WO2021253372A1 - 一种高兼容性的PCR-free建库和测序方法 - Google Patents

一种高兼容性的PCR-free建库和测序方法 Download PDF

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WO2021253372A1
WO2021253372A1 PCT/CN2020/096987 CN2020096987W WO2021253372A1 WO 2021253372 A1 WO2021253372 A1 WO 2021253372A1 CN 2020096987 W CN2020096987 W CN 2020096987W WO 2021253372 A1 WO2021253372 A1 WO 2021253372A1
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dna
product
reaction solution
linker
stranded
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PCT/CN2020/096987
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English (en)
French (fr)
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赵霞
沈寒婕
刘鹏娟
李巧玲
席阳
江媛
陈芳
蒋慧
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深圳华大智造科技有限公司
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Priority to EP20940612.3A priority Critical patent/EP4170028A4/en
Priority to CN202080099449.1A priority patent/CN115715323A/zh
Priority to PCT/CN2020/096987 priority patent/WO2021253372A1/zh
Publication of WO2021253372A1 publication Critical patent/WO2021253372A1/zh
Priority to US18/067,540 priority patent/US20230265500A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the invention relates to the technical field of molecular biology high-throughput sequencing, in particular to a highly compatible PCR-free library construction and sequencing method, which is suitable for samples in which the DNA is constructed without PCR amplification.
  • Next-generation sequencing technology is the most commonly used technology in modern molecular biology high-throughput sequencing research.
  • Next-generation sequencing DNA sequencing mainly includes two processes: library preparation and computer sequencing.
  • library preparation standard PCR is generally used to amplify randomly interrupted genomic fragments.
  • PCR amplification preference if there are factors such as complex secondary structure or poor thermal stability, it will cause template PCR amplification preference. Therefore, not all sequences can be equally reflected in the PCR amplification library.
  • PCR-free and conventional PCR libraries when they are sequenced on the computer, but the library construction process does not require PCR.
  • PCR-free libraries can improve the data reading distribution and have more uniform sequence coverage.
  • the mainstream direction also requires PCR amplification to amplify the subsequent detection signals. Even if the PCR-free process of the library building process can be achieved, the PCR problem introduced during the sequencing process cannot be avoided.
  • PCR library has the shortcomings of poor detection accuracy and sensitivity of coverage, GC bias, and InDel, and the library building process is relatively long. Full automation is necessary for instruments and laboratories. The requirements are high, and the cost of construction, labor, and depreciation is high.
  • Illumina Truseq DNA PCR-free sample preparation kit can theoretically complete library construction in one day and is compatible with the ever-increasing read length of the Illumina sequencing platform. However, this kit is only compatible with 1-2 ⁇ g physical interrupted French library construction, which is mainly used for human resequencing.
  • PCR-free kits on the market are mainly concentrated in foreign kit companies. The higher requirement for the initial quantity is an important reason for restricting its application. Improving the efficiency of library construction is the key point to reduce the initial quantity of the library. At present, the lowest starting amount for building a library on the market is 50ng of gDNA and 5ng of cfDNA.
  • the purpose of the present invention is to provide a new, fast and efficient method for preparing a PCR-free sequencing library that can be applied to the BGI gene autonomous sequencing platform in view of the deficiencies of the prior art.
  • This method does not carry out PCR amplification, but directly forms a computerizable library by single-stranded circularization after linker ligation, thereby reducing base errors, data bias and repetitive sequences introduced by PCR.
  • the present invention claims a PCR-free high-throughput sequencing method.
  • PCR-free high-throughput sequencing method claimed in the present invention can be the following method A or method B or method C:
  • the method A may include the following steps:
  • the nucleic acid to be sequenced is fragmented according to the size or not to obtain the target size DNA fragment;
  • the method B may include the following steps:
  • the method C may include the following steps:
  • (C1) The nucleic acid to be sequenced is fragmented according to the size, and the ends are repaired at the same time to obtain the target size DNA fragment;
  • the fragmentation process is to digest the nucleic acid to be sequenced with a fragmentation enzyme.
  • the fragmentation enzyme may be as Ultra TM II FS DNA Module, Qiagen5X WGS Fragmentation Mix, or self-developed interrupting enzyme.
  • a barcode is included in the linker.
  • one of the linkers includes two barcodes.
  • the linkers including two barcodes are respectively added to both ends of the nucleic acid to be sequenced to form a library structure with double tags.
  • the linker is formed by annealing two partially complementary nucleic acid single strands, and the tag sequence is located in the non-complementary region of the two nucleic acid single strands.
  • the nucleic acid to be sequenced may be DNA or RNA.
  • the DNA is genomic DNA or naturally occurring small molecule DNA or DNA fragments obtained by amplification.
  • the naturally occurring small molecule DNA may be cfDNA.
  • the amplified DNA fragments may be MDA products, cDNA products, amplicons and the like.
  • the starting sample of the present invention can be extended to directly build a database of blood or saliva samples other than DNA samples.
  • the fragmentation treatment method may be a physical interruption method or an enzyme interruption method.
  • the physical interruption method can be such as ultrasonic interruption.
  • the target size DNA fragment for example, genomic DNA
  • the target size DNA fragment is selected through the magnetic bead fragment
  • the size of the DNA fragment is described (for example, cfDNA, the DNA fragment is small and the main band is concentrated), there is no need to continue the fragmentation process for the nucleic acid to be sequenced.
  • the nucleic acid to be sequenced is RNA
  • reverse transcription is required to obtain DNA
  • performing the fragmentation treatment is to fragment RNA or to fragment the DNA obtained by reverse transcription of the RNA.
  • the range of the target size DNA fragment can be 150bp-800bp (such as 300-500bp).
  • step (A2) performing the end repair and A addition reaction can be completed in one step, and specifically can be performed according to a method including the following steps: mixing the end repair and A addition reaction solution with the (A1) product, and reacting, The product (A2) is obtained.
  • the terminal repair and addition A reaction solution contains T4 polynucleotide kinase buffer (T4 PNK buffer), adenylate deoxyribonucleic acid (dATP), deoxyribonucleic acid mixture (dNTPs), T4 DNA polymerase (T4 DNA polymerase), T4 polynucleotide kinase (T4 PNK), Taq DNA polymerase (rTaq).
  • T4 PNK buffer T4 polynucleotide kinase buffer
  • dATP adenylate deoxyribonucleic acid
  • dNTPs deoxyribonucleic acid mixture
  • T4 DNA polymerase T4 DNA polymerase
  • T4 PNK T4 polynucleotide kinase
  • Taq DNA polymerase rTaq
  • the ratio of the T4 polynucleotide kinase (T4 PNK) and the Taq DNA polymerase (rTaq) is 50 nmol: 12.5 nmol (each dNTP): 6U: 10U: 2-5U.
  • the end repair and A addition reaction solution is composed of 10 ⁇ T4 polynucleotide kinase buffer (10 ⁇ T4 PNK buffer), adenylate deoxyribonucleic acid solution with a concentration of 100 mM ( dATP), 4 types of deoxyribonucleic acid mixtures (dNTPs) each with a deoxyribonucleic acid concentration of 25mM, T4 DNA polymerase at a concentration of 3U/ ⁇ L, and T4 polymerase at a concentration of 10U/ ⁇ L Taq DNA polymerase (T4 PNK) and Taq DNA polymerase (rTaq) with a concentration of 5 U/ ⁇ L are mixed in a volume ratio of 5:0.5:0.5:2:1:(0.4-1).
  • 10 ⁇ T4 polynucleotide kinase buffer 10 ⁇ T4 PNK buffer
  • dNTPs deoxyribonucleic acid mixtures
  • T4 DNA polymerase at a concentration of 3U/ ⁇ L
  • step (A2) the volume ratio of the terminal repair and A addition reaction solution and the product (A1) may be 1:4.
  • step (A2) the reaction conditions after mixing the terminal repair and A addition reaction solution with the product (A1) can be 1) 14°C for 15 minutes; 37°C for 25 minutes; 65°C for 15 minutes; 4°C incubation.
  • the temperature of the PCR instrument cover is: 70°C; or 2) incubate at 37°C for 10 minutes; incubate at 72°C for 15 minutes, and cool down to 4°C at a rate of 0.1 seconds.
  • step (B1) the fragmentation treatment and the end repair and A addition reaction are completed in one step: the fragmentation treatment and the end repair and A addition reaction solution are mixed with the nucleic acid to be sequenced and reacted to obtain The (B1) product.
  • a reaction solution contains fragmentation enzyme, fragmentation enzyme reaction buffer, adenylate deoxyribonucleic acid, deoxyribonucleic acid mixture, T4 DNA polymerase, Taq DNA polymerase and TE buffer liquid.
  • the adenylate deoxyribonucleic acid, the deoxyribonucleic acid mixture, the T4 DNA polymerase and the Taq DNA polymerase The ratio can be 170nmol:57.5nmol:3U:5U.
  • the dosage of the interrupting enzyme is determined according to the instructions or the results of multiple experiments.
  • step (B1) the conditions for reacting after mixing the fragmentation treatment and end repair and adding A reaction solution with the nucleic acid to be sequenced may be incubation at 37°C for 10-20 min; incubation at 65°C for 30 min; cooling down To 4°C.
  • the temperature of the thermal cycler cover is 70°C.
  • step (C1) the fragmentation treatment and the end repair reaction are completed in one step: the fragmentation treatment and end repair reaction solution is mixed with the nucleic acid to be sequenced and reacted to obtain the (C1) product .
  • the fragmentation treatment and end repair reaction solution contains fragmentation enzyme, fragmentation enzyme reaction buffer, deoxyribonucleic acid mixture, DNA polymerase I, MgCl 2 and enzyme-free pure water.
  • the mixing ratio of the deoxyribonucleic acid mixture, the DNA polymerase I and the MgCl 2 may be 75 nmol (each dNTP): 20 U: 0.3 ⁇ mol.
  • the dosage of the interrupting enzyme is determined according to the instructions or the results of multiple experiments.
  • the conditions for reacting after mixing the fragmentation treatment and end repair reaction solution with the nucleic acid to be sequenced may be 37° C. for 30 min; 4° C. incubation.
  • the temperature of the thermal cycler cover is 70°C. After the reaction is over, place the sample on ice and add 30 ⁇ l TE.
  • the A addition reaction solution used when performing the A addition reaction on the product (C1) contains T4 polynucleotide kinase buffer (T4 PNK buffer), adenylate deoxyribonucleic acid (dATP) , Deoxyribonucleic acid mixture (dNTPs), Taq DNA polymerase (rTaq).
  • T4 PNK buffer T4 polynucleotide kinase buffer
  • dATP adenylate deoxyribonucleic acid
  • dNTPs Deoxyribonucleic acid mixture
  • rTaq Taq DNA polymerase
  • the ratio of the adenylate deoxyribonucleic acid (dATP), the deoxyribonucleic acid mixture (dNTPs), and the Taq DNA polymerase (rTaq) is 50 nmol : 8.75nmol (each dNTP): 1U.
  • the A-adding reaction solution consists of 10 ⁇ T4 polynucleotide kinase buffer (10 ⁇ T4 PNK buffer), adenylate deoxyribonucleic acid solution (dATP) with a concentration of 100 mM, 4 kinds of DNA mixtures (dNTPs) with each DNA concentration of 25mM, Taq DNA polymerase (rTaq) with a concentration of 5U/ ⁇ L, and enzyme-free pure water in a volume ratio of 5:0.5:0.35:0.2 :Mixed in the ratio of 1.
  • 10 ⁇ T4 polynucleotide kinase buffer 10 ⁇ T4 PNK buffer
  • dATP adenylate deoxyribonucleic acid solution
  • dNTPs DNA mixtures
  • rTaq Taq DNA polymerase
  • enzyme-free pure water in a volume ratio of 5:0.5:0.35:0.2 :Mixed in the ratio of 1.
  • the linker is formed by annealing the B chain and the T chain.
  • the 3'end of the B chain is complementary to the 5'end of the T chain, and the other parts of the two chains are not complementary, and the 3'end of the B chain has a protruding dT;
  • the B chain and / Or the non-complementary paired part of the T chain contains a tag sequence (ie barcode) for labeling different samples;
  • the 5'ends of the B chain and the T chain are both modified with a phosphate group or connected with a 3' A single-stranded oligonucleotide with U at the end.
  • the linker When the linker contains a single-stranded oligonucleotide with a U at the 3'end, it needs to be treated with USER enzyme.
  • the steps of USER enzyme treatment can be carried out while ligating, or after the linker has been added and purified.
  • joint may be any of the following:
  • Linker 1 It is formed by annealing the single-stranded DNA shown in SEQ ID No. 1 modified with a phosphate group at the 5'end and the single-stranded DNA shown in SEQ ID No. 2 modified with a phosphate group at the 5'end.
  • Phosphorylated linker B chain /Phos/GAACGACATGGCTACGATCCGACTT (SEQ ID No. 1);
  • the phosphorylated linker T chain /Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAANNNNNNNNNNNN CNN CAACTCCTTGGCTCACA (SEQ ID No. 2).
  • the joint 1 is a bifurcated joint. After the joint is connected, cyclization or a one-step reaction of cyclization and rolling ring replication can be directly carried out.
  • Joint 2 formed by annealing the single-stranded DNA shown in SEQ ID No. 3 or SEQ ID No. 4 and the single-stranded DNA shown in SEQ ID No. 2.
  • Linker B chain with U base (Design 1): TTGTCTTCCUGAACGACATGGCTACGATCCGACTT (SEQ ID No. 3);
  • Linker B chain with U base (design 2): TTGTCTTCCTAAGUGAACGACATGGCTACGATCCGACTT (SEQ ID No. 4);
  • Phosphorylated linker T chain /Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAANNNNNNNNCAACTCCTTGGCTCACA (SEQ ID No. 2).
  • the linker 2 is a Bubble U-shaped linker, and the linker can be subjected to USER enzyme treatment while being connected, and then can be directly subjected to a one-step reaction of cyclization or cyclization and rolling circle replication.
  • Joint 3 formed by annealing the single-stranded DNA shown in SEQ ID No. 5 and the single-stranded DNA shown in SEQ ID No. 6.
  • Linker B chain TTGTCTTCCUTCTCAGTACGTCAGCAGTTNNNNNNNNCAACTCCTTGGCTCA CAGAACGACATGGCTACGATCCGACTT (SEQ ID No. 5);
  • Phosphorylated linker T chain /Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAANNNNNNNNCTGATAAGGTCGCCATGCC (SEQ ID No. 6).
  • the joint 3 is a double Barcode U-shaped joint, and the joint can be subjected to USER enzyme treatment while being connected, and then can be directly subjected to a one-step reaction of cyclization or cyclization and rolling circle replication.
  • Joint 4 formed by annealing the single-stranded DNA shown in SEQ ID No. 7 and the single-stranded DNA shown in SEQ ID No. 6.
  • Phosphorylated linker B chain /Phos/TCTCAGTACGTCAGCAGTTNNNNNNNNCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT (SEQ ID No. 7);
  • Phosphorylated linker T chain /Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAANNNNNNNNCTGATAAGGTCGCCATGCC (SEQ ID No. 6).
  • the linker 4 is a double-barcode bifurcated linker sequence. After the linker is connected, cyclization or a one-step reaction of cyclization and rolling circle replication can be directly carried out.
  • the bifurcated parts of the two chains of the linker each have a barcode sequence, which is added to the library through a ligation reaction ( Figure 1). After PCR, there is a barcode on both ends of the library.
  • Figure 1 After PCR, there is a barcode on both ends of the library.
  • barcode sequences at both ends of the library and through the combination of the barcode sequences at both ends, the types of barcodes are greatly increased, and the mixed sequencing of a large number of libraries is realized.
  • the horizontal and vertical barcodes are added to the two ends of the library, and the barcodes at the two ends are combined to increase the types of barcodes.
  • the barcode sequences at both ends of a library can be the same or different. In the example shown in Figure 2, only 8 barcode sequences need to be designed. By combining the two ends, 64 combinations can be realized, which solves the need to design a large variety of barcodes in a single barcode library.
  • the sequence can realize the corresponding multi-library mixed sequencing problem.
  • Each linker sequence is from left to right from 5'end to 3'end, "//" indicates a modified group, and "phos" indicates phosphorylation.
  • the 10 base sequence of the linker T chain NNNNNNNN represents the barcode sequence, and N can be A or T or C or G.
  • the tag sequence is used to label different samples. Samples with different tag sequences can be used to construct a mixed library.
  • the linker sequence is not limited to the above sequence. Even if the sequence is modified, as long as the structure can be sequenced on the BGI/MGI platform, similar effects can be achieved.
  • the library can also add unique molecular identifiers (UMI) while completing the connection of the linker.
  • UMI can be used as an identifier for each sample chain, and is mostly used for the detection of low-frequency mutations. UMI is usually adjacent to the sample or adjacent to the barcode. When sequencing, it shares a sequencing primer with the sample or barcode for continuous sequencing; it can also be separated from both the sample and the barcode and use independent sequencing primers for sequencing.
  • steps (A3), (B2) and (C3) connecting the linker to the product (A2) or (B1) or (C2) can be carried out according to a method including the following steps:
  • the product (A2) or (B1) or (C2) is mixed with the connection reaction solution and reacted to obtain the product (A3) or (B2) or (C3).
  • the ligation reaction solution contains T4 polynucleotide kinase buffer (T4 PNK buffer), adenylate ribonucleic acid (ATP), PEG8000, T4 DNA ligase (T4 DNA ligase) and enzyme-free pure water.
  • T4 PNK buffer T4 polynucleotide kinase buffer
  • ATP adenylate ribonucleic acid
  • PEG8000 PEG8000
  • T4 DNA ligase T4 DNA ligase
  • enzyme-free pure water enzyme-free pure water.
  • the ratio of the adenylate ribonucleic acid (ATP), the PEG8000, and the T4 DNA ligase (T4 DNA ligase) is 0.8 ⁇ mol adenylate ribonucleic acid: 10 -16 ⁇ L 50% PEG8000 (such as Rigaku products): 1200-3000U T4 DNA ligase, such as 0.8 ⁇ mol adenylate ribonucleic acid: 16 ⁇ L 50% PEG8000: 3000U T4 DNA ligase.
  • the ligation reaction solution consists of 10 ⁇ T4 polynucleotide kinase buffer (10 ⁇ T4 PNK buffer), adenylate ribonucleic acid (ATP) at a concentration of 0.1M, and a concentration of 50% PEG8000 (such as Rigaku products), 600U/ ⁇ L T4 DNA ligase (T4 DNA ligase) and enzyme-free pure water are mixed in a volume ratio of 3:0.8:16:5:0.2.
  • 10 ⁇ T4 polynucleotide kinase buffer 10 ⁇ T4 PNK buffer
  • ATP adenylate ribonucleic acid
  • PEG8000 such as Rigaku products
  • 600U/ ⁇ L T4 DNA ligase T4 DNA ligase
  • enzyme-free pure water enzyme-free pure water
  • the linker, the (A2) or (B1) or (C2) product and the connection reaction solution are mixed to form a linker solution containing the linker ,
  • the (A2) or (B1) or (C2) product and the connection reaction liquid are in a volume ratio of 1-5:50:25-29 (specifically, 5:50:25 or 1:50:29 ) Mixing; the concentration of the linker in the linker solution is 6 ⁇ M or 1 ⁇ M.
  • the reaction conditions after mixing the linker, the (A2) or (B1) or (C2) product and the ligation reaction solution may be 25°C 10-30min (such as 30min); keep at 4°C.
  • the thermal cover of the thermal cycler is: 30°C.
  • the present invention designs joints compatible with the DNBSEQ TM series of sequencing platforms and BGI’s multiple independent high-throughput sequencing platforms, such as single barcode joints and double barcode joints.
  • the joint sequence contains the barcode sequence and can simultaneously label multiple samples.
  • the samples with different tag sequences are used to construct a mixed library.
  • the present invention adopts an optimized linker connection system, which can still ensure high-efficiency linking efficiency when the input amount of linker is low (for example, the molar ratio of linker to DNA is 5:1 to 50:1). This advantage is to avoid This solves the problem of linker contamination caused by too many linkers, and can improve the efficiency of DNA template conversion into a sequencing library with both ends plus linkers.
  • steps (A4), (B3) and (C4) the step of purifying the product (A3) or (B2) or (C3) is further included before single-stranded circularization and rolling circle replication are performed;
  • the purification is magnetic bead purification (such as XP magnetic beads or various domestic purified magnetic beads).
  • TE buffer to the (A3) or (B2) or (C3) product, then add XP magnetic beads (Beckman Coulter) or various domestic purified magnetic beads for purification, and then dissolve and recover the product with TE buffer.
  • XP magnetic beads Beckman Coulter
  • steps (A4), (B3) and (C4) the product (A3) or (B2) or (C3) is subjected to single-stranded circularization and rolling circle replication to form DNA nanospheres, which can be specified by any of the following One realization:
  • step (a1) the single-stranded cyclization can be carried out according to method I or method II:
  • the method I includes the following steps:
  • I-1 Place the product of (A3) or (B2) or (C3) at 95°C and incubate for 3 minutes; incubate at 4°C for 10 minutes to obtain the incubated product;
  • step I-2 Mix the single-stranded cyclization reaction solution 1 with the incubation product obtained in step I-1, and react to obtain a cyclization product.
  • the single-stranded circularization reaction solution 1 contains TA buffer, adenylate ribonucleic acid (ATP), mediating fragments, T4 DNA ligase and enzyme-free pure water.
  • the mediating fragment is the reverse complementary of the 5'end and the 5'end of the B strand constituting the linker (after removing the single-stranded oligonucleotide whose 3'end is base U), and the 3'end is The 3'end of the T strand constituting the linker is a single-stranded DNA that is reverse-complementary.
  • the linker is the linker 1 or the linker 2 described above
  • the nucleotide sequence of the mediation fragment is shown in SEQ ID No. 8.
  • the ratio of the adenylate ribonucleic acid (ATP), the mediating fragment, and the T4 DNA ligase (T4 DNA ligase) is 60 nmol: 62.5 pmol: 600U.
  • the single-stranded cyclization reaction solution 1 consists of 10 ⁇ TA buffer, adenylate ribonucleic acid (ATP) at a concentration of 100 mM, the mediation fragment at a concentration of 25 ⁇ M, and a concentration of 600U/ ⁇ L T4 DNA ligase (T4 DNA ligase) and enzyme-free pure water are mixed in a volume ratio of 6:0.6:2.5:1:1.9.
  • the volume ratio of the single-stranded cyclization reaction solution 1 and the post-incubation product may be 48:12.
  • the reaction conditions after mixing the single-stranded cyclization reaction solution 1 and the incubation product may be 37°C for 60 minutes; 4°C incubation.
  • the thermal cover of the thermal cycler is 42°C.
  • the method II includes the following steps: after mixing the product (A3) or (B2) or (C3) with the mediating fragment and NaOH solution, incubating for 5 minutes at room temperature; then mixing with the Tris-HCL solution and adding The single-stranded mild reaction solution 2 reacts to obtain a cyclized product.
  • the concentration of the NaOH solution is 2M
  • the concentration of the Tris-HCL solution is 1M
  • the pH is 6.8.
  • the ratio of the NaOH, the mediation fragment, and the Tris-HCL is 5 ⁇ mol: 100 pmol: 5 ⁇ mol.
  • the single-stranded circularization reaction solution 2 contains TA buffer, adenylate ribonucleic acid (ATP) and T4 DNA ligase (T4 DNA ligase).
  • the ratio of the adenylate ribonucleic acid (ATP) and the T4 DNA ligase (T4 DNA ligase) is 60 nmol: 240 U.
  • the single-stranded cyclization reaction solution 2 consists of 10 ⁇ TA buffer, adenylate ribonucleic acid (ATP) with a concentration of 100 mM and T4 DNA ligase (T4) with a concentration of 600 U/ ⁇ L.
  • ATP adenylate ribonucleic acid
  • T4 DNA ligase
  • DNA ligase is mixed in a volume ratio of 6:0.6:0.4.
  • the (A3) or (B2) or (C3) product, the mediation fragment with a concentration of 20 ⁇ M, the NaOH solution with a concentration of 2M, 1M Tris-HCL and the The volume ratio of the single-stranded cyclization reaction solution 2 is 48:5:2.5:5:7.
  • the conditions for carrying out the reaction can be 37°C for 30 minutes; 4°C incubation.
  • the thermal cover of the thermal cycler is 42°C.
  • step (a1) the digestion of linear single strands can be performed according to a method including the following steps:
  • the third step the digestion reaction solution is mixed with the cyclized product obtained in the second step, and reacted to obtain the digested product.
  • the digestion reaction solution contains TA buffer, ExoI enzyme, ExoIII enzyme and enzyme-free water.
  • the ratio of the ExoI enzyme to the ExoIII enzyme may be 4U:1U.
  • the digestion reaction solution consists of 10 ⁇ TA buffer, ExoI enzyme at a concentration of 20 U/ ⁇ L, ExoIII enzyme at a concentration of 10 U/ ⁇ L, and enzyme-free water in a volume ratio of 0.4: 2: It is mixed in a ratio of 1:0.6.
  • the volume ratio of the digestion reaction liquid and the cyclization product may be 4:60 or 4:67.5.
  • the reaction conditions may be 37° C. for 30 min.
  • This step may also include: adding EDTA to the reaction product and mixing.
  • the purification in step (a1) is purification with magnetic beads.
  • XP magnetic beads can be used to purify and recover the product from the previous step, and then the product can be dissolved in TE buffer.
  • the online sequencing can be specifically completed by using the BGISEQ, MGISEQ or DNBSEQ TM series sequencer platform.
  • the present invention claims a DNA library construction method suitable for PCR-free high-throughput sequencing.
  • the method for constructing a DNA library suitable for PCR-free high-throughput sequencing as claimed in the present invention may include the steps (A1)-(A4) or (B1)-(B3) or (C1)- in the first aspect above. (C4).
  • the present invention claims a DNA library constructed using the method described in the second aspect above.
  • the present invention claims a joint.
  • the joint claimed in the present invention is the joint described in the first aspect above.
  • the present invention claims a complete set of products.
  • the present invention claims a system.
  • the system claimed in the present invention contains the complete set of products described in the fifth aspect above and reagents and/or equipment for DNBSEQ TM sequencing.
  • the present invention claims that the DNA library described in the third aspect above or the linker described in the fourth aspect above or the kit product described in the fifth aspect above or the system described in the sixth aspect above perform PCR-free high-throughput sequencing. In the application.
  • the present invention claims the application of the linker described in the fourth aspect above or the set of products described in the fifth aspect above in the construction of the DNA library described in the third aspect above.
  • Figure 1 is a schematic diagram of the combination of barcodes at both ends of the library when a dual barcode adapter is used.
  • Figure 2 is a schematic diagram of the double barcode adapter after being added to the nucleic acid to be sequenced through a ligation reaction.
  • Figure 3 shows the results of the whole genome interruption of two Human gDNA samples in Example 1.
  • Lane 1 and Lane 2 are two parallel repeats of 1 ⁇ g NA12878g DNA digestion product.
  • Figure 4 shows the detection result of the 6% TBU glue of the single-stranded loop library in Example 1.
  • Lane 1 and Lane 2 are two parallel repeats of the product after ssCir digestion.
  • Figure 5 shows the PCR result of the ligation product in Example 2.
  • Lane 1 and Lane 2 are two parallel repeats of the PCR product of the ligation product.
  • Fig. 6 shows the results of analyzing and counting the 5M and 30M sequencing data in Example 2 respectively.
  • a is the effective comparison rate;
  • b is the repetition rate;
  • c is the GC content.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative experiments in the following examples are all set to repeat the experiment three times, and the results are averaged.
  • Example 1 Autonomous platform PCR-free library construction kit (enzyme digestion method) construction of human whole genome library and sequencing
  • the final DNA fragment is 300-500bp.
  • reaction sample into a PCR machine for reaction, and the reaction conditions: 14°C for 15min; 37°C for 25min; 65°C for 15min; 4°C forever; PCR instrument cover temperature: 70°C.
  • linker sequence used in this scheme is as follows (the sequence in this example is from 5'to 3'from left to right, "//" indicates a modified group, "phos” indicates phosphorylation, and an underline indicates 10 bases Tag sequence.)
  • Phosphorylated linker B chain /Phos/GAACGACATGGCTACGATCCGACTT (SEQ ID No. 1);
  • Phosphorylated linker T chain /Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAA NNNNNNNNNNNN CNN CAACTCCTTGGCTCACA (SEQ ID No. 2).
  • N can be A or T or C or G.
  • Linker A 25 ⁇ M
  • linker B chain 20 ⁇ L 100 ⁇ M linker B chain, 20 ⁇ L 100 ⁇ M linker T chain and 40 ⁇ L 2 ⁇ adpter buffer (components: 50mM Tris-HCl (pH8.0), 0.1mM EDTA, 50mM NaCl) are mixed to form linker A (25 ⁇ M)
  • linker A 25 ⁇ M
  • linker B 6 ⁇ M
  • the ligation reaction solution prepared above, the mixed solution of connector B and the product of step 4 is vortexed to mix, and centrifuged instantaneously. Then put the reaction sample into the PCR machine for reaction, the reaction conditions: 25°C for 30 min; 4°C hold, and the thermal cover of the PCR machine: 30°C. After the reaction, add 20 ⁇ L of TE buffer, then add 50 ⁇ L of XP magnetic beads for purification, and 50 ⁇ L of TE buffer to dissolve and recover the product.
  • the 20 ⁇ M mediated fragment has a corresponding complementary sequence for connecting the two ends of the single strand.
  • the sequence is as follows (the sequence in this example is from left to right from 5'to 3'): GCCATGTCGTTCTGTGAGCCAAGG (SEQ ID No. 8) .
  • the linear digested single-stranded loop product obtained in the previous step was quantified with Qubit ssDNA Assay Kit.
  • Figure 3 shows the results of whole genome interruption of two Human gDNA samples (the results of parallel and repeated library construction of NA12878 standard DNA).
  • Figure 4 shows the detection result of the 6% TBU gel of the single-stranded loop library. It can be seen from Figures 3 and 4 that the results of restriction digestion and library construction are normal, indicating that the library construction system is compatible with other restriction digestion methods.
  • Table 6 shows the sequencing quality of the WGS PCR free library (NEB enzyme digestion method) of the Human sample finally obtained from library construction and sequencing in this example.
  • Table 6 shows that the WGS PCR free library (NEB digestion method) of Human samples has good sequencing quality on BGI's autonomous high-throughput sequencing platform MGISEQ-2000RS PE150.
  • Covaris break (PE100) is a PCR-free control, using the same library building system, even if the enzyme digestion method is used to build the library, the effect is equivalent to that of the physical method.
  • Table 7 shows the results of the SNP and Indel mutation detection analysis results of the NA12878 WGS PCR free library (NEB enzyme digestion method) finally obtained by the library construction and sequencing in this example.
  • Table 7 shows that the PCR free library (NEB digestion method) of the present invention has obvious advantages over PCR libraries in indel calling, and the overall performance is similar to the data of NovaSeq PCR free PE150 on the Illumina platform.
  • Covaris break is a PCR-free control, using the same library building system, even if the enzyme digestion method is used to build the library, the effect is equivalent to that of the physical method.
  • BGISEQ-500 physical PCR is a PCR-dependent control.
  • Experimental purpose use MGI PCR-free kit to construct a plasma sample library.
  • Source of experimental samples 20 plasma samples, including 2 samples with chromosomal abnormalities.
  • 10 ⁇ T4 polynucleotide kinase buffer Enzematics 5 ⁇ L T4 polynucleotide kinase (10U/ ⁇ L) (Enzematics) 1 ⁇ L Deoxyribonucleic acid mixture (25mM each) (Enzematics) 0.5 ⁇ L Taq DNA polymerase (5U/ ⁇ L) (Takara) 0.4 ⁇ L Adenosine deoxyribonucleic acid (100mM) (Enzematics) 0.5 ⁇ L T4 DNA polymerase (3U/ ⁇ L) (Enzymatics) 2 ⁇ L Enzyme-free pure water (Sigma) 0.6 ⁇ L total capacity 10 ⁇ L
  • linker sequence used in this scheme is as follows (the sequence in this example is from 5'to 3'from left to right, "//" indicates a modified group, "phos” indicates phosphorylation, and an underline indicates 10 bases Tag sequence.)
  • Phosphorylated linker B chain /Phos/GAACGACATGGCTACGATCCGACTT (SEQ ID No. 1);
  • Phosphorylated linker T chain /Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAA NNNNNNNNNNNN CNN CAACTCCTTGGCTCACA (SEQ ID No. 2).
  • N can be A or T or C or G.
  • Adapter preparation 20 ⁇ L 100 ⁇ M linker B chain, 20 ⁇ L 100 ⁇ M linker T chain and 40 ⁇ L 2 ⁇ adapter buffer (components: 50mM Tris-HCl (pH8.0), 0.1mM EDTA, 50mM NaCl) and mixed to prepare linker A (25 ⁇ M) , After being placed at room temperature for more than half an hour, it can be diluted to use concentration or stored at -20°C. When in use, linker A (25 ⁇ M) was diluted with TE to linker B (1 ⁇ M).
  • the ligation reaction solution prepared above and the mixed solution of joint B and step 4 are vortexed to mix, and centrifuged instantaneously. Then put the reaction sample into the PCR machine for reaction, the reaction conditions: 25°C for 30 min; 4°C hold, and the thermal cover of the PCR machine: 30°C. After the reaction, add 20 ⁇ L of TE buffer, then add 50 ⁇ L of XP magnetic beads (Beckman Coulter) for purification, and 22 ⁇ L of TE buffer to dissolve and recover the product. Take 15 ⁇ l of each sample, mix multiple samples in equal volume, then add a double volume of XP magnetic beads (Beckman Coulter) for purification, and dissolve and recover the product in 22 ⁇ L TE buffer.
  • Method 1 Refer to Example 1, steps 6 to step 9 of whole-genome library construction and sequencing, sample single-strand circularization, linear digestion, DNA nanosphere preparation, and BGISEQ-500SE50+10 on-machine sequencing.
  • the sequencing process is performed on the computer and data analysis according to the BGISEQ-500SE50+10 standard operating procedure.
  • Method 2 Take the constructed ligation product and perform one-step DNA nanosphere preparation and BGISEQ-500SE50+10 on-machine sequencing. The sequencing process is performed on the computer and data analysis according to the BGISEQ-500SE50+10 standard operating procedure. Method 2 is adopted in this embodiment.
  • Figure 6 shows the results of analysis and statistics by intercepting 5M and 30M sequencing data respectively. It can be seen that the effective comparison rate of all samples in a meets the requirements of BGI prenatal testing kit; the repetition rate of all samples in b meets the requirements of BGI prenatal testing kit; the GC of all samples in c The content meets the requirements of BGI's prenatal testing kit.
  • Example 3 Construction of a human whole genome library by an autonomous platform PCR-free library building kit (enzyme digestion method)
  • each tube with an initial amount of 1 ⁇ g, use dsDNA Fragmentase for restriction digestion and end repair plus A.
  • the interruption system is 50 ⁇ L. Take out the corresponding reagents in advance to dissolve and mix, and turn the enzyme reagents upside down to ensure that they are mixed and placed on ice.
  • the reaction system was prepared on ice as shown in Table 11.
  • the final DNA fragment is 300-500bp.
  • linker sequence used in this scheme is as follows (the sequence in this example is from 5'to 3'from left to right, "//" indicates a modified group, "phos” indicates phosphorylation, and an underline indicates 10 bases Tag sequence.)
  • Phosphorylated linker B chain /Phos/GAACGACATGGCTACGATCCGACTT (SEQ ID No. 1);
  • Phosphorylated linker T chain /Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAA NNNNNNNNNNNN CNN CAACTCCTTGGCTCACA (SEQ ID No. 2).
  • N can be A or T or C or G.
  • Linker A 20 ⁇ L 100 ⁇ M linker B chain, 20 ⁇ L 100 ⁇ M linker T chain and 40 ⁇ L 2 ⁇ adpterbuffer (components: 50mM Tris-HCl (pH8.0), 0.1mM EDTA, 50mM NaCl) are mixed to form linker A (25 ⁇ M), After being placed at room temperature for more than half an hour, it can be diluted to use concentration or stored at -20°C. When in use, linker A (25 ⁇ M) was diluted with TE to linker B (6 ⁇ M).
  • the ligation reaction solution prepared above, the mixed solution of connector B and the product of step 4 is vortexed to mix, and centrifuged instantaneously. Then put the reaction sample into the PCR machine for reaction, the reaction conditions: 25°C for 30 min; 4°C hold, and the thermal cover of the PCR machine: 30°C. After the reaction, add 20 ⁇ L of TE buffer, then add 50 ⁇ L of XP magnetic beads for purification, and 50 ⁇ L of TE buffer to dissolve and recover the product.
  • the 20 ⁇ M mediated fragment has a corresponding complementary sequence for connecting the two ends of the single strand.
  • the sequence is as follows (the sequence in this example is from left to right from 5'to 3'): GCCATGTCGTTCTGTGAGCCAAGG (SEQ ID No. 8) .
  • the linear digested single-stranded loop product obtained in the previous step was quantified with Qubit ssDNA Assay Kit.
  • Fragment selection product concentration 2.6 ng/ ⁇ l
  • Single-stranded ring concentration 2.16 ng/ ⁇ l
  • Example 4 Construction of a human whole genome library by an autonomous platform PCR-free library building kit (enzyme digestion method)
  • the final DNA fragment is 300-500bp.
  • reaction sample into the PCR machine for reaction, the reaction conditions: 65°C for 30min; 4°C forever; PCR instrument cover temperature: 70°C.
  • linker sequence used in this scheme is as follows (the sequence in this example is from 5'to 3'from left to right, "//" indicates a modified group, "phos” indicates phosphorylation, and an underline indicates 10 bases Tag sequence.)
  • Phosphorylated linker B chain /Phos/GAACGACATGGCTACGATCCGACTT (SEQ ID No. 1);
  • Phosphorylated linker T chain /Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAA NNNNNNNNNNNN CNN CAACTCCTTGGCTCACA (SEQ ID No. 2).
  • N can be A or T or C or G.
  • Linker A 25 ⁇ M
  • linker B chain 20 ⁇ L 100 ⁇ M linker B chain, 20 ⁇ L 100 ⁇ M linker T chain and 40 ⁇ L 2 ⁇ adpter buffer (components: 50mM Tris-HCl (pH8.0), 0.1mM EDTA, 50mM NaCl) are mixed to form linker A (25 ⁇ M)
  • linker A 25 ⁇ M
  • linker B 6 ⁇ M
  • the ligation reaction solution prepared above, the mixed solution of connector B and the product of step 4 is vortexed to mix, and centrifuged instantaneously. Then put the reaction sample into the PCR machine for reaction, the reaction conditions: 25°C for 30 min; 4°C hold, and the thermal cover of the PCR machine: 30°C. After the reaction, add 20 ⁇ L of TE buffer, then add 50 ⁇ L of XP magnetic beads for purification, and 50 ⁇ L of TE buffer to dissolve and recover the product.
  • reaction sample into the PCR machine for reaction, the reaction conditions: 37°C for 30 min; 4°C hold, and the thermal cover of the PCR machine: 42°C.
  • the 20 ⁇ M mediated fragment has a corresponding complementary sequence for connecting the two ends of the single strand.
  • the sequence is as follows (the sequence in this example is from left to right from 5'to 3'): GCCATGTCGTTCTGTGAGCCAAGG (SEQ ID No. 8) .
  • the linear digested single-stranded loop product obtained in the previous step was quantified with Qubit ssDNA Assay Kit.
  • Total fragment selection product 184 ng Single-stranded ring concentration 2.2 ng/ ⁇ l
  • Example 5 Self-contained platform PCR-Free library building kit (enzyme digestion method) combined with double barcode linker to construct a human whole genome library and sequencing
  • each tube of 1 ⁇ g starting amount use dsDNA Fragmentase (NEB company, catalog number M0348) for restriction digestion, the interruption system is 50 ⁇ L.
  • 10X Fragmentase Reaction Buffer v2 Take out 10X Fragmentase Reaction Buffer v2 in advance to dissolve and votex and mix well. Mix dsDNA Fragmentase votex and place on ice. Prepare the reaction system on ice as shown in Table 22.
  • the final DNA fragment is 300-500bp.
  • reaction sample into the PCR machine for reaction, and the reaction conditions: 14°C for 15min; 37°C for 25min; 65°C for 15min; 4°C forever; PCR instrument cover temperature: 70°C.
  • linker sequence used in this scheme is as follows (the sequence in this example is from 5'to 3'from left to right, "//" indicates a modified group, "phos” indicates phosphorylation, and an underline indicates 10 bases Tag sequence.)
  • the phosphorylated linker B chain /Phos/TCTCAGTACGTCAGCAGTTNNNNNNNNCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT (SEQ ID No. 7);
  • Phosphorylated linker T chain /Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAANNNNNNNNCTGATAAGGTCGCCATGCC (SEQ ID No. 6).
  • N can be A or T or C or G.
  • Linker A 25 ⁇ M
  • linker B chain 20 ⁇ L 100 ⁇ M linker B chain, 20 ⁇ L 100 ⁇ M linker T chain and 40 ⁇ L 2 ⁇ adaptorbuffer (components: 50mM Tris-HCl (pH8.0), 0.1mM EDTA, 50mM NaCl) are mixed to form linker A (25 ⁇ M), After being placed at room temperature for more than half an hour, it can be diluted to use concentration or stored at -20°C. When in use, linker A (25 ⁇ M) was diluted with TE to linker B (6 ⁇ M).
  • the ligation reaction solution prepared above, the mixed solution of connector B and the product of step 4 is vortexed to mix, and centrifuged instantaneously. Then put the reaction sample into the PCR machine for reaction, the reaction conditions: 25°C for 30 min; 4°C hold, and the thermal cover of the PCR machine: 30°C. After the reaction, add 20 ⁇ L of TE buffer, then add 50 ⁇ L of XP magnetic beads for purification, and 50 ⁇ L of TE buffer to dissolve and recover the product.
  • the 20 ⁇ M mediated fragment has a corresponding complementary sequence for connecting the two ends of the single strand.
  • the sequence is as follows (the sequence in this example is from left to right from 5'to 3'): TGCTGACGTACTGAGAGGCATGGCGACCT (SEQ ID No. 8) .
  • the linear digested single-stranded loop product obtained in the previous step was quantified with Qubit ssDNA Assay Kit.
  • Table 27 shows the results of the product concentration detection in each step.
  • Table 28 shows the sequencing quality of the WGS PCR free library (enzyme digestion method) of the Human sample finally obtained from library construction and sequencing in this example.
  • Table 28 shows that the Human sample WGS PCR free library (enzyme digestion method) has good sequencing quality on BGI's autonomous high-throughput sequencing platform MGISEQ-2000RS PE150.
  • Fragment selection product concentration 3.35 ng/ ⁇ l Total fragment selection product 150.75 ng Single-stranded ring concentration 1.25 ng/ ⁇ l
  • Covaris break (PE100) is a PCR-free control. Using the same library building system, even the double barcode enzyme digestion method is equivalent to the physical method.
  • Table 29 shows that the PCR free library (enzyme digestion method) of the present invention has obvious advantages over PCR libraries in indel calling, and the overall performance is similar to the data of NovaSeq PCR free PE150 on the Illumina platform.
  • Covaris break (PE100) is a PCR-free control, using the same library construction system, even if the enzyme digestion method is used to build the library, the effect is equivalent to that of the physical method.
  • the present invention provides a PCR-free library construction scheme, which avoids the problems of base errors, data bias and repetitive sequences introduced by PCR in the library construction process, and the scheme is compatible with different interruption methods and low initial amount of library construction.
  • the PCR-free library building technology of the present invention combined with the rolling circle replication nanosphere preparation technology truly realizes the PCR-free process of the sample from library building to sequencing, forming a full-process PCR-free.
  • the invention adopts the end repair and linker efficiency optimization system, single-stranded circularization and rolling circle replication one-step method to realize the improvement of the library building efficiency of an autonomous platform PCR-free library building kit and reduce the required amount of starting DNA.
  • the database construction system of the present invention is compatible with different types of initial samples, including but not limited to genomic DNA, interrupted DNA, DNA or DNA interrupted products obtained by whole-genome amplification methods, amplicon DNA, cfDNA, RNA DNA after reverse transcription, etc.
  • the present invention has the following beneficial effects: 1) Wide applicability: The present invention is applicable to all species with known or unknown reference sequences, and can be adopted by general molecular biology laboratories. Physical interruption and enzyme digestion interruption are compatible with library building.
  • Compatible with different types of samples including but not limited to genomic DNA, interrupted DNA, DNA or DNA interrupted products obtained by whole-genome amplification methods, amplicon DNA, cfDNA, DNA after RNA reverse transcription, etc. 2) Simple operation and short time for database construction.
  • the invention adopts a one-tube method for end repair and A addition reaction, skips magnetic bead purification and directly connects the joints, saves conventional PCR amplification and purification operations, and greatly saves library building time.
  • a library preparation method that combines circularization and rolling circle replication in a one-step reaction can further shorten the library construction and sequencing time.
  • High library construction efficiency Through optimized end repair-add A formula and system, optimized joint connection system, and a library preparation method that combines cyclization and rolling circle replication at the same time, pooling of low starting volume samples can be realized. Sequencing can realize PCR-free library construction of 200 ⁇ l plasma DNA.
  • the present invention realizes that there is no amplification reaction for real library construction and sequencing, and can improve the accuracy and sensitivity of SNP and InDel detection. In particular, the detection performance of InDel is excellent, and it can be rushed. Super-competitive Illumina platform.

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Abstract

本发明公开了一种PCR-free建库和测序方法。本发明提供了一种PCR-free高通量测序方法，包括如下步骤：将待测序核酸根据大小进行或不进行片段化处理，得到目的大小DNA片段；末端修复和加A反应；连接含有标签序列的接头；进行单链环化和滚环复制，形成DNA纳米球；上机测序。

Description

一种高兼容性的PCR-free建库和测序方法 技术领域
本发明涉及分子生物学高通量测序技术领域，具体涉及一种高兼容性的PCR-free建库和测序方法，该方法适用于DNA不经PCR扩增就进行文库构建的样品。
背景技术
二代测序技术(NGS)是现代分子生物学高通量测序研究中最常用的技术。二代测序对DNA测序主要包括文库制备和上机测序两个过程。在文库制备中,一般通过标准PCR来扩增随机打断的基因组片段。但对于一些特殊模板，若存在二级结构复杂或热稳定性不好等因素，会造成模板PCR扩增偏好，因此并非所有序列都能在PCR扩增文库中同等体现。特别是对一些高GC或高AT含量的模板，有时很难用PCR方法进行文库构建。PCR-free和常规PCR文库上机测序时无明显区别，只是建库过程不需要做PCR，PCR-free文库理论上可以改善数据读取分布并有更均一的序列覆盖度。二代测序技术的上机测序部分，其主流方向还需经过PCR扩增来放大后续检测信号，即使能做到建库过程的PCR-free仍不能避免测序过程中引入的PCR问题。
目前自主平台的大部分建库试剂盒都是基于PCR建库，PCR建库存在coverage、GC bias、InDel检测准确性和灵敏度差的缺点，且建库流程较长，全自动化对仪器和实验室要求高，建库、人工、折旧成本较高。Illumina的Truseq DNA PCR-free样品制备试剂盒理论上可在一天内完成文库构建，并可与Illumina测序平台不断增加读长相兼容。但此试剂盒只兼容1-2μg起始的物理打断法文库构建，主要用于人重测序，不兼容酶切打断法和FFPE，cfDNA等低起始量样本，应用领域和灵活性较窄。此外，Illumina平台采用的是桥式PCR进行文库放大，即使建库采用的PCR-free，测序之前的文库放大仍然是PCR的形式，不是真正意义上的PCR-free。目前市面PCR-free试剂盒及优缺点总结如表1所示。
表1 目前市面PCR-free试剂盒及优缺点总结
Figure PCTCN2020096987-appb-000001
Figure PCTCN2020096987-appb-000002
常规PCR建库时间长、成本较高，且无法避免PCR带来的碱基偏好性，从而 引起碱基错误、数据偏向及重复序列，且PCR方法在Indel calling方面相比PCR-free表现较差。目前，市面上的PCR-free试剂盒主要集中在国外试剂盒公司。起始量要求较高，是限制其应用的一个重要原因，提高建库效率，是降低建库起始量的关键点。目前市面最低建库起始量为50ng的gDNA和5ng的cfDNA。大部分建库试剂盒的兼容性较差，表现在：1)有些只能用于物理打断法；2)有些能兼容的样本类型只限于正常基因组DNA，而对FFPE，cfDNA，严重降解的DNA不兼容；3)有些未提供配套的酶打断试剂盒。
发明公开
本发明目的在于针对现有技术的不足，提供一种新的快速高效的可应用于华大基因自主测序平台的PCR-free测序文库的制备方法。此方法不进行PCR扩增而在接头连接后直接经单链环化制成可上机文库，从而减少PCR引入的碱基错误、数据偏向及重复序列。
第一方面，本发明要求保护一种PCR-free高通量测序方法。
本发明要求保护的PCR-free高通量测序方法，可为如下方法A或方法B或方法C：
所述方法A，可包括如下步骤：
(A1)将待测序核酸根据大小进行或不进行片段化处理，得到目的大小DNA片段；
(A2)对(A1)产物进行末端修复和加A反应；
(A3)对(A2)产物连接接头；
(A4)对(A3)产物进行单链环化和滚环复制，形成DNA纳米球；
(A5)上机测序。
所述方法B，可包括如下步骤：
(B1)将待测序核酸根据大小进行片段化处理，同时进行末端修复和加A反应得到目的大小DNA片段；
(B2)对(B1)产物连接接头；
(B3)对(B2)产物进行单链环化和滚环复制，形成DNA纳米球；
(B4)上机测序。
所述方法C，可包括如下步骤：
(C1)将待测序核酸根据大小进片段化处理，同时进行末端修复，得到目的大小DNA片段；
(C2)对(C1)产物进行加A反应；
(C3)对(C2)产物连接接头；
(C4)对(C3)产物进行单链环化和滚环复制，形成DNA纳米球；
(C5)上机测序。
在步骤(B1)和(C1)中，所述片段化处理是利用片段化酶消化所述待测序核酸。
进一步地，所述片段化酶可如
Figure PCTCN2020096987-appb-000003
Ultra TM II FS DNA Module、Qiagen5X WGS Fragmentation Mix，或自研的打断酶。
在所述方法A、所述方法B和所述方法C中，所述接头中均包括标签序列(barcode)。优选的，一个所述接头中包括两个标签序列(barcode)。在所述待测序核酸两端分别加上包括两个标签序列(barcode)的所述接头，形成带有双标签(barcode)的文库结构。
进一步地，所述接头是由两条部分互补的核酸单链退火形成的，所述标签序列位于两条核酸单链的非互补区域。
在步骤(A1)、(B1)和(C1)中，所述待测序核酸可为DNA或者RNA。
进一步地，所述DNA为基因组DNA或者天然存在的小分子DNA或经扩增所得的DNA片段。
所述天然存在的小分子DNA可如cfDNA。所述经扩增所得的DNA片段可如MDA产物，cDNA产物，扩增子等。
本发明的起始样本可以扩展到非DNA样本的血液或唾液样本直接建库。
在步骤(A1)中，所述片段化处理方式可为物理打断法或酶打断法。
其中，所述物理打断法可如超声波打断。
当所述待测序核酸大于所述目的大小DNA片段时(如为基因组DNA)，需对其进行打断处理，经过磁珠片段选择所述目的大小DNA片段；当所述待测序核酸不大于所述目的大小DNA片段时(如为cfDNA，DNA片段小而且主带集中)，无需对所述待测序核酸继续片段化处理。
当所述待测序核酸为RNA时，需要反转录得到DNA；进行所述片段化处理为对RNA进行片段化处理或者对所述RNA反转录得到的DNA进行片段化处理。
在步骤(A1)，(B1)和(C1)中，所述目的大小DNA片段的范围可为150bp-800bp(如300-500bp)。
在步骤(A2)中，进行所述末端修复和加A反应可一步完成的，具体可按照包括如下步骤的方法进行：将末端修复与加A反应液与所述(A1)产物混合，反应，得到所述(A2)产物。
其中，所述末端修复与加A反应液含有T4多聚核苷酸激酶缓冲液(T4 PNK buffer)、腺苷酸脱氧核糖核酸(dATP)、脱氧核糖核酸混合液(dNTPs)、T4 DNA聚合酶(T4 DNA polymerase)、T4多聚核苷酸激酶(T4 PNK)、Taq DNA聚合酶(rTaq)。
进一步地，在所述末端修复与加A反应液中，所述腺苷酸脱氧核糖核酸(dATP)、所述脱氧核糖核酸混合液(dNTPs)、所述T4 DNA聚合酶(T4 DNA polymerase)、所述T4多聚核苷酸激酶(T4 PNK)和所述Taq DNA聚合酶(rTaq)的配比为50nmol:12.5nmol(每种dNTP):6U:10U:2-5U。
在本发明的具体实施方式中，所述末端修复与加A反应液由10×T4多聚核苷酸激酶缓冲液(10×T4 PNK buffer)、浓度为100mM的腺苷酸脱氧核糖核酸 溶液(dATP)、每种脱氧核糖核酸浓度均为25mM的4种脱氧核糖核酸混合液(dNTPs)、浓度为3U/μL的T4 DNA聚合酶(T4 DNA polymerase)、浓度为10U/μL的T4多聚核苷酸激酶(T4 PNK)、浓度为5U/μL的Taq DNA聚合酶(rTaq)按照体积比为5：0.5：0.5：2：1：(0.4-1)的比例混合而成。
在步骤(A2)中，将所述末端修复与加A反应液与所述(A1)产物混合时两者的体积比可为1：4。
在步骤(A2)中，将所述末端修复与加A反应液与所述(A1)产物混合后进行反应的条件可为1)14℃15min；37℃25min；65℃15min；4℃保温。PCR仪盖温为：70℃；或者2)37℃孵育10min；72℃孵育15min,以0.1秒的速率降温至4℃。
在步骤(B1)中，进行所述片段化处理和所述末端修复和加A反应是一步完成的：将片段化处理和末端修复与加A反应液与所述待测序核酸混合，反应，得到所述(B1)产物。
所述片段化处理和末端修复与加A反应液含有片段化酶、片段化酶反应缓冲液、腺苷酸脱氧核糖核酸、脱氧核糖核酸混合液、T4 DNA聚合酶、Taq DNA聚合酶和TE缓冲液。
进一步地，在所述片段化处理和末端修复与加A反应液中，所述腺苷酸脱氧核糖核酸、所述脱氧核糖核酸混合液、所述T4 DNA聚合酶和所述Taq DNA聚合酶的配比可为170nmol:57.5nmol:3U:5U。所述打断酶的用量根据说明书或者经多次实验测试结果确定。
进一步地，步骤(B1)中，将所述片段化处理和末端修复与加A反应液与所述待测序核酸混合后进行反应的条件可为37℃孵育10-20min；65℃孵育30min；降温至4℃。PCR仪盖温为：70℃。
在步骤(C1)中，进行所述片段化处理和所述末端修复反应是一步完成的：将片段化处理和末端修复反应液与所述待测序核酸混合，反应，得到所述(C1)产物。
所述片段化处理和末端修复反应液含有片段化酶、片段化酶反应缓冲液、脱氧核糖核酸混合液、DNA聚合酶I、MgCl 2和无酶纯水。
进一步地，在所述片段化处理和末端修复反应液中，所述脱氧核糖核酸混合液、所述DNA聚合酶I和所述MgCl 2的配比可为75nmol(每种dNTP):20U:0.3μmol。所述打断酶的用量根据说明书或者经多次实验测试结果确定。
进一步地，步骤(C1)中，将所述片段化处理和末端修复反应液与所述待测序核酸混合后进行反应的条件可为37℃30min；4℃保温。PCR仪盖温为：70℃。反应结束后立即区样品于冰上，补充TE 30μl。
在步骤(C2)中，对所述(C1)产物进行加A反应时采用的加A反应液含有T4多聚核苷酸激酶缓冲液(T4 PNK buffer)、腺苷酸脱氧核糖核酸(dATP)、脱氧核糖核酸混合液(dNTPs)、Taq DNA聚合酶(rTaq)。
进一步地，在所述加A反应液中，所述腺苷酸脱氧核糖核酸(dATP)、所述脱氧核糖核酸混合液(dNTPs)、和所述Taq DNA聚合酶(rTaq)的配比为50nmol:8.75nmol(每种dNTP):1U。
在本发明的具体实施方式中，所述加A反应液由10×T4多聚核苷酸激酶缓冲液(10×T4 PNK buffer)、浓度为100mM的腺苷酸脱氧核糖核酸溶液(dATP)、每种脱氧核糖核酸浓度均为25mM的4种脱氧核糖核酸混合液(dNTPs)、浓度为5U/μL的Taq DNA聚合酶(rTaq)和无酶纯水按照体积比为5：0.5：0.35：0.2：1的比例混合而成。
在步骤(A3)、(B2)和(C3)中，所述接头由B链和T链退火后形成。所述B链的3’端与所述T链的5’端互补配对，且两条链的其他部分不互补配对，且所述B链的3’末端有突出的dT；所述B链和/或所述T链中不互补配对的部分含有用于标记不同样品的标签序列(即barcode)；所述B链和所述T链的5’端均修饰有磷酸基团或连接有3’末端为U的一段单链寡核苷酸。
当所述接头中含有3’末端为U的一段单链寡核苷酸时，还需要对其进行USER酶处理。进行USER酶处理的步骤可以边连接边进行，也可以加完接头纯化后再进行。
进一步地，所述接头可为如下任一：
接头1：由5’端修饰有磷酸基团的SEQ ID No.1所示单链DNA和5’端修饰有磷酸基团的SEQ ID No.2所示单链DNA退火后形成。
磷酸化的接头B链：/Phos/GAACGACATGGCTACGATCCGACTT(SEQ ID No.1)；
磷酸化的接头T链：/Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAANNNNNNNNNN CAACTCCTTGGCTCACA(SEQ ID No.2)。
所述接头1为分叉型接头，接头连接之后可以直接进行环化或环化与滚环复制一步反应。
接头2：由SEQ ID No.3或SEQ ID No.4所示单链DNA和SEQ ID No.2所示单链DNA退火后形成。
带U碱基的接头B链(设计1)：TTGTCTTCCUGAACGACATGGCTACGATCCGACTT(SEQ ID No.3)；
带U碱基的接头B链(设计2)：TTGTCTTCCTAAGUGAACGACATGGCTACGATCCGACTT(SEQ ID No.4)；
磷酸化的接头T链：/Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAANNNNNNNNNNCAACTCCTTGGCTCACA(SEQ ID No.2)。
所述接头2为Bubble U型接头，接头可边连接边进行USER酶处理，之后可以直接进行环化或环化与滚环复制一步反应。
接头3：由SEQ ID No.5所示单链DNA和SEQ ID No.6所示单链DNA退火后形成。
接头B链：TTGTCTTCCUTCTCAGTACGTCAGCAGTTNNNNNNNNNNCAACTCCTTGGCTCA CAGAACGACATGGCTACGATCCGACTT(SEQ ID No.5)；
磷酸化的接头T链：/Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAANNNNNNNNNNCTGATAAGGTCGCCATGCC(SEQ ID No.6)。
所述接头3为双Barcode U型接头，接头可边连接边进行USER酶处理，之后可以直接进行环化或环化与滚环复制一步反应。
接头4：由SEQ ID No.7所示单链DNA和SEQ ID No.6所示单链DNA退火后形成。
磷酸化的接头B链：/Phos/TCTCAGTACGTCAGCAGTTNNNNNNNNNNCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT(SEQ ID No.7)；
磷酸化的接头T链：/Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAANNNNNNNNNNCTGATAAGGTCGCCATGCC(SEQ ID No.6)。
所述接头4为双barcode分叉型接头序列，接头连接之后可以直接进行环化或环化与滚环复制一步反应。
接头两条链分叉部分各带有一个barcode序列，通过连接反应加到文库上(图1)，PCR之后文库两端各带有一个barcode，优点是存在在加接头之后样品即可混合的可能性，并且PCR free文库可以通用。
在文库的两端加上barcode序列，并且通过两端barcode序列的组合，极大提高了barcode的种类，实现大量文库的混合上机测序。如图2所示，横列和竖列的barcode分别加到文库两端，两端barcode相互组合提高了barcode的种类。一个文库两端的barcode序列既可以相同又可以不同，图2的例子中，只需设计8种barcode序列，通过两端的组合，可以实现64种组合，解决了单barcode文库里需要设计非常多种barcode序列才能实现相应多文库混合测序的问题。
另外在每一个barcode在两端都是唯一使用的情况下，利用两端barcode的唯一对应关系，能够大幅度消除文库构建或者测序中发生的barcode跳转的错误结果。
各接头序列，均是从左到右为5’端至3’端，“//”示修饰基团，“phos”示磷酸化。其中接头T链10碱基序列NNNNNNNNNN代表标签(Barcode)序列，N可为A或T或C或G。标签序列用于标记不同样品。可将带不同标签序列的样品构建混合文库。
所述接头序列不局限于以上的序列，即使对序列进行修改，只要保证结构能在BGI/MGI平台上测序，也可以达到类似的效果即可。
此外，通过对所述接头的序列的改动，如在接头序列中加入相应的NNN(N可为A或T或C或G，在具体实施例中N的长度可根据实验目的进行设置)标签，文库在完成连接所述接头的同时，也能够加入独特分子标记(Unique Molecular  Identifiers，UMI)。UMI能够作为每一条样品链的标识，多用于低频突变的检测。UMI通常与样品相邻或与barcode相邻，测序时和样品或barcode公用一条测序引物连续测序；也可与样品和barcode都隔开，测序时使用独立的测序引物。
在步骤(A3)、(B2)和(C3)中，对所述(A2)或(B1)或(C2)产物连接上所述接头可按照包括如下步骤的方法进行：将所述接头、所述(A2)或(B1)或(C2)产物和连接反应液混合，反应，得到所述(A3)或(B2)或(C3)产物。
其中，所述连接反应液含有T4多聚核苷酸激酶缓冲液(T4 PNK buffer)、腺苷酸核糖核酸(ATP)、PEG8000、T4 DNA连接酶(T4 DNA ligase)和无酶纯水。
进一步地，在所述连接反应液中，所述腺苷酸核糖核酸(ATP)、所述PEG8000、所述T4 DNA连接酶(T4 DNA ligase)的配比为0.8μmol腺苷酸核糖核酸：10-16μL 50％PEG8000(如Rigaku产品)：1200-3000U T4 DNA连接酶,如0.8μmol腺苷酸核糖核酸：16μL 50％PEG8000：3000U T4 DNA连接酶。
在本发明的具体实施方式中，所述连接反应液由10×T4多聚核苷酸激酶缓冲液(10×T4 PNK buffer)、浓度为0.1M的腺苷酸核糖核酸(ATP)、浓度为50％的PEG8000(如Rigaku产品)、浓度为600U/μL的T4 DNA连接酶(T4 DNA ligase)和无酶纯水按照体积比为3：0.8：16：5：0.2的比例混合而成。
在步骤(A3)、(B2)和(C3)中，将所述接头、所述(A2)或(B1)或(C2)产物和所述连接反应液混合为将含有所述接头的接头溶液、所述(A2)或(B1)或(C2)产物和所述连接反应液按照体积比为1-5：50：25-29的比例(具体如5：50：25或者1：50：29)混合；所述接头在所述接头溶液中的浓度为6μM或1μM。
在步骤(A3)、(B2)和(C3)中，将所述接头、所述(A2)或(B1)或(C2)产物和所述连接反应液混合后进行反应的条件可为25℃10-30min(如30min)；4℃保温。PCR仪热盖为：30℃。
本发明设计了可兼容于DNBSEQ TM系列测序平台和华大多种自主高通量测序平台的接头，如单barcode接头和双barcode接头，接头序列含有Barcode序列，可同时对多个样品进行标记，可将带不同标签序列的样品构建混合文库。本发明采用优化的接头连接体系，可以在接头投入量较低(如接头与DNA的摩尔比例为5:1到50:1)的情况下，仍然保证高效的连接效率，这样的好处是既避免了接头过多带来的接头污染问题，又能提高DNA模板转化成双端都加上接头的测序文库的效率。
在步骤(A4)、(B3)和(C4)中，在对所述(A3)或(B2)或(C3)产物进行单链环化和滚环复制之前还包括进行纯化的步骤；
进一步地，所述纯化为磁珠纯化(如：XP磁珠或各种国产纯化磁珠)。
向所述(A3)或(B2)或(C3)产物加入TE缓冲液，再加入XP磁珠(Beckman Coulter公司)或各种国产纯化磁珠进行纯化，然后用TE缓冲液溶解回收产物。
在步骤(A4)、(B3)和(C4)中，对所述(A3)或(B2)或(C3)产物进行单链环化和滚环复制，形成DNA纳米球，具体可通过如下任一实现：
(a1)依次进行单链环化、消化线性单链、纯化、滚环复制，得到DNA纳米球；
(a2)单链环化和滚环复制一步完成，得到DNA纳米球。
在步骤(a1)中，所述单链环化可按照方法I或方法II进行：
所述方法I包括如下步骤：
I-1：将所述(A3)或(B2)或(C3)产物置于95℃孵育3min；4℃孵育10min，得到孵育后产物；
I-2：将单链环化反应液1与步骤I-1所得的所述孵育后产物混合，反应，得到环化产物。
其中，所述单链环化反应液1含有TA缓冲液、腺苷酸核糖核酸(ATP)、介导片段、T4 DNA连接酶(T4 DNA ligase)和无酶纯水。所述介导片段为5’端与构成所述接头的所述B链的5’端(去除3’末端为碱基U的一段寡核苷酸单链后)反向互补，3’端与构成所述接头的所述T链的3’端反向互补的单链DNA。
在本发明中，当所述接头为前文所述接头1或所述接头2时，所述介导片段的核苷酸序列如SEQ ID No.8所示。
进一步地，在所述单链环化反应液1中，所述腺苷酸核糖核酸(ATP)、所述介导片段、所述T4 DNA连接酶(T4 DNA ligase)的配比为60nmol:62.5pmol:600U。
在本发明的具体实施方式中，所述单链环化反应液1由10×TA缓冲液、浓度为100mM的腺苷酸核糖核酸(ATP)、浓度为25μM的所述介导片段、浓度为600U/μL的T4 DNA连接酶(T4 DNA ligase)和无酶纯水按照体积比为6：0.6：2.5：1：1.9的比例混合而成。
将所述单链环化反应液1与所述孵育后产物混合时两者的体积比可为48：12。
将所述单链环化反应液1与所述孵育后产物混合后进行反应的条件可为37℃60min；4℃保温。PCR仪热盖为：42℃。
所述方法II包括如下步骤：将所述(A3)或(B2)或(C3)产物与所述介导片段、NaOH溶液混合后，室温孵育5min；然后再和Tris-HCL溶液混合，再加入单链缓和反应液2，反应，得到环化产物。
其中，所述NaOH溶液浓度为2M,所述Tris-HCL溶液浓度为1M,pH6.8。
进一步地，所述NaOH、所述介导片段、所述Tris-HCL的配比为5μmol:100pmol:5μmol。
其中，所述单链环化反应液2含有TA缓冲液、腺苷酸核糖核酸(ATP)和T4 DNA连接酶(T4 DNA ligase)。
在本发明中，在所述单链环化反应液2中，所述腺苷酸核糖核酸(ATP)、所述T4 DNA连接酶(T4 DNA ligase)的配比为60nmol:240U。
在本发明的具体实施方式中，所述单链环化反应液2由10×TA缓冲液、浓度为100mM的腺苷酸核糖核酸(ATP)和浓度为600U/μL的T4 DNA连接酶(T4 DNA ligase)按照体积比为6：0.6：0.4的比例混合而成。
在本发明的具体实施方式中，所述(A3)或(B2)或(C3)产物、浓度为20μM的所述介导片段、浓度为2M的所述NaOH溶液、1M Tris-HCL和所述单链环化反应液2的体积比为48：5：2.5：5：7。
在所述方法II中，进行所述反应的条件可为37℃30min；4℃保温。PCR仪热盖为：42℃。
在步骤(a1)中，所述消化线性单链可按照包括如下步骤的方法进行：
第三步：将消化反应液与第二步所得的所述环化产物混合，反应，得到消化后产物。
其中，所述消化反应液含有TA缓冲液、ExoI酶、ExoIII酶和无酶水。
进一步地，在所述消化反应液中，所述ExoI酶、所述ExoIII酶的配比可为4U：1U。
在本发明的具体实施方式中，所述消化反应液由10×TA缓冲液、浓度为20U/μL的ExoI酶、浓度为10U/μL的ExoIII酶和无酶水按照体积比为0.4：2：1：0.6的比例混合而成。
将所述消化反应液与所述环化产物混合时两者的体积比可为4：60或4：67.5。
将所述消化反应液与所述环化产物混合后进行反应的条件可为37℃孵育30min。
该步骤中还可包括：向反应产物中加入EDTA，混匀。
步骤(a1)中所述纯化为用磁珠纯化。具体地，可使用XP磁珠纯化回收上一步的产物，然后用TE缓冲液溶解产物。
在步骤(A5)、(B4)和(C5)中，所述上机测序具体可利用BGISEQ，MGISEQ或DNBSEQ TM系列测序仪平台完成。
第二方面，本发明要求保护一种适用于PCR-free高通量测序的DNA文库构建方法。
本发明所要求保护的适用于PCR-free高通量测序的DNA文库构建方法，可包括前文第一方面中的步骤(A1)-(A4)或(B1)-(B3)或(C1)-(C4)。
第三方面，本发明要求保护利用前文第二方面中所述方法构建得到的DNA文库。
第四方面，本发明要求保护一种接头。
本发明所要求保护的接头为前文第一方面中所述的接头。
第五方面，本发明要求保护一种成套产品。
本发明所要求保护的成套产品含有前文第四方面所述接头和如下中的全部或部分：
(b1)前文第一方面中所述的末端修复与加A反应液；
(b2)前文第一方面中所述的片段化处理和末端修复与加A反应液；
(b3)前文第一方面中所述的片段化处理和末端修复反应液；
(b4)前文第一方面中所述的连接反应液；
(b5)前文第一方面中所述的单链环化反应液1或单链环化反应液2；
(b6)前文第一方面中所述的消化反应液。
第六方面，本发明要求保护一种系统。
本发明所要求保护的系统含有前文第五方面所述成套产品和DNBSEQ TM测序用试剂和/或设备。
第七方面，本发明要求保护前文第三方面所述DNA文库或前文第四方面所述接头或前文第五方面所述成套产品或前文第六方面所述系统在进行PCR-free高通量测序中的应用。
第八方面，本发明要求保护前文第四方面所述接头或前文第五方面所述成套产品在构建前文第三方面所述DNA文库中的应用。
附图说明
图1为使用双barcode接头时文库两端barcode的组合示意图。
图2为双barcode接头通过连接反应加到待测序核酸上后的示意图。
图3为实施例1中2个Human gDNA样品全基因组打断结果。泳道1和泳道2为1μg NA12878gDNA酶切法打断产物的两个平行重复。
图4为实施例1中单链环文库6％TBU胶检测结果。泳道1和泳道2为ssCir消化后产物的两个平行重复。
图5为实施例2中连接产物的PCR结果。泳道1和泳道2为连接产物的PCR产物的两个平行重复。
图6为实施例2中分别截取5兆和30兆测序数据进行分析统计的结果。a为有效比对率；b为重复率；c为GC含量。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
实施例1、自主平台PCR-free建库试剂盒(酶切法)构建人全基因组文库及测序
实验目的：用MGIPCR-free试剂盒配方结合NEB公司打断酶试剂盒构建 Human gDNA样品全基因组文库。
实验样本来源：NA12878标准品DNA(货号：NA12878，公司：CORIELL INSTITUTE)。
1、DNA样品NEB酶切法打断
分别取DNA标准品(TE溶解)，每管1μg起始量，使用
Figure PCTCN2020096987-appb-000004
Ultra TM II FS DNA Module(NEB公司)进行酶切打断，打断体系为35μL。提前取出NEBNext Ultra II FS Reaction Buffer溶解并votex混匀，NEBNext Ultra II FS Enzyme Mix上下颠倒确保混匀后置于冰上。于冰上配制反应体系如表1。
表1 DNA样品NEB酶切法打断反应体系
组分 用量
NEBNext Ultra II FS Reaction Buffer 7μL
gDNA(TE溶解) XμL
TE 26-XμL
总体积 33μL
用枪吹打混匀后，每个样品加入2μl NEBNext Ultra II FS Enzyme Mix,用枪温柔吹打混匀6-8次，禁止Votex；快速离心后迅速置thermocycler开始反应，反应条件：4℃forever；37℃10min；65℃30min；4℃forever；PCR仪盖温为：70℃。反应结束后立即区样品于冰上，补充TE 65μl。
2、DNA片段化选择
(1)取打断后样本100μL转移至新1.5ml不粘管中，加入60μL XP磁珠，震荡混匀并室温结合10分钟，置于磁力架上结合2分钟(至液体澄清)，小心吸取上清到新的1.5ml EP管中(该步保留上清)。向上清中加入15μLXP磁珠，震荡混匀并室温结合10分钟，置于磁力架上结合2分钟(至液体澄清)，吸弃上清。
(2)在磁力架上向不粘管里加入500μL 75％乙醇，盖紧管盖并混匀，弃掉上清；500μL 75％乙醇重复洗1次，用小量程的移液器尽可能弃掉残留的乙醇，室温晾干。
(3)用42μL TE重悬磁珠，震荡混匀并室温结合10分钟，置于磁力架上结合2分钟(至液体澄清)，小心吸出40μL上清至新的1.5mL EP管中，准备进行下一步反应或保存至-20℃冰箱
3、样本定量检测和样品均一化
取上述纯化后的DNA 2μL用于Qubit dsDNA HS定量检测。按照Qubit测定的浓度均一化片段选择的DNA，统一调整至150ng，使用1xTE补齐使总体积至40μL。如需保存可将均一化样品置于-20℃冰箱。
最终所得DNA片段为300-500bp。
4、末端修复与加A
首先，按照表2配制末端修复与加A反应液。
表2 末端修复与加A反应液组成
组分 用量
T4 10×PNK buffer(Enzymatics) 5μL
dATP(100mM)(Enzymatics) 0.5μL
dNTPs(each 25mM)(Enzymatics) 0.5μL
T4 DNA polymerase(3U/μL)(Enzymatics) 2μL
T4 PNK(10U/μL)(Enzymatics) 1μL
rTaq(5U/μL)(Enzymatics) 1μL
总体积 10μL
将10μL以上配制好的末端修复与加A反应液加到40μL步骤3获得的样本产物中，涡旋混匀，瞬时离心。总体积为50μL。将反应样品置入PCR仪中反应，反应条件：14℃15min；37℃25min；65℃15min；4℃forever；PCR仪盖温为：70℃。
5、接头连接
本方案中使用的接头序列如下(本实施例中的序列从左到右为5’端至3’端，“//”示修饰基团，“phos”示磷酸化，下划线示10个碱基的标签序列。)
磷酸化的接头B链：/Phos/GAACGACATGGCTACGATCCGACTT(SEQ ID No.1)；
磷酸化的接头T链：/Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAA NNNNNNNNNNCAACTCCTTGGCTCACA(SEQ ID No.2)。
其中，N可为A或T或C或G。
接头配制：20μL 100μM接头B链、20μL 100μM接头T链和40μL 2×adpter buffer(组分：50mM Tris-HCl(pH8.0)，0.1mM EDTA，50mM NaCl)进行混合配制成接头A(25μM)，在室温放置半小时以上之后可稀释成使用浓度或放置-20℃保存。使用时将接头A(25μM)用TE稀释成接头B(6μM)。
将5μL配制好的接头B(6μM)加入步骤4的产物中，充分混匀。
按照表3配制连接反应液。
表3 连接反应液组成
组分 用量
10×T4 PNK buffer(Enzymatics) 3μL
0.1MATP(Thermo) 0.8μL
50％PEG8000(Rigaku) 16μL
T4 DNA ligase(600U/μL)(Enzymatics) 5μL
无酶纯水(Sigma) 0.2μL
总体积 25μL
将上述配制好的连接反应液与接头B和步骤4产物的混合液涡旋混匀，瞬时离心。然后将反应样品置入PCR仪中反应，反应条件：25℃30min；4℃hold， PCR仪热盖为：30℃。反应完之后，加入20μL TE缓冲液，再加入50μL XP磁珠进行纯化，50μL TE缓冲液溶解回收产物。
6、单链环化
将48μL上述纯化产物置于95℃孵育3min；4℃孵育10min。
按照表4配制单链环化反应液。
表4 单链环化反应液组成
组分 用量
10×TA buffer(Epicentre) 6μL
100mM ATP(Thermo) 0.6μL
20μM介导片段 2.5μL
T4 DNA ligase(600U/μL)(Enzymatics) 1μL
无酶纯水(Sigma) 1.9μL
总体积 12μL
将上述配制好的12μL单链环化反应液与上述48μL热变性产物涡旋混匀，瞬时离心。然后将反应样品置入PCR仪中反应，反应条件：37℃60min；4℃hold，PCR仪热盖为：42℃。
其中，20μM介导片段具有相应互补序列用于连接单链两端，其序列如下(本实施例中的序列从左到右为5’端至3’端)：GCCATGTCGTTCTGTGAGCCAAGG(SEQ ID No.8)。
7、消化线性单链
按照表5配制消化反应液。
表5 消化反应液组成
组分 用量
10×TA buffe(Epicentre) 0.4μL
ExoI(20U/μL)，Enzymatics 2μL
ExoIII(10U/μL)，Enzymatics 1μL
无酶纯水 0.6μL
总体积 4μL
将上述配制好的4μL消化反应液加入上一步骤的60μL反应产物中，混匀，置于37℃孵育30min。加入3μLEDTA(500Mm,Ambion)，混匀。使用120μLXP磁珠纯化回收，30μL TE缓冲液溶解产物。
8、单链环定量
将上一步骤获得的线性消化的单链环产物用Qubit ssDNA Assay Kit定量。
9、测序
取构建的单链环状DNA文库进行DNA纳米球制备、MGISEQ-2000 PE150上机测序。测序过程按照MGISEQ-2000 PE150标准操作流程进行上机操作及数据分析。
10、本实施例建库和测序结果
图3所示为2个Human gDNA样品全基因组打断结果(NA12878标准品DNA的平行重复建库结果)。图4所示为单链环文库6％TBU胶检测结果。由图3和图4可见，酶切打断和建库结果均正常，说明该建库体系可兼容其他酶切打断方法。
本实施例建库测序最终所得Human样品WGS PCR free文库(NEB酶切法)测序质量如表6所示。表6显示Human样品WGS PCR free文库(NEB酶切法)在华大基因自主高通量测序平台MGISEQ-2000RS PE150具有较好测序质量。
表6 Human样品WGS PCR free文库(NEB酶切法)测序质量
Figure PCTCN2020096987-appb-000005
注：Covaris打断(PE100)为PCR-free对照，用相同的建库体系，即使酶切法建库也和物理法效果相当。
本实施例建库测序最终所得NA12878 WGS PCR free文库(NEB酶切法)SNP、Indel变异检测分析结果如表7所示。表7显示本发明PCR free文库(NEB酶切法)在Indel calling方面相比PCR文库具有明显优势，总体表现和Illumina平台的NovaSeq PCR free PE150数据相似。
表7 NA12878 WGS PCR free文库(NEB酶切法)SNP、Indel变异检测分析结果
Figure PCTCN2020096987-appb-000006
注：Covaris打断(PE100)为PCR-free对照，用相同的建库体系，即使酶切法建库也和物理法效果相当。BGISEQ-500物理PCR(PE100)为依赖于PCR的对照。
实施例2、20例cfDNA文库构建及测序
实验目的：用MGI PCR-free试剂盒构建血浆样品文库。
实验样本来源：20例血浆样品，包括2例染色体异常的样品。
1、样品收集及处理：
取静脉血2ml，1600g，4℃离心10分钟，将血细胞和血浆分开，血浆再以16000g，4℃离心10分钟，进一步去除残留的白细胞。从200μL血浆中提取DNA，最后将DNA溶于40μL TE溶液。
2、末端修复-加腺苷酸脱氧核糖核酸
按照表8配制末端修复和加A反应液。
表8 末端修复和加A反应液组成
10×T4多聚核苷酸激酶缓冲液(Enzematics公司) 5μL
T4多聚核苷酸激酶(10U/μL)(Enzematics公司) 1μL
脱氧核糖核酸混合液(25mM each)(Enzematics公司) 0.5μL
Taq DNA聚合酶(5U/μL)(Takara公司) 0.4μL
腺苷酸脱氧核糖核酸(100mM)(Enzematics公司) 0.5μL
T4 DNA聚合酶(3U/μL)(Enzymatics公司) 2μL
无酶纯水(Sigma) 0.6μL
总体积 10μL
将10μL以上配制好的末端修复与加A反应液加入40μL的DNA中，混匀，置于37℃混匀孵育10min；72℃混匀孵育15min,以0.1秒的速率降温至4℃。
3、接头连接：
本方案中使用的接头序列如下(本实施例中的序列从左到右为5’端至3’端，“//”示修饰基团，“phos”示磷酸化，下划线示10个碱基的标签序列。)
磷酸化的接头B链：/Phos/GAACGACATGGCTACGATCCGACTT(SEQ ID No.1)；
磷酸化的接头T链：/Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAA NNNNNNNNNNCAACTCCTTGGCTCACA(SEQ ID No.2)。
其中，N可为A或T或C或G。
接头配制：20μL 100μM接头B链、20μL 100μM接头T链和40μL 2×adapter buffer(组分：50mM Tris-HCl(pH8.0)，0.1mM EDTA，50mM NaCl)进行混合配制成接头A(25μM)，在室温放置半小时以上之后可稀释成使用浓度或放置-20℃保存。使用时将接头A(25μM)用TE稀释成接头B(1μM)。
将1μL配制好的接头B(1μM)加入步骤3的产物中，充分混匀。
按照表9配制连接反应液。
表9 连接反应液组成
组分 用量
10×T4 PNK buffer(Enzymatics) 3μL
0.1M ATP(Thermo) 0.8μL
50％PEG8000(Rigaku) 16μL
T4 DNA ligase(600U/μL)(Enzymatics) 5μL
无酶纯水(Sigma) 4.2μL
总体积 29μL
将上述配制好的连接反应液与接头B和步骤4的混合液涡旋混匀，瞬时离心。然后将反应样品置入PCR仪中反应，反应条件：25℃30min；4℃hold，PCR仪热盖为：30℃。反应完之后，加入20μL TE缓冲液，再加入50μL XP磁珠(Beckman Coulter公司)进行纯化，22μL TE缓冲液溶解回收产物。每个样品取15μl，进行多个样品等体积混合，然后加入一倍体积的XP磁珠(Beckman Coulter公司)进行纯化，22μL TE缓冲液溶解回收产物。
4、测序
本方法可以采取多种方式进行DNA纳米球制备
方式1：参考实施例1，全基因组文库构建及测序的步骤6-步骤9，进行样品单链环化、线性消化和DNA纳米球制备、BGISEQ-500SE50+10上机测序。测序过程按照BGISEQ-500SE50+10标准操作流程进行上机操作及数据分析。
方式2：取构建的连接产物，进行一步法DNA纳米球制备、BGISEQ-500SE50+10上机测序。测序过程按照BGISEQ-500SE50+10标准操作流程进行上机操作及数据分析。本实施例采用方式2。
5、本实施例建库和测序结果
取1μl连接产物进行PCR，验证连接接头之后的片段大小，结果如图5所示。由图可见，加完接头后的PCR产物为250bp左右，符合理论值：cfDNA片段大小为160bp,接头总长度约84bp,加完接头后的总片段长度为260bp左右。
各步骤产物浓度检测结果如表10所示。可见，该方法可以用于该类型样本建库。
表10 各步骤产物浓度检测结果
连接产物浓度 0.32 ng/μl
连接产物总量 12.8 ng
DNA纳米球浓度 12.3 ng/μl
图6为分别截取5兆和30兆测序数据进行分析统计的结果。可见，a中所有样品的有效比对率都达到了华大产前检测试剂盒的要求；b中所有样品的重复率都达到了华大产前检测试剂盒的要求；c中所有样品的GC含量都达到了华大产前检测试剂盒的要求。
实施例3、自主平台PCR-free建库试剂盒(酶切法)构建人全基因组文库
实验目的：用MGIPCR-free试剂盒配方结合NEB公司打断酶试剂盒构建Human gDNA样品全基因组文库。
实验样本来源：NA12878标准品DNA(货号：NA12878，公司：CORIELL INSTITUTE)。
1、DNA样品酶切法打断和末端修复与加A
分别取DNA标准品(TE溶解)，每管1μg起始量，使用dsDNA Fragmentase进行酶切打断和末端修复加A，打断体系为50μL。提前取出相应试剂溶解并混匀，酶试剂上下颠倒确保混匀后置于冰上。于冰上配制反应体系如表11。
表11 DNA样品打断末端修复加A反应体系
Figure PCTCN2020096987-appb-000007
最后加入DNA样本，然后用枪吹打混匀后，或者Votex混匀；快速离心后迅速置thermocycler开始反应，反应条件：4℃forever；37℃20min；65℃30min；4℃forever；PCR仪盖温为：70℃。反应结束后立即区样品于冰上，补充TE 50μl。
2、DNA片段化选择
(1)取打断后样本100μL转移至新1.5ml不粘管中，加入60μL XP磁珠，震荡混匀并室温结合10分钟，置于磁力架上结合2分钟(至液体澄清)，小心吸取上清到新的1.5ml EP管中(该步保留上清)。向上清中加入15μLXP磁珠，震荡混匀并室温结合10分钟，置于磁力架上结合2分钟(至液体澄清)，吸弃上清。
(2)在磁力架上向不粘管里加入500μL 75％乙醇，盖紧管盖并混匀，弃掉上清；500μL 75％乙醇重复洗1次，用小量程的移液器尽可能弃掉残留的乙醇，室温晾干。
(3)用42μL TE重悬磁珠，震荡混匀并室温结合10分钟，置于磁力架上结合2分钟(至液体澄清)，小心吸出40μL上清至新的1.5mL EP管中，准备进行下一步反应或保存至-20℃冰箱
3、样本定量检测和样品均一化
取上述纯化后的DNA 2μL用于Qubit dsDNA HS定量检测。按照Qubit测定的浓度均一化片段选择的DNA，统一调整至150ng，使用1xTE补齐使总体积至40μL。如需保存可将均一化样品置于-20℃冰箱。
最终所得DNA片段为300-500bp。
5、接头连接
本方案中使用的接头序列如下(本实施例中的序列从左到右为5’端至3’端，“//”示修饰基团，“phos”示磷酸化，下划线示10个碱基的标签序列。)
磷酸化的接头B链：/Phos/GAACGACATGGCTACGATCCGACTT(SEQ ID No.1)；
磷酸化的接头T链：/Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAA NNNNNNNNNNCAACTCCTTGGCTCACA(SEQ ID No.2)。
其中，N可为A或T或C或G。
接头配制：20μL 100μM接头B链、20μL 100μM接头T链和40μL 2×adpterbuffer(组分：50mM Tris-HCl(pH8.0)，0.1mM EDTA，50mM NaCl)进行混合配制成接头A(25μM)，在室温放置半小时以上之后可稀释成使用浓度或放置-20℃保存。使用时将接头A(25μM)用TE稀释成接头B(6μM)。
将5μL配制好的接头B(6μM)加入步骤4的产物中，充分混匀。
按照表12配制连接反应液。
表12 连接反应液组成
组分 用量
10×T4 PNK buffer(Enzymatics) 3μL
0.1MATP(Thermo) 0.8μL
50％PEG8000(Rigaku) 16μL
T4 DNA ligase(600U/μL)(Enzymatics) 5μL
无酶纯水(Sigma) 0.2μL
总体积 25μL
将上述配制好的连接反应液与接头B和步骤4产物的混合液涡旋混匀，瞬时离心。然后将反应样品置入PCR仪中反应，反应条件：25℃30min；4℃hold，PCR仪热盖为：30℃。反应完之后，加入20μL TE缓冲液，再加入50μL XP磁珠进行纯化，50μL TE缓冲液溶解回收产物。
6、单链环化
将48μL上述纯化产物置于95℃孵育3min；4℃孵育10min。
按照表13配制单链环化反应液。
表13 单链环化反应液组成
组分 用量
10×TA buffer(Epicentre) 6μL
100mM ATP(Thermo) 0.6μL
20μM介导片段 2.5μL
T4 DNA ligase(600U/μL)(Enzymatics) 1μL
无酶纯水(Sigma) 1.9μL
总体积 12μL
将上述配制好的12μL单链环化反应液与上述48μL热变性产物涡旋混匀， 瞬时离心。然后将反应样品置入PCR仪中反应，反应条件：37℃60min；4℃hold，PCR仪热盖为：42℃。
其中，20μM介导片段具有相应互补序列用于连接单链两端，其序列如下(本实施例中的序列从左到右为5’端至3’端)：GCCATGTCGTTCTGTGAGCCAAGG(SEQ ID No.8)。
7、消化线性单链
按照表14配制消化反应液。
表14 消化反应液组成
组分 用量
10×TA buffe(Epicentre) 0.4μL
ExoI(20U/μL)，Enzymatics 2μL
ExoIII(10U/μL)，Enzymatics 1μL
无酶纯水 0.6μL
总体积 4μL
将上述配制好的4μL消化反应液加入上一步骤的60μL反应产物中，混匀，置于37℃孵育30min。加入3μLEDTA(500Mm,Ambion)，混匀。使用120μLXP磁珠纯化回收，30μL TE缓冲液溶解产物。
8、单链环定量
将上一步骤获得的线性消化的单链环产物用Qubit ssDNA Assay Kit定量。
各步骤产物浓度检测结果如表15所示。可见，将打断和末端修复加A合并后，也适用于PCR-free建库。
表15 各步骤产物浓度检测结果
片段选择产物浓度 2.6 ng/μl
片段选择产物总量 130 ng
单链环浓度 2.16 ng/μl
实施例4、自主平台PCR-free建库试剂盒(酶切法)构建人全基因组文库
实验目的：用MGIPCR-free试剂盒配方结合NEB公司打断酶试剂盒构建Human gDNA样品全基因组文库。
实验样本来源：NA12878标准品DNA(货号：NA12878，公司：CORIELL INSTITUTE)。
1、DNA样品酶切法打断和末端修复
分别取DNA标准品(TE溶解)，每管1μg起始量，使用dsDNA Fragmentase(NEB公司，货号M0348)进行酶切打断和末端修复，体系为50μL。提前取出10X Fragmentase Reaction Buffer v2溶解并votex混匀，dsDNA Fragmentasevotex混匀后置于冰上。于冰上配制反应体系如表16。
表16 DNA样品酶切法打断和末端修复反应体系
组分 用量
10X Fragmentase Reaction Buffer v2(NEB) 5μL
dsDNA Fragmentase(NEB) 3μL
dNTPs(each 25mM)(Enzymatics) 3μL
DNA polymerase I(10U/μL)(NEB) 2μL
1M MgCl 2(Sigma) 0.3μL
无酶纯水(Sigma公司) 6.7μL
总体积 20μL
用枪吹打混匀后，每个样品加入20μl gDNA样本(总量1ug),用枪温柔吹打混匀6-8次，或Votex；快速离心后迅速置thermocycler开始反应，反应条件：37℃30min；4℃forever；PCR仪盖温为：70℃。反应结束后立即区样品于冰上，补充TE 30μl。
2、DNA片段化选择
(1)取打断后样本100μL转移至新1.5ml不粘管中，加入52μL XP磁珠，震荡混匀并室温结合10分钟，瞬时离心后置于磁力架上结合2分钟(至液体澄清)，小心吸取上清到新的1.5ml EP管中(该步保留上清)。向上清中加入15μLXP磁珠，震荡混匀并室温结合10分钟，置于磁力架上结合2分钟(至液体澄清)，吸弃上清。
(2)在磁力架上向不粘管里加入500μL 75％乙醇，盖紧管盖并混匀，弃掉上清；500μL 75％乙醇重复洗1次，用小量程的移液器尽可能弃掉残留的乙醇，室温晾干。
(3)用42μL TE重悬磁珠，震荡混匀并室温结合10分钟，瞬时离心后置于磁力架上结合2分钟(至液体澄清)，小心吸出40μL上清至新的1.5mL EP管中，准备进行下一步反应或保存至-20℃冰箱。
3、样本定量检测和样品均一化
取上述纯化后的DNA 2μL用于Qubit dsDNA HS定量检测。按照Qubit测定的浓度均一化片段选择的DNA，统一调整至150ng以内，使用1×TE补齐使总体积至40μL。如需保存可将均一化样品置于-20℃冰箱。
最终所得DNA片段为300-500bp。
4、加A
首先，按照表17配制加A反应液。
表17 加A反应液组成
组分 用量
T4 10×PNK buffer(Enzymatics) 5μL
dATP(100mM)(Enzymatics) 0.5μL
dNTPs(each 25mM)(Enzymatics) 0.35μL
rTaq(5U/μL)(Enzymatics) 0.2μL
无酶纯水(Sigma公司) 1μL
将10μL以上配制好的加A反应液加到40μL步骤3获得的样本产物中，涡旋混匀，瞬时离心。总体积为50μL。将反应样品置入PCR仪中反应，反应条件：65℃30min；4℃forever；PCR仪盖温为：70℃。
5、接头连接
本方案中使用的接头序列如下(本实施例中的序列从左到右为5’端至3’端，“//”示修饰基团，“phos”示磷酸化，下划线示10个碱基的标签序列。)
磷酸化的接头B链：/Phos/GAACGACATGGCTACGATCCGACTT(SEQ ID No.1)；
磷酸化的接头T链：/Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAA NNNNNNNNNNCAACTCCTTGGCTCACA(SEQ ID No.2)。
其中，N可为A或T或C或G。
接头配制：20μL 100μM接头B链、20μL 100μM接头T链和40μL 2×adpter buffer(组分：50mM Tris-HCl(pH8.0)，0.1mM EDTA，50mM NaCl)进行混合配制成接头A(25μM)，在室温放置半小时以上之后可稀释成使用浓度或放置-20℃保存。使用时将接头A(25μM)用TE稀释成接头B(6μM)。
将5μL配制好的接头B(6μM)加入步骤4的产物中，充分混匀。
按照表18配制连接反应液。
表18 连接反应液组成
组分 用量
10×T4 PNK buffer(Enzymatics) 3μL
0.1MATP(Thermo) 0.8μL
50％PEG8000(Rigaku) 16μL
T4 DNA ligase(600U/μL)(Enzymatics) 5μL
无酶纯水(Sigma) 0.2μL
总体积 25μL
将上述配制好的连接反应液与接头B和步骤4产物的混合液涡旋混匀，瞬时离心。然后将反应样品置入PCR仪中反应，反应条件：25℃30min；4℃hold，PCR仪热盖为：30℃。反应完之后，加入20μL TE缓冲液，再加入50μL XP磁珠进行纯化，50μL TE缓冲液溶解回收产物。
6、单链环化
在48μL上述纯化产物中加入5μL 20μM介导片段，2.5μL 2M NaOH(sigma)涡旋混匀后室温放置5min；然后再加入5μL 1M Tris-HCl(pH6.8)，涡旋混匀后再加入表19的单链环化反应液。
表19 单链环化反应液组成
组分 用量
10×TA buffer(Epicentre) 6μL
100mM ATP(Thermo) 0.6μL
T4 DNA ligase(600U/μL)(Enzymatics) 0.4μL
总体积 7μL
然后将反应样品置入PCR仪中反应，反应条件：37℃30min；4℃hold，PCR仪热盖为：42℃。
其中，20μM介导片段具有相应互补序列用于连接单链两端，其序列如下(本实施例中的序列从左到右为5’端至3’端)：GCCATGTCGTTCTGTGAGCCAAGG(SEQ ID No.8)。
7、消化线性单链
按照表20配制消化反应液。
表20 消化反应液组成
组分 用量
10×TA buffe(Epicentre) 0.4μL
ExoI(20U/μL)，Enzymatics 2μL
ExoIII(10U/μL)，Enzymatics 1μL
无酶纯水 0.6μL
总体积 4μL
将上述配制好的4μL消化反应液加入上一步骤的67.5μL反应产物中，混匀，置于37℃孵育30min。加入3μLEDTA(500Mm,Ambion)，混匀。使用120μLXP磁珠纯化回收，30μL TE缓冲液溶解产物。
8、单链环定量
将上一步骤获得的线性消化的单链环产物用Qubit ssDNA Assay Kit定量。
各步骤产物浓度检测结果如表21所示。可见，将打断和末端修复加A合并后，也适用于PCR-free建库。
表21 各步骤产物浓度检测结果
片段选择产物浓度 4.6 ng/μl
片段选择产物总量 184 ng
单链环浓度 2.2 ng/μl
实施例5、自主平台PCR-Free建库试剂盒(酶切法)结合双barcode接头构建人全基因组文库及测序
实验目的：用MGIPCR-Free试剂盒配方结合双barcode接头以及NEB公司打断酶构建Human gDNA样品全基因组文库。
实验样本来源：NA12878标准品DNA(货号：NA12878，公司：CORIELL INSTITUTE)。
1、DNA样品酶切法打断
分别取DNA标准品(TE溶解)，每管1μg起始量，使用dsDNA Fragmentase (NEB公司，货号M0348)进行酶切打断，打断体系为50μL。提前取出10X Fragmentase Reaction Buffer v2溶解并votex混匀，dsDNA Fragmentasevotex混匀后置于冰上。于冰上配制反应体系如表22。
表22 DNA样品酶切法打断反应体系
组分 用量
10X Fragmentase Reaction Buffer v2(NEB) 3μL
gDNA(TE溶解) XμL
TE 27-XμL
总体积 27μL
用枪吹打混匀后，每个样品加入3μl dsDNA Fragmentase,用枪温柔吹打混匀6-8次，或Votex；快速离心后迅速置thermocycler开始反应，反应条件：37℃25min；65℃15min；4℃forever；PCR仪盖温为：70℃。反应结束后立即区样品于冰上，补充TE 70μl。
2、DNA片段化选择
(1)取打断后样本100μL转移至新1.5ml不粘管中，加入60μL XP磁珠，震荡混匀并室温结合10分钟，瞬时离心后置于磁力架上结合2分钟(至液体澄清)，小心吸取上清到新的1.5ml EP管中(该步保留上清)。向上清中加入15μLXP磁珠，震荡混匀并室温结合10分钟，置于磁力架上结合2分钟(至液体澄清)，吸弃上清。
(2)在磁力架上向不粘管里加入500μL 75％乙醇，盖紧管盖并混匀，弃掉上清；500μL 75％乙醇重复洗1次，用小量程的移液器尽可能弃掉残留的乙醇，室温晾干。
(3)用42μL TE重悬磁珠，震荡混匀并室温结合10分钟，瞬时离心后置于磁力架上结合2分钟(至液体澄清)，小心吸出40μL上清至新的1.5mL EP管中，准备进行下一步反应或保存至-20℃冰箱。
3、样本定量检测和样品均一化
取上述纯化后的DNA 2μL用于Qubit dsDNA HS定量检测。按照Qubit测定的浓度均一化片段选择的DNA，统一调整至150ng，使用1×TE补齐使总体积至40μL。如需保存可将均一化样品置于-20℃冰箱。
最终所得DNA片段为300-500bp。
4、末端修复与加A
首先，按照表23配制末端修复与加A反应液。
表23 末端修复与加A反应液组成
组分 用量
T4 10×PNK buffer(Enzymatics) 5μL
dATP(100mM)(Enzymatics) 0.5μL
dNTPs(each 25mM)(Enzymatics) 0.5μL
T4 DNA polymerase(3U/μL)(Enzymatics) 2μL
T4 PNK(10U/μL)(Enzymatics) 1μL
rTaq(5U/μL)(Enzymatics) 1μL
无酶纯水(Sigma公司) 1μL
将10μL以上配制好的末端修复与加A反应液加到40μL步骤3获得的样本产物中，涡旋混匀，瞬时离心。总体积为50μL。将反应样品置入PCR仪中反应，反应条件：14℃15min；37℃25min；65℃15min；4℃forever；PCR仪盖温为：70℃。
5、接头连接
本方案中使用的接头序列如下(本实施例中的序列从左到右为5’端至3’端，“//”示修饰基团，“phos”示磷酸化，下划线示10个碱基的标签序列。)
磷酸化的接头B链：/Phos/TCTCAGTACGTCAGCAGTTNNNNNNNNNNCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT(SEQ ID No.7)；
磷酸化的接头T链：/Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAANNNNNNNNNNCTGATAAGGTCGCCATGCC(SEQ ID No.6)。
其中，N可为A或T或C或G。
接头配制：20μL 100μM接头B链、20μL 100μM接头T链和40μL 2×adaptorbuffer(组分：50mM Tris-HCl(pH8.0)，0.1mM EDTA，50mM NaCl)进行混合配制成接头A(25μM)，在室温放置半小时以上之后可稀释成使用浓度或放置-20℃保存。使用时将接头A(25μM)用TE稀释成接头B(6μM)。
将5μL配制好的接头B(6μM)加入步骤4的产物中，充分混匀。
按照表24配制连接反应液。
表24 连接反应液组成
组分 用量
10×T4 PNK buffer(Enzymatics) 3μL
0.1MATP(Thermo) 0.8μL
50％PEG8000(Rigaku) 16μL
T4 DNA ligase(600U/μL)(Enzymatics) 5μL
无酶纯水(Sigma) 0.2μL
总体积 25μL
将上述配制好的连接反应液与接头B和步骤4产物的混合液涡旋混匀，瞬时离心。然后将反应样品置入PCR仪中反应，反应条件：25℃30min；4℃hold，PCR仪热盖为：30℃。反应完之后，加入20μL TE缓冲液，再加入50μL XP磁珠进行纯化，50μL TE缓冲液溶解回收产物。
6、单链环化
将48μL上述纯化产物置于95℃孵育3min；4℃孵育10min。
按照表25配制单链环化反应液。
表25 单链环化反应液组成
组分 用量
10×TA buffer(Epicenter) 6μL
100mM ATP(Thermo) 0.6μL
20μM介导片段 2.5μL
T4 DNA ligase(600U/μL)(Enzymatics) 1μL
无酶纯水(Sigma) 1.9μL
总体积 12μL
将上述配制好的12μL单链环化反应液与上述48μL热变性产物涡旋混匀，瞬时离心。然后将反应样品置入PCR仪中反应，反应条件：37℃60min；4℃hold，PCR仪热盖为：42℃。
其中，20μM介导片段具有相应互补序列用于连接单链两端，其序列如下(本实施例中的序列从左到右为5’端至3’端)：TGCTGACGTACTGAGAGGCATGGCGACCT(SEQ ID No.8)。
7、消化线性单链
按照表26配制消化反应液。
表26 消化反应液组成
组分 用量
10×TA buffe(Epicenter) 0.4μL
ExoI(20U/μL)，Enzymatics 2μL
ExoIII(10U/μL)，Enzymatics 1μL
无酶纯水 0.6μL
总体积 4μL
将上述配制好的4μL消化反应液加入上一步骤的60μL反应产物中，混匀，置于37℃孵育30min。加入3μLEDTA(500Mm,Ambion)，混匀。使用120μLXP磁珠纯化回收，30μL TE缓冲液溶解产物。
8、单链环定量
将上一步骤获得的线性消化的单链环产物用Qubit ssDNA Assay Kit定量。
9、测序
取构建的单链环状DNA文库进行DNA纳米球制备、MGISEQ-2000 PE150上机测序。测序过程按照MGISEQ-2000 PE150标准操作流程进行上机操作及数据分析。
10、本实施例建库和测序结果
各步骤产物浓度检测结果如表27所示。本实施例建库测序最终所得Human样品WGS PCR free文库(酶切法)测序质量如表28所示。表28显示Human样 品WGS PCR free文库(酶切法)在华大基因自主高通量测序平台MGISEQ-2000RS PE150具有较好测序质量。
表27 各步骤产物浓度检测结果
片段选择产物浓度 3.35 ng/μl
片段选择产物总量 150.75 ng
单链环浓度 1.25 ng/μl
表28 本实施例建库测序最终所得Human样品WGS PCR free文库(酶切法)测序质量
Figure PCTCN2020096987-appb-000008
注：Covaris打断(PE100)为PCR-free对照，用相同的建库体系，即使双barcode酶切法建库也和物理法效果相当。
本实施例建库测序最终所得NA12878 WGS PCR free文库(NEB酶切法)SNP、Indel变异检测分析结果如表29所示。表29显示本发明PCR free文库(酶切法)在Indel calling方面相比PCR文库具有明显优势，总体表现和Illumina平台的NovaSeq PCR free PE150数据相似。
表29 NA12878 WGS PCR free文库(酶切法)SNP、Indel变异检测分析结果
Figure PCTCN2020096987-appb-000009
注：Covaris打断(PE100)为PCR-free对照，用相同的建库体系，即使酶切法建库也和物理法效果相当。
工业应用
本发明提供了PCR-free建库方案，避免建库过程中因PCR引入的碱基错误、数据偏向及重复序列等问题，而且该方案可兼容不同打断方式和低起始量建库。本发明的PCR-free建库技术结合滚环复制纳米球制备技术，真正实现了样本从建库到测序的PCR-free过程，形成了全流程的PCR-free。本发明采用末端修复和接头效率优化体系、单链环化与滚环复制一步法配合实现自主平台PCR-free建库试剂盒建库效率提升，并降低起始DNA的要求量。本发明的建库体系可兼容不同类型起始的样本，包括但不限于基因组DNA，打断的DNA，采用全基因组扩增方法得到的DNA或DNA打断产物，扩增子DNA，cfDNA，RNA反转录之后的DNA等。具体而言，与现有技术相比本发明具有以下有益效果：1)适用性广：本发明适用于所有已知或未知参考序列的物种，可为一般分子生物学实验室采用。物理打断和酶切打断兼容建库。不同类型样本兼容，包括但不限于基因组DNA，打断的DNA，采用全基因组扩增方法得到的DNA或DNA打断产物，扩增子DNA，cfDNA，RNA反转录之后的DNA等。2)操作简单、建库时间短。本发明在末端修复和加A反应采用一管法进行，并略过磁珠纯化直接进行接头连接，且省去常规PCR扩增和纯化操作，大大节约建库时间。同时结合环化与滚环复制一步反应的文库制备方法，可以进一步缩短建库测序时间。3)建库效率高：通过优化 的末端修复-加A配方和体系、优化的接头连接体系、同时结合环化与滚环复制一步反应的文库制备方法，可以实现低起始量样本pooling建库测序，可以实现200μl血浆DNA的PCR-free建库。4)提高自主平台测序数据准确性：本发明实现了真正的建库和测序均无扩增反应，可提高SNP和InDel检出的准确性和灵敏度，特别是InDel的检出性能优秀，可赶超竞品illumina平台。

Claims (32)

  1. 一种PCR-free高通量测序方法，包括如下步骤：
    (A1)将待测序核酸根据大小进行或不进行片段化处理，得到目的大小DNA片段；
    (A2)对(A1)产物进行末端修复和加A反应；
    (A3)对(A2)产物连接接头；
    (A4)对(A3)产物进行单链环化和滚环复制，形成DNA纳米球；
    (A5)上机测序。
  2. 一种PCR-free高通量测序方法，包括如下步骤：
    (B1)将待测序核酸根据大小进行片段化处理，同时进行末端修复和加A反应得到目的大小DNA片段；
    (B2)对(B1)产物连接接头；
    (B3)对(B2)产物进行单链环化和滚环复制，形成DNA纳米球；
    (B4)上机测序。
  3. 一种PCR-free高通量测序方法，包括如下步骤：
    (C1)将待测序核酸根据大小进行片段化处理，同时进行末端修复反应得到目的大小DNA片段；
    (C2)对(C1)产物进行加A反应；
    (C3)对(C2)产物连接接头；
    (C4)对(C3)产物进行单链环化和滚环复制，形成DNA纳米球；
    (C5)上机测序。
  4. 根据权利要求3所述的方法，其特征在于：步骤(B1)和(C1)中，所述片段化处理是利用片段化酶消化所述待测序核酸。
  5. 根据权利要求1-3中任一所述的方法，其特征在于：所述接头中包括标签序列。
  6. 根据权利要求5所述的方法，其特征在于：一个所述接头中包括两个标签序列。
  7. 根据权力要求6所述的方法，其特征在于：所述接头是由两条部分互补的核酸单链退火形成的，所述标签序列位于两条核酸单链的非互补区域。
  8. 根据权利要求1-3中任一所述的方法，其特征在于：步骤(A1),步骤(B1)和步骤(C1)中，所述待测序核酸为DNA或者RNA。
  9. 根据权利要求8所述的方法，其特征在于：所述DNA为基因组DNA或者天然存在的小分子DNA或经扩增所得的DNA片段。
  10. 根据权利要求1所述的方法，其特征在于：步骤(A1)中，所述片段化处理方式为物理打断法或酶打断法。
  11. 根据权利要求8所述的方法，其特征在于：当所述待测序核酸为RNA时， 需要反转录得到DNA；进行所述片段化处理为对RNA进行片段化处理或者对所述RNA反转录得到的DNA进行片段化处理。
  12. 根据权利要求1所述的方法，其特征在于：步骤(A2)中，进行所述末端修复和加A反应是一步完成的：将末端修复与加A反应液与所述(A1)产物混合，反应，得到所述(A2)产物；
    所述末端修复与加A反应液含有T4多聚核苷酸激酶缓冲液、腺苷酸脱氧核糖核酸、脱氧核糖核酸混合液、T4 DNA聚合酶、T4多聚核苷酸激酶、Taq DNA聚合酶。
  13. 根据权利要求12所述的方法，其特征在于：步骤(A2)中，将所述末端修复与加A反应液与所述(A1)产物混合时两者的体积比为1：4。
  14. 根据权利要求2所述的方法，其特征在于：步骤(B1)中，进行所述片段化处理和所述末端修复和加A反应是一步完成的：将片段化处理和末端修复与加A反应液与所述待测序核酸混合，反应，得到所述(B1)产物；
    所述片段化处理和末端修复与加A反应液含有片段化酶、片段化酶反应缓冲液、腺苷酸脱氧核糖核酸、脱氧核糖核酸混合液、T4 DNA聚合酶、Taq DNA聚合酶和TE缓冲液。
  15. 根据权利要求3所述的方法，其特征在于：步骤(C1)中，进行所述片段化处理和所述末端修复反应是一步完成的：将片段化处理和末端修复反应液与所述待测序核酸混合，反应，得到所述(C1)产物；
    所述片段化处理和末端修复反应液含有片段化酶、片段化酶反应缓冲液、脱氧核糖核酸混合液、DNA聚合酶I和MgCl 2。
  16. 根据权利要求1-25中任一所述的方法，其特征在于：步骤(A3)、(B2)和(C3)中，所述接头由B链和T链退火后形成；
    所述B链的3’端与所述T链的5’端互补配对，且两条链的其他部分不互补配对，且所述B链的3’末端有突出的dT；所述B链和/或所述T链中不互补配对的部分含有用于标记不同样品的标签序列；所述B链和所述T链的5’端均修饰有磷酸基团或连接有3’末端为U的一段单链寡核苷酸。
  17. 根据权利要求16所述的方法，其特征在于：所述接头为如下任一：
    接头1：由5’端修饰有磷酸基团的SEQ ID No.1所示单链DNA和5’端修饰有磷酸基团的SEQ ID No.2所示单链DNA退火后形成；
    接头2：由SEQ ID No.3或SEQ ID No.4所示单链DNA和SEQ ID No.2所示单链DNA退火后形成；
    接头3：由SEQ ID No.5所示单链DNA和SEQ ID No.6所示单链DNA退火后形成；
    接头4：由SEQ ID No.7所示单链DNA和SEQ ID No.6所示单链DNA退火后形成。
  18. 根据权利要求1-17中任一所述的方法，其特征在于：步骤(A3)、(B2) 和(C3)中，对所述(A2)或(B1)或(C2)产物连接所述接头是按照包括如下步骤的方法进行的：将所述接头、所述(A2)或(B1)或(C2)产物和连接反应液混合，反应，得到所述(A3)或(B2)或(C3)产物；
    所述连接反应液含有T4多聚核苷酸激酶缓冲液、腺苷酸核糖核酸、PEG8000、T4 DNA连接酶和无酶纯水。
  19. 根据权利要求18所述的方法，其特征在于：步骤(A3)、(B2)和(C3)中，将所述接头、所述(A2)或(B1)或(C2)产物和所述连接反应液混合为将含有所述接头的接头溶液、所述(A2)或(B1)或(C2)产物和所述连接反应液按照体积比为1-5：50：25-29的比例混合；所述接头在所述接头溶液中的浓度为6μM或1μM。
  20. 根据权利要求18或19所述的方法，其特征在于：步骤(A3)、(B2)和(C3)中，将所述接头、所述(A2)或(B1)或(C2)产物和所述连接反应液混合后进行反应的条件为25℃10-30min；4℃保温。
  21. 根据权利要求1-20中任一所述的方法，其特征在于：步骤(A4)、(B3)和(C4)中，在对所述(A3)或(B2)或(C3)产物进行单链环化和滚环复制之前还包括进行纯化的步骤。
  22. 根据权利要求1-21中任一所述的方法，其特征在于：步骤(A4)、(B3)和(C4)中，对所述(A3)或(B2)或(C3)产物进行单链环化和滚环复制，形成DNA纳米球，通过如下任一实现：
    (a1)依次进行单链环化、消化线性单链、纯化、滚环复制，得到DNA纳米球；
    (a2)单链环化和滚环复制一步完成，得到DNA纳米球。
  23. 根据权利要求22所述的方法，其特征在于：步骤(a1)中，所述单链环化按照方法I或方法II进行；
    所述方法I包括如下步骤：
    I-1：将所述(A3)或(B2)或(C3)产物置于95℃孵育3min；4℃孵育10min，得到孵育后产物；
    I-2：将单链环化反应液1与步骤I-1所得的所述孵育后产物混合，反应，得到环化产物；
    所述单链环化反应液1含有TA缓冲液、腺苷酸核糖核酸、介导片段、T4 DNA连接酶和无酶纯水；
    所述介导片段为5’端与构成所述接头的所述B链的5’端反向互补，3’端与构成所述接头的所述T链的3’端反向互补的单链DNA；
    所述方法II包括如下步骤：将所述(A3)或(B2)或(C3)产物与所述介导片段、NaOH溶液混合后室温放置5min，然后加入Tris-HCl溶液混合，再加入单链缓和反应液2，反应，得到环化产物；
    所述单链环化反应液2含有TA缓冲液、腺苷酸核糖核酸和T4 DNA连接酶。
  24. 根据权利要求23所述的方法，其特征在于：当所述接头为权利要求17中所述接头1或所述接头2时，所述介导片段的核苷酸序列如SEQ ID No.8所示。
  25. 根据权利要求22-24中任一所述的方法，其特征在于：步骤(a1)中，所述消化线性单链按照包括如下步骤的方法进行：将消化反应液与所述环化产物混合，反应，得到消化后产物；
    所述消化反应液含有TA缓冲液、ExoI酶、ExoIII酶和无酶水。
  26. 一种适用于PCR-free高通量测序的DNA文库构建方法，包括权利要求1-32任一中的步骤(A1)-(A4)或(B1)-(B3)或(C1)-(C4)。
  27. 利用权利要求26所述方法构建得到的DNA文库。
  28. 接头，为权利要求16或17中所述的接头。
  29. 成套产品，含有权利要求28所述接头和如下中的全部或部分：
    (b1)权利要求12或13中所述的末端修复与加A反应液；
    (b2)权利要求14中所述的片段化处理和末端修复与加A反应液；
    (b3)权利要求15中所述的片段化处理和末端修复反应液；
    (b4)权利要求18中所述的连接反应液；
    (b5)权利要求23中所述的单链环化反应液1或所述的单链环化反应液2；
    (b6)权利要求25中所述的消化反应液。
  30. 系统，含有权利要求29所述成套产品和DNBSEQ测序用试剂和/或设备。
  31. 权利要求27所述DNA文库或权利要求28所述接头或权利要求29所述成套产品或权利要求30所述系统在进行PCR-free高通量测序中的应用。
  32. 权利要求28所述接头或权利要求29所述成套产品在构建权利要求27所述DNA文库中的应用。
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