment,

Alternatively, the reaction initiation system comprises water and 0.2. Mu.M-1. Mu.M of the second set of nucleotides, 0.1pM-10pM sequencing template, 0.5. Mu.M-1.5. Mu.M sequencing primer, 0.05U/. Mu.L-0.2U/. Mu.L of the second nucleic acid polymerase, 2mM-4mM catalytic competing metal ion 、45mM-55mM NaCl、55mM-65mM(NH₄)₂SO₄、0.045％-0.055％Tween-20、0.08mM-1mM EDTA、4.5％-5.5％DMSO and 45mM-55mM Tris, pH 7.5-9. In some examples, the concentration of each nucleotide in the second set of nucleotides is, for example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1. Mu.M, the concentration of the sequencing template is, for example, 0.1, 0.5, 1, 2,3, 4, 5, 6, 7, 8, 9, 10pM, the concentration of the sequencing primer is, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5. Mu.M, the concentration of the second nucleic acid polymerase is, for example, 0.05, 0.07, 0.09, 0.12, 0.14, 0.16, 0.18, 0.2U/. Mu.L, the concentration of the catalytic competing metal ion is, for example, 2.5, 3.5, 4mM, the concentration of NaCl is, for example, 45, 46, 48, 49, 50, 51, 53, 55mM, (NH ₄)₂SO₄ concentration such as 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65mM, tween-20 concentration such as 0.045%, 0.046%, 0.047%, 0.048%, 0.049%, 0.05%, 0.051%, 0.052%, 0.053%, 0.054%, 0.055%, EDTA concentration such as 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1mM, DMSO concentration such as 4.5%, 4.6%, 4.7%, 4.8, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5.5%, tris concentration such as 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55mM, pH such as 7.5, 7.7, 7.8, 8.8, 8.8.8, 8.8.8.8).

In some examples, the sequencing method further comprises, after determining the identity of the nucleotide of the test site, cleaving the reversible terminator structure to determine the identity of the nucleotide of the next site of the test site. The present application is not particularly limited to reagents that cleave the reversible terminator structure, including but not limited to N3 cleavage reagents, such as: the 3' -end blocking group and the fluorescent group are removed by using a cutting reagent with THPP, TECP and other reducing agents as main components.

In some of these examples, the method of determining the kind of nucleotide of the next site is the same as the method of determining the kind of nucleotide of the site to be detected.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to the guidelines given in the present invention, and may be according to the experimental manual or conventional conditions in the art, the conditions suggested by the manufacturer, or the experimental methods known in the art.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

Embodiments are described below:

Instrument: large-scale self-developed principle prototypes, or other instruments supporting second generation sequencing; and (3) a chip: a large-scale self-developed chip, or other gene sequencing chip, or Illumina company Miseq micro v's sequencing chip; the library is optionally human whole genome or phix or other DNA fragment combinations.

Reagent: single molecules or monoclonal molecular clusters are immobilized or generated on the surface in a random or lattice mode, a sequencing primer combined with a template cluster is added, and the 3' end of the sequencing primer is provided with normally-extendable nucleotides (labeled and unlabeled reversible terminator nucleotides). The next base of the sequencing primer paired with the template contains A, T, C, G bases. The formation of the non-covalent five-membered complex and the extension of the covalent reversible terminator are performed at the same site. Based on the cluster generation reagent in Miseq v < 2 > kit, a Phix on-machine library with a certain concentration is prepared, the cluster generation and R1 chain primer hybridization are carried out on a Miseq Micro Flow Cell chip, and the generated R1 chain is sequenced by preparing a reagent related to SBB+SBS sequencing.

The flow chart of the formation of the non-covalent five-membered complex and the extension of the covalent reversible terminator is shown in FIG. 1, and is illustrated below in conjunction with FIG. 1:

in the first step, a pre-wash buffer B is added to the reaction tank, and the reaction tank environment is suitable for the formation of the following non-covalent five-membered complex.

The reaction solution of the non-covalent five-membered complex comprises 45mM-55mMTris、45mM-55mMNaCl、55mM-65mM(NH₄)₂SO₄、0.045％-0.055％Tween-20、0.08mM-0.12mM EDTA、0.45mM-0.55mM CaCl₂、0.8M-1.2M betaine, 0mM-55mM LiCl, 0% -0.12% hydroxylamine solution, 0.2. Mu.M-1. Mu.M Hot dNTP-532 (fluorescence labeled reversible terminator nucleotide A, T, C, G, each nucleotide concentration of 0.2. Mu.M-1. Mu.M), 0.05U/. Mu.L-0.2U/. Mu.L 9 DEG N enzyme, 0% -5.5% DMSO and no nucleic acid water, pH7.5-9, 25 ℃.

The difference between the pre-wash buffer B and the non-covalent five-membered complex reaction solution is that it does not contain metal ions (Na, ca, li), betaine, nucleotides and 9 DEG N enzyme.

In the second step, the non-covalent five-membered complex reaction solution containing the Hot dATP-AF532 is added into a reaction tank, reacted for 10s-180s at 25.7-65.6 ℃, and the next base of a sequencing primer (the initial concentration of the reaction can be 0.1pM-10 pM) paired with a sequencing template (the initial concentration of the reaction can be 0.5 mu M-1.5 mu M) is A, so that the non-covalent five-membered complex containing the base A is formed, and the structure diagram is shown in figure 2.

And thirdly, flushing out redundant reagents which do not form stable non-covalent five-membered complexes (the non-covalent five-membered complexes are dynamic complexes) by using a scanning buffer (a scanning buffer B in the figure 1), wherein the scanning buffer is prepared by reducing fluorescent labeled reversible terminator nucleotides and 9 DEG N enzyme in a non-covalent five-membered complex reaction solution, but adding 45mM-55mM sodium ascorbate, wherein the sodium ascorbate has the effect of not quenching a fluorescent signal too fast when the laser shooting is carried out. If the next base of the sequencing primer paired with the sequencing template is the cluster point of A, fluorescence can be generated under the laser excitation of AF 532.

And fourthly, adding a break-up buffer solution, and rapidly breaking up five-membered complexes generated by the sequencing template, the sequencing primer, the nucleotide, the divalent metal and the polymerase within 2 seconds and cleaning, wherein hybridization between the sequencing primer and the sequencing template is not affected. In addition, the scattering buffer solution contains 3.5M-4.5M guanidine thiocyanate, 40mM-45mM sodium citrate, 8mM-12mM EDTA and sterilized water (pH 7.5-8.5, 25 ℃), and has good scattering effect, and particularly, the scattering buffer solution can be washed out only for 1.8s by adopting the best formula tested by us (comprising 4M guanidine thiocyanate, 42mM sodium citrate, 10mM EDTA and sterilized water, pH 8.0).

Fifthly, adding a Hot dATP-AF532 fluorescence reversible termination base extension reaction solution, wherein the reversible terminator extension reaction solution comprises 45mM-55mM Tris、45mM-55mM NaCl、55mM-65mM(NH₄)₂SO₄、2mM-4mM MgSO₄、0.08mM-1mM EDTA、0.045％-0.055％Tween-20、4.5％-5.5％ DMSO、0.2μM-1μM Cold dGTP、0.2μM-1μM Cold dTTP、0.2μM-1μM Hot dATP-532、0.2μM-1μM Cold dCTP、0.05U/μL-0.2U/μL 9°N enzyme and non-nucleic acid water (pH 7.5-9, 25 ℃), the concentration of a sequencing template can be 0.1pM-10pM, and the concentration of a sequencing primer can be 0.5 mu M-1.5 mu M sequencing primer. The photographing result shows that the fluorescence of the non-covalent five-membered complex of the reversible termination base of the Hot dATP-AF532 fluorescence of the same site is the same as the fluorescence of the covalent extension.

Sixth, the base 3' -end blocking group is excised by N3 cleavage reagent (corresponding to the cleavage buffer in FIG. 1), and the formation of the co-sited non-covalent five-membered complex and the extension of the covalent reversible terminator proceed in the next round.

TABLE 1

Example 1 formation of non-covalent five-membered Complex with 3' -end blocked and unblocked sequencing primer

The method comprises the following steps:

1. The sequencing reaction system included a second generation sequencer and chip (chip specifically refers to a chip that had completed the generation of a library cluster using Miseq kit and hybridized to the R1 sequencing primer ", the library was denatured with 0.2N NaOH and diluted to a concentration of 0.25pM with HT1 diluent). The library used in this example was a human whole genome library, and the sequence of the sequencing primers used was as follows: 5'-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-3' (SEQ ID NO. 1).

2. Preparing a sequencing reaction solution:

The 3' end of the sequencing primer is a normally extendable nucleotide (labeled and unlabeled reversible terminator nucleotides). The next base of the sequencing primer paired with the template contains A, T, C, G bases. In this example, taking the fluorescent labeled A base as an example, the formation of a noncovalent five-membered complex is performed under the conditions that the 3 '-end of the sequencing primer is blocked and unblocked, and all bases in this example are bases with the 3' -end reversibly blocked unless otherwise specified.

Non-covalent five-membered complex reaction solution: AF532 fluorophore-modified dA bases (0.2. Mu.M), fluorophore-free modified dC, dT, dG (0.2. Mu.M each), 0.2U/. Mu.L of 9℃N enzyme, buffer comprising 50mM Tris, 50mM NaCl, 60mM (NH ₄)₂SO₄、0.5mM CaCl₂, 1M betaine, 1mM EDTA, 0.05% Tween-20, 5% DMSO and nuclease-free water, reaction time of 60s during pH8.8, reaction temperature of 58 ℃.

Reversible terminator extension reagents include: dATP, dCTP, dTTP, dGTP (0.2. Mu.M each) without fluorescent modification, 0.2U/. Mu.L 9℃N enzyme 、50mM Tris、50mM NaCl、60mM(NH₄)2SO₄、3mM MgSO₄、1mM EDTA、0.05％ Tween-20、5％ DMSO and water without ribozyme, pH8.8. In the reaction process, the reaction time is 60s, and the reaction temperature is 61-65 ℃.

3. Formation of non-covalent five-membered complexes with 3' end blocking of sequencing primer: firstly, the reversible terminator extension reagent prepared by the 3 'closed base without the fluorescent group modification is used for extension to form a 3' closed primer. The chip was then rinsed with pre-wash buffer B, which had a reduced amount of labeled/unlabeled nucleotides, 9°n enzyme, metal ions and betaine compared to the non-covalent five-membered complex reaction. Then adding a non-covalent five-membered complex reaction solution, and reacting for 1min at room temperature to form a stable non-covalent five-membered complex by using a sequencing template, a sequencing primer, a nucleic acid polymerase, calcium ions and fluorescent molecule labeled nucleotides.

4. Signal detection of non-covalent five-membered complex under 3' -end blocking condition of sequencing primer: and adding a scanning buffer solution B to wash out redundant residual reagents which do not form stable non-covalent five-membered complexes, wherein compared with a non-covalent five-membered complex reaction solution, the components of the scanning buffer solution B are reduced in marked/unmarked nucleotide and 9 DEG N enzyme, 50mM sodium ascorbate is added, and the sodium ascorbate is used for protecting fluorescent signals from quenching too fast when the fluorescent signals are photographed by laser, such that cluster point fluorescent signals can be detected, fluorescent molecular markers A base, a sequencing template, a sequencing primer, polymerase and calcium ions are displayed, and the stable non-covalent five-membered complexes are formed. FIG. 3A is a diagram showing the result of forming a non-covalent five-membered complex of the A base component in the case of 3' end closure.

5. Cleaning of non-covalent five-membered complex under 3' -end closure condition of sequencing primer: the five-membered complex formed by the template, sequencing primer, nucleotide, divalent metal (Ca ²⁺) and polymerase (FIG. 3, FIG. B is a non-covalent five-membered complex break-up result in FIG. A) can be broken up and washed by adding a break-up buffer, but still keep hybridization of the sequencing primer and sequencing template unaffected.

6. Formation of five-membered Complex with 3' -end of sequencing primer unblocked: the 3' -end is directly utilized as a sequencing primer capable of extending the nucleotide normally, or the 3' -end blocking primer is subjected to the removal of the 3' -end blocking group by a cutting reagent taking a reducing agent such as THPP or TECP as a main component, and then the chip is washed by a pre-washing buffer solution B, so that compared with a non-covalent five-membered complex reaction solution, the components of the sequencing primer are reduced in marked/unmarked nucleotide, 9 DEG N enzyme, metal ion and betaine. Then adding a non-covalent five-membered complex reaction solution, and reacting for 1min at room temperature to form a stable non-covalent five-membered complex by using a sequencing template, a sequencing primer, a nucleic acid polymerase, calcium ions and fluorescent molecule labeled nucleotides. After the reaction described in the step 4, the fluorescent signal of the cluster A can be detected as shown in the graph C in FIG. 3, and after the reaction described in the step 5, the fluorescent signal of the cluster A disappears as shown in the graph D in FIG. 3, so that a stable noncovalent five-membered complex can be formed under the condition that the 3' -end of the primer is not blocked.

Example 2 identical site non-covalent five-membered Complex Signal and reversible terminator extension Signal identity

The method comprises the following steps:

1. The sequencing reaction system includes, but is not limited to, the above-described instruments and chips (chip specifically refers to a chip that has been used to complete the generation of a library cluster and hybridization of the R1 sequencing primer using Miseq kit ", the library was denatured using 0.2N NaOH and diluted to a concentration of 0.25pM using HT1 diluent). The library used in this example was a human whole genome library, and the sequence of the sequencing primers used was as follows: 5'-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-3' (SEQ ID NO. 1).

2. Non-covalent five-membered complex signal: the 3' end of the sequencing primer is a normally extendable nucleotide (labeled and unlabeled reversible terminator nucleotides). The next base of the sequencing primer paired with the sequencing template comprises A, T, C, G bases. In this example, sequencing primers with unblocked 3' -ends were used.

Firstly, a pre-washing buffer solution B is added to maintain the environment in the chip, and compared with a non-covalent five-membered complex reaction solution, the components of the pre-washing buffer solution B reduce labeled nucleotide, 9 DEG N enzyme, betaine and metal ions. Then adding non-covalent five-membered complex reagent (pH8.0 ℃) which comprises 50mM Tris、50mM NaCl、60mM(NH₄)₂SO₄、0.05％ Tween-20、0.1mM EDTA、0.5mM CaCl₂、1M betaine, 50mM LiCl, 0.1% hydroxylamine solution, 0.8 mu M Hot dNTP-532/0.8 mu M Cold dNTP, 0.05U/. Mu.L 9 DEG N enzyme and non-nucleic acid water. The fluorescent labeled single base dNTPs are selected for testing, and the reaction is carried out for 1min at room temperature. The cluster point signal of the stable point can be detected as shown in FIG. 4, which shows that the template, the sequencing primer, the nucleic acid polymerase, the calcium ion and the fluorescent molecule marked nucleotide form a stable non-covalent five-membered complex; and then adding a break-up buffer solution to break up the five-membered complex, cleaning the complex, and extending the reversible terminator at the same site.

3. Extension of reversible terminator: after the above reaction, the same fluorescent base was extended at the same site with a reversible terminator (the temperature of the extension reaction was 61 to 65 ℃ C. For 60 s), and the reversible terminator extension reagent was similar to that of example 1 except that the combination of the labeled/unlabeled fluorescent reversible terminator bases was different. After the extension reaction, adding a scanning buffer solution A to wash out a reversible terminator reaction solution which does not participate in the reaction, and collecting signals; the scan buffer a composition includes: 1M Tris, 400mM NaCl, 60mM sodium ascorbate, 0.25mM EDTA, 0.05% Tween-20, ultrapure water.

4. Cleavage of reversible terminator fluorophore and 3' -end blocking (N3 cleavage reagent): removing the 3 '-end blocking group and the fluorescent group by using a cutting reagent taking THPP reducer as a main component, so that the 3' -end of the primer can be used for forming a non-covalent five-membered complex and extending a reversible terminator in the next period;

5. By the detection scheme, the non-covalent five-membered complexes of ACT bases (corresponding to A diagram, C diagram and E diagram in FIG. 4) are respectively compared, signals of reversible terminator extension (corresponding to B diagram, D diagram and F diagram in FIG. 4) show that the signals of the non-covalent five-membered complexes at the same site are consistent with the signals of reversible terminator extension, and the formed non-covalent five-membered complexes have base recognition specificity. In FIG. 4, A is the signal detection result of the five-membered complex with A base, B is the signal detection result of the extension of the reversible terminator with A base, C is the signal detection result of the five-membered complex with C base, D is the signal detection result of the extension of the reversible terminator with C base, E is the signal detection result of the five-membered complex with T base, and F is the signal detection result of the extension of the reversible terminator with T base.

Example 3 detection of non-covalent five-membered Complex Signal at different temperature conditions

The method comprises the following steps:

1. because the formation of the non-covalent five-membered complex does not have covalent bond formation, experimental tests under different conditions can be repeatedly performed in the same period and signals can be collected multiple times, and in this way, different reaction conditions can be selected to determine which conditions are more conducive to the formation and detection of the non-covalent five-membered complex. This example tests the effect of different reaction temperatures on the formation of non-covalent five-membered complexes.

2. The sequencing reaction system includes, but is not limited to, the above-described instruments and chips (the chip specifically refers to a chip that has been clustered and hybridized with the R1 sequencing primer using Miseq kit ", the template was diluted to a concentration of 0.5pM using HT1 diluent), and in this example, a single library of known sequences was used, all synthesized by Dai sequencing biotechnology Co., ltd. In the bead sea.

The sequence of the sequencing primer used is as follows:

5'-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGT-3'(SEQ ID NO.1)。

The sequence of the sequencing template used is as follows:

Sequencing template 1:

5'-AATGATACGGCGACCACCGAGATCTACACACTCTTTCCCTACACGACGCTCTTCCGATCTAGA TGAGTGAGAGCATCAACTTCTCTCACAACCTAGGCCAGTAAGTAGTGCATCTCGTATGCCGTCTTCT GCTTG-3'(SEQ ID NO.2).

sequencing template 2:

5'-AATGATACGGCGACCACCGAGATCTACACACTCTTTCCCTACACGACGCTCTTCCGATCTTCT ACTCACTCTCGTAGTTGAAGAGAGTGTTGGATCCGGTCATTCATCACGATCTCGTATGCCGTCTTCTG CTTG-3'(SEQ ID NO.3).

wherein, test A, G base uses the above template 1 and test T, C base uses the above template 2;

3. After hybridization of the long cluster and the sequencing primer, the sequencing template 1 is subjected to a non-covalent five-membered complex test with the 3' end not blocked. The primer is then two bases A and G, and the template is enriched in A and G, so that it can be used to test the effect of different temperatures on the formation of five-membered complexes of fluorescent labeled A and G bases. After hybridization of the long cluster and sequencing primer of the template 2, a non-covalent five-membered complex with unblocked 3' -end is tested. The sequencing primer is then two bases, T and C, and is enriched in T and C, and thus can be used to test the effect of different temperatures on the formation of non-covalent five-membered complexes of fluorescently labeled T or C bases. The library is a single known sequence, and theoretically, clusters in a chip are all the same cluster points, and the signal detection of the five-membered complex of single base at different temperatures can be completed by preparing a non-covalent five-membered complex reaction solution containing only one fluorescence labeling dNTP; preparing a Hot dATP non-covalent five-membered complex reaction solution (0.8 mu M Hot dATP-AF532, 0.8 mu M Cold dCTP, 0.8 mu M Cold dGTP, 0.8 mu M Cold dTTP) respectively, a Hot dCTP non-covalent five-membered complex reaction solution (0.8 mu M Hot dCTP-AF532, 0.8 mu M Cold dATP, 0.8 mu M Cold dTTP), a Hot dTTP non-covalent five-membered complex reaction solution (0.8 mu M Hot dTTP-AF532, 0.8 mu M Cold dCTP, 0.8 mu M Cold dGTP, 0.8 mu M Cold dATP), a Hot dG non-covalent five-membered complex reaction solution (0.8 mu M Hot dGTP-AF532, 0.8 mu M Cold dCTP, 0.8 mu M Cold dTTP) and other components consistent with example 2, namely, including 50mM Tris、50mM NaCl、60mM(NH₄)₂SO₄、0.05％ Tween-20、0.1mM EDTA、0.5mM CaCl₂、1M, 50% aqueous solution of 1. Mu.8% of a hydroxylase, 0.05% of an aqueous solution of a hydroxylase; 4. in the same test period, after washing the chip with the pre-washing buffer solution B, respectively adding non-covalent five-membered complex reaction solution corresponding to the Hot base according to the known base sequence, carrying out reaction under a certain temperature setting condition, adding the scanning buffer solution, photographing to collect signals, then scattering and washing to remove the non-covalent five-membered complex signals, changing the reaction conditions for forming the non-covalent five-membered complex repeatedly for other reaction temperatures, and repeatedly forming the non-covalent five-membered complex in the same cycle until different temperatures are tested (see the table 2 below); then using a reversible terminator (the base combination is consistent with the noncovalent five-membered complex, and the other components are consistent with the embodiment 2) to carry out extension and then cutting, and entering the next cycle; repeating the experiment to test different circulations, and completing 3 groups of repeated experiments of different bases at different temperatures;

In the reaction for forming the non-covalent five-membered complex, the reaction temperature is shown in table 2, and the reaction time is 60s;

In the extension reaction, the reaction temperature is 61-65 ℃ and the reaction time is 60s;

5. The temperature conditions tested are shown in the following table (wherein the temperature set value refers to the temperature parameter value set on the sequencer, and the actual temperature value refers to the temperature value measured when the temperature correction is performed by factory of the instrument):

TABLE 2

6. The results show a set of test results including each base test as shown in fig. 5, 6, 7 and 8. It is known that the optimal reaction temperature of the non-covalent five-membered complexes A and G is 36.2-45.8 ℃ and the optimal reaction temperature of the non-covalent five-membered complexes T and C is 36.2 ℃.

Example 4 detection of non-covalent five-membered Complex Signal under different reaction time conditions

The method comprises the following steps:

1. The sequencing reaction system (including reagents, reaction temperature and extension reaction time) was the same as in example 2 above, but the effect of different reaction times on pentad complex formation was tested in the same test cycle. In the same test period, after washing the chip with the pre-washing buffer solution B, respectively adding five-membered complex reaction solutions corresponding to the Hot bases according to the known base sequences, carrying out reaction at a set time, adding a scanning buffer solution for photographing, changing reaction conditions for repeating the formation of the five-membered complex after washing out the signals of the five-membered complex, collecting the signals, and repeating the formation of the five-membered complex in the same cycle until different time gradients are tested (see in the table below); then using a reversible terminator to extend and then cutting, and entering the next cycle; repeating the experiment to test different periods, and completing the repeated experiment of different bases at different times; 2. the reaction times tested included: 10s, 20s, 30s, 60s, 120s, 180s; the results are shown in FIG. 9:

in FIG. 9, panel A shows a template co-cycling five-membered complex A,10s; drawing B is template co-circulating five-membered complex A,20s; c is template and the five-membered complex A circulates together, 30s; d, the template is used for circulating the five-membered complex A together for 60s; e is template and the five-membered complex A,120s circulates together; and F, the template is used for circulating the five-membered complex A and 180s together. Therefore, the reaction time is below 30s, the fluorescent signal of the cluster point is weak, when the reaction time is between 60 and 180s, the fluorescent signal is not obviously different, and the reaction time can be controlled between 30 and 60s.

Example 5 stability adjustment of non-covalent five-membered Complex reaction solution

The method comprises the following steps:

1. In the embodiment, the composition components of the related reagent formed by the non-covalent five-membered complex are screened and optimized; testing the different reagent compositions to determine conditions under which a five-membered complex can be stably formed;

2. The reagent synchronization of the pre-washing buffer B and the scanning buffer B needs to be considered except the reagent of the reaction solution; the pre-wash buffer B functions include: (1) Washing off the liquid remained in the previous step of reaction, (2) providing environmental support for the formation of the five-membered complex; the functions of scan buffer B include: (1) Flushing out redundant reagent which does not form stable five-membered complex residues, removing background signals, and improving signal to noise ratio, (2) containing a photo-protecting agent, so that fluorescent signals are not quenched too fast when being photographed by laser, and stable collection of the five-membered complex signals is ensured; wherein the scanning buffer B needs to synchronously increase/decrease components such as betaine stabilizing additive and the like with the five-membered complex reaction liquid;

3. The experimental group 1 is defined as a control group, and the non-covalent five-membered complex reaction solution of the experimental group 1 is ：50mM Tris、50mM NaCl、60mM(NH₄)₂SO₄、0.05％ Tween-20、0.1mM EDTA、0.5mM CaCl₂、0.1％ hydroxylamine solution, 0.05U/. Mu.L of 9 DEG N enzyme and non-nucleic acid water. On the basis of the reaction solution as a basic version, other experimental groups test the stability influence of the solution of other components (including 50mM LiCl, 1M betaine and the like) on the non-covalent five-membered complex;

4. In addition, the enzyme concentration can be in the range from 0.05U/mu L to 0.2U/mu L to obtain good effect, and the proper proportion of Hot dNTP and Cold dNTP in the reaction solution in the five-membered complex can also lead the sequencing to have longer reading length;

The reaction system (including the reaction reagents, the reaction time and the reaction temperature) according to this example is the same as that of example 2 except that no special description is given;

5. The result arrangement is shown in fig. 10.

In FIG. 10, panel A shows five-membered complex A and C base signals for the control group; panel B shows the signal from panel 2 (50 mM LiCl); panel C shows the signal from panel 3 (1M betaine); panel D shows the signal from panel 4 (50 mM LiCl+1M betaine); and E graph shows the same period reversible terminator extension signal. From this, both LiCl and betaine added on the control group basis can enhance cluster stability and fluorescence signal.

Example 6 optimization of the reagent to break up and rinse away five-membered complexes

The method comprises the following steps:

1. After forming a stable pentad complex and collecting signals, reagents need to be added to break up pentad complexes generated by templates, primers, nucleotides, divalent metal ions and polymerase, but reagents that still leave hybridization of sequencing primers and templates unaffected include, but are not limited to, ligands that complex metal ions including, but are not limited to EDTA, EGTA, NTA, HPTA, and the like. High salt reagents may also be used, with varying salt ion concentrations from 150mM to 1.5M, or protein denaturing reagents including, but not limited to, guanidine hydrochloride, guanidine thiocyanate, SDS, and the like.

2. The break up buffer may be in a variety of combinations including, but not limited to:

Scheme 1:4M guanidine hydrochloride, 42mM sodium citrate, 10mM EDTA;

Scheme 2:60mM Tris,1M NaCl, 4mM EDTA, 0.02% SDS, 0.05% Tween-20;

Scheme 3:4M guanidine thiocyanate, 42mM sodium citrate, 10mM EDTA;

scheme 4:50mM Tris, 50mM NaCl, 60mM (NH 4) ₂ SO4, 0.05% Tween-20, 0.1% hydroxylamine, 0.01% CTAB, 10mM EDTA, etc.;

3. the stable pentad complex can be broken up by different reagents, but the required flushing volume and time are different, as in fig. 11, scheme 2 requires an action time of about 36s or more, whereas scheme 3 requires only 1.8s at maximum, preferably scheme 3 reagents are chosen as break up reagents. However, in this case, the bridge-type amplified clusters are generated by other cluster generation methods, and other schemes of the dispersing agent may be preferable.

In fig. 11, panel a shows five-membered complex signal acquisition; FIG. B is the state after scheme 2 is flushed for 36s to break up the five-membered complex; c, collecting a five-membered complex signal; panel D shows the state after 1.8s of washing of scheme 3 to break up the five-membered complex.

Example 7, specific embodiments of the non-covalent five-membered complex and reversible terminator extension sequencing protocol

1. Generating a base-specific pairing stable non-covalent five-membered complex by using a template, a sequencing primer, a labeled or non-labeled nucleotide, a non-catalytic competitive metal ion and polymerase to obtain a labeled binary signal I, performing one-step covalent synthesis by using single base extension labeled or non-labeled reversible termination to obtain a labeled binary signal II, and combining the signals in two steps: 1,1;1,0;0,1; and 0,0 to recognize four different bases;

2. the repeated flow of the sequencing protocol may include the following liquid flow patterns (all of the reagents, reaction conditions, etc. used are not specifically described with reference to example 2, the pre-wash buffer a and pre-wash buffer B are identical in composition):

(i) The chip reaction tank is flushed by the pre-washing buffer solution B, so that the reaction tank environment is suitable for the formation of the following non-covalent five-membered complex;

(ii) Adding the fluorescent-labeled reversible termination base non-covalent five-membered complex reaction solution into a reaction tank, and reacting for 30s at 35 ℃ to form a non-covalent five-membered complex;

(iii) The scanning buffer solution is used for flushing out redundant reagents which do not form stable non-covalent five-membered complex (the non-covalent five-membered complex is a dynamic complex), the proportion of the scanning buffer solution reduces fluorescent marked reversible terminator nucleotides and 9 DEG N enzyme in the non-covalent five-membered complex reaction solution, but increases sodium ascorbate, and the functions of the scanning buffer solution include that fluorescent signals are not quenched too fast when the laser is photographed;

(iv) Adding a break-up buffer (using the optimal formulation of example 6) can break up five-membered complexes generated by templates, sequencing primers, nucleotides, divalent metals and polymerase rapidly (within 2 s) and wash, but still keep hybridization of sequencing primers and templates unaffected;

(v) Adding a pre-washing buffer solution A to wash a chip reaction tank, so that the environment of the reaction tank is suitable for the extension of the following reversible terminator base;

(vi) Adding a fluorescence reversible termination base extension reaction solution to extend, wherein the reaction solution contains Mg ²⁺ which can form a bond by base complementation and pairing for extension;

(vi) Adding a pre-washing buffer solution A to wash out redundant reversible terminator reaction solution;

(vii) The scanning buffer solution A is added for signal acquisition, wherein the vitamin C acid is added, so that signal quenching can be reduced, and stable signal acquisition is ensured;

(viii) Pre-washing buffer solution C for washing;

(ix) N3 cleavage reagent (corresponding to the cleavage buffer in FIG. 1) cleaves the fluorophore and exposes the 3' end so that the next cycle can be performed; this cycle is repeated until sequencing is complete.

3. There are a number of ways to combine and match the acquisition schemes for signals, including but not limited to the following examples:

a. Each round of single base luminescence (the luminescent base types can be exchanged with each other), the other components of the SBB+A noncovalent five-membered complex reaction solution (0.8. Mu.M Hot dATP-AF532, 0.8. Mu.M Cold dCTP, 0.8. Mu.M Cold dGTP, 0.8. Mu.M Cold dTTP), the SBB+T noncovalent five-membered complex reaction solution (0.8. Mu.M Hot dTTP-AF532, 0.8. Mu.M Cold dCTP, 0.8. Mu.M Cold dGTP, 0.8. Mu.M Cold dATP) were identical to those of example 2, i.e., including 50mM Tris、50mM NaCl、60mM(NH₄)₂SO₄、0.05％ Tween-20、0.1mM EDTA、0.5mM CaCl₂、1M betaine, 50mM LiCl, 0.1% hydroxylamine solution, 0.05U/. Mu.L 9℃N enzyme, and no nucleic acid water. The SBS reversible terminator extends the reaction liquid, one or two bases (one of which is the same as the non-covalent five-membered complex and the other of which is different) are selected as Hot bases, the other is Cold bases, and the other reaction liquid components are 50mM Tris、50mM NaCl、60mM(NH₄)₂SO₄、3mM MgSO₄、1mM EDTA、0.05％Tween-20、5％ DMSO、0.05U/μL 9°N enzyme, non-nucleic acid water and pH value is 8.8.

TABLE 3 Table 3

One or two bases with fluorescent markers are selected from the extension;

TABLE 4 Table 4

If the Cold base is selected for extension, the influence of scars can be reduced, the base marked by fluorescence can also be selected, and the reading frequency is increased to verify the accuracy;

b. Each round of double base luminescence (the luminescent base types can be exchanged with each other):

TABLE 5

2 Kinds of base band scars;

TABLE 6

The final SBS can be provided with fluorescent groups or not, and no scar exists;

TABLE 7

Each base is measured for 2 times, so that the accuracy of base recognition can be improved;

TABLE 8

C. Three bases per round of luminescence (the base types of luminescence can be exchanged with each other):

TABLE 9

Finally, the 3 bases have scars; if no scar is desired, the fourth rotation to SBB is performed and SBS is performed with full Cold.

4. For the combination collocation of 2 bases, 2 bases can emit light and collect signals in the form of SBB in the first round of reaction, 1 new base and 1 base in the first round of extension reaction are tried to emit light and collect signals in the form of SBS in the same period, and specifically, the following scheme (G base is selected as non-luminous base) can be tried:

Table 10

TABLE 11

Scheme for the production of a semiconductor device	A	T	C	G
					1	(1,0)	(0,1)	(1,1)	(0,0)
2	(1,1)	(0,1)	(1,0)	(0,0)
					3	(1,1)	(1,0)	(0,1)	(0,0)
4	(1,0)	(1,1)	(0,1)	(0,0)
					5	(0,1)	(1,1)	(1,0)	(0,0)
6	(0,1)	(1,0)	(1,1)	(0,0)

In fig. 12, a diagram is a diagram of a scheme 1sbb AC, b diagram is a scheme 1SBS tc, c diagram is a scheme 2sbb AC, d diagram is a scheme 2SBS at, e diagram is a scheme 3sbb at, f diagram is a scheme 3SBS AC, g diagram is a scheme 4sbb at, h diagram is a scheme 4SBS tc, i diagram is a scheme 5sbb tc, j diagram is a scheme 5SBS at, k diagram is a scheme 6sbb tc, and l diagram is a scheme 6SBS AC.

The application realizes the gene sequencing method of five-membered complex and reversible terminator digital identification base, and realizes the design of the microfluidic chip with high accuracy, high speed sequencing chemistry, low cost and high flux by simplifying the hardware sequencing instrument of the extreme single-excitation light source and the monochromatic fluorescence light path system.

The technical features of the above-described embodiments and examples may be combined in any suitable manner, and for brevity of description, all of the possible combinations of the technical features of the above-described embodiments and examples are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be considered to be within the scope described in the present specification.

The above examples merely represent a few embodiments of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the above teachings, and equivalents thereof are intended to fall within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims

1. A method of sequencing a nucleic acid molecule, the method comprising the steps of:

Providing a first set of nucleotides, wherein the nucleotides in the first set of nucleotides, which are paired with a site to be detected of a sequencing template, the sequencing template, a sequencing primer, a first nucleic acid polymerase and non-catalytic competing metal ions react to form a non-covalent five-membered complex; the first set of nucleotides comprises a first dATP or analogue thereof, a first dTTP or analogue thereof, a first dCTP or analogue thereof, and a first dGTP or analogue thereof, each nucleotide or analogue thereof independently having a preset identity; detecting a preset mark correspondingly contained in the non-covalent five-membered complex, and obtaining first mark information;

Wherein,

The conditions satisfied by the first set of nucleotides and the second set of nucleotides include: combination tag 1, combination tag 2, combination tag 3, and combination tag 4 are defined as follows, different from each other;

the combined identifier 4 is a preset identifier of a first dGTP or an analogue thereof in the first group of nucleotides and a preset identifier of a second dGTP or an analogue thereof in the second group of nucleotides;

The preset mark comprises a mark and a non-mark;

the preset mark is marked, the mark information is marked as 1, the preset mark is unmarked, the mark information is marked as 0, and binary combination is carried out by 1 and 0;

The sequencing method further comprises the steps of after determining the type of the nucleotide of the to-be-detected site, cutting off the reversible terminator structure, and determining the type of the nucleotide of the next site of the to-be-detected site;

The method of determining the kind of the nucleotide of the next site is the same as the method of determining the kind of the nucleotide of the site to be detected.

2. The method of sequencing a nucleic acid molecule of claim 1, wherein the label comprises one or more of a fluorescent label, a chemiluminescent label, a bioluminescent label, an electrochemiluminescent label, an electrical signal label, and a magnetic signal.

3. The method of sequencing a nucleic acid molecule of claim 2, wherein the fluorescent label is selected from one or more of AF532, ATTO532, cy3B, cy, cy5, ATTO647N, ATTO647, and AF 647.

4. The method of sequencing a nucleic acid molecule of claim 2, wherein the predetermined identity of the second dATP or analogue thereof or/and the second dGTP or analogue thereof in the second set of nucleotides is a label.

5. The method of sequencing a nucleic acid molecule of any one of claims 1 to 4, wherein the non-catalytic competing metal ions are selected from one or more of calcium ions, strontium ions and barium ions.

6. The method of sequencing a nucleic acid molecule of any one of claims 1 to 4, wherein the catalytic competing metal ion is magnesium ion.

7. The method of sequencing a nucleic acid molecule of any one of claims 1 to 4, wherein the reversible terminator structure is selected from one of 3' -benzyl azide, 3' -R-SS-and 3' -NH ₂ -O-.

8. The method of sequencing a nucleic acid molecule of any one of claims 1 to 4, wherein the first nucleic acid polymerase and the second nucleic acid polymerase are each independently selected from one of Taq DNA polymerase, KOD DNA polymerase, 9°n DNA polymerase, and Klenow enzyme.

9. The method of sequencing a nucleic acid molecule of any one of claims 1 to 4, wherein the reagent that breaks up the non-covalent five-membered complex comprises a ligand that complexes the non-catalytic competing metal ion.

10. The method of sequencing a nucleic acid molecule of claim 9, wherein the ligand is selected from one or more of EDTA, EGTA, NTA and HPTA.

11. The method of sequencing a nucleic acid molecule of claim 10, wherein the reagent that breaks up the non-covalent five-membered complex comprises guanidine thiocyanate, sodium citrate, and EDTA.

12. The method of sequencing a nucleic acid molecule of claim 11, wherein the reagent for breaking up the non-covalent five-membered complex comprises water and 3.5M-4.5M guanidine thiocyanate, 40mM-45mM sodium citrate and 8mM-12mM edta, at a ph of 7.5-8.5.

13. The method of sequencing a nucleic acid molecule of any one of claims 1 to 4, wherein the conditions under which the non-covalent five-membered complex is formed by reaction comprise one or more of the following (1) and (2):

(2) The initial reaction system comprises water, the first group of nucleotides, a sequencing template, a sequencing primer, 0.05U/. Mu.L-0.2U/. Mu.L of first nucleic acid polymerase, non-catalytic competing metal ions, naCl, (NH ₄)₂SO₄, tween-20, EDTA, betaine and Tris.

14. The method of sequencing a nucleic acid molecule of claim 13, wherein the reaction initiation system comprises water and 0.2 μm-1 μm of the first set of nucleotides, 0.1pM-10pM sequencing template, 0.5 μm-1.5 μm sequencing primer, 0.05U/-0.2U/. Mu.l of the first nucleic acid polymerase, 0.45mM-0.55mM non-catalytic competing metal ion 、45mM-55mM NaCl、55mM-65mM (NH₄)₂SO₄、0.045%-0.055% Tween-20、0.08mM-1mM EDTA、0.8M-1.2 M betaine, 0 mM-55 mM LiCl, 0% -0.12% hydroxylamine, 45 mM-55 mM Tris and 0% -5.5% dmso, and a ph of 7.5-9.

15. The method of sequencing a nucleic acid molecule of any one of claims 1 to 4, wherein the conditions for covalent extension comprise one or more of the following (1) and (2):

(2) The initial reaction system comprises water, the first group of nucleotides, a sequencing template, a sequencing primer, 0.05U/. Mu.L-0.2U/. Mu.L of a second nucleic acid polymerase, catalytic competing metal ions, naCl, (NH ₄)₂SO₄, tween-20, EDTA, DMSO and Tris.

16. The method of sequencing a nucleic acid molecule of claim 15, wherein the reaction initiation system comprises water and 0.2 μm-1 μm of the second set of nucleotides, 0.1pM-10pM sequencing template, 0.5 μm-1.5 μm sequencing primer, 0.05U/-0.2U/-2L of the second nucleic acid polymerase, 2mM-4mM catalytic competing metal ions 、45mM-55mM NaCl、55mM-65mM (NH₄)₂SO₄、0.045%-0.055% Tween-20、0.08mM-1mM EDTA、4.5% -5.5%DMSO and 45mM-55mM tris, at a ph of 7.5-9.