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WO2023137667A1 - 一种接头及其在构建dnb文库中的应用 - Google Patents

一种接头及其在构建dnb文库中的应用 Download PDF

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Publication number
WO2023137667A1
WO2023137667A1 PCT/CN2022/073026 CN2022073026W WO2023137667A1 WO 2023137667 A1 WO2023137667 A1 WO 2023137667A1 CN 2022073026 W CN2022073026 W CN 2022073026W WO 2023137667 A1 WO2023137667 A1 WO 2023137667A1
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linker
adapter
nucleic acid
library
primer
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French (fr)
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王业钦
杨林
欧日晶
杨贵芳
吕硕
聂自豪
张韶红
张艳艳
陈芳
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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Priority to PCT/CN2022/073026 priority patent/WO2023137667A1/zh
Publication of WO2023137667A1 publication Critical patent/WO2023137667A1/zh
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    • 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
    • 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
    • C12Q1/6869—Methods for sequencing

Definitions

  • the invention belongs to the field of bioengineering, and in particular relates to a linker and its application in constructing a DNB library.
  • PCR amplification process which is highly enriched and highly adaptable to micro-sample and hotspot SNPs, and can obtain high-quality data at a very low cost.
  • PCR Free PCR-free amplification
  • the second is PCR-free amplification (PCR Free), which avoids errors introduced by amplification and can obtain real sample information.
  • the complete PCR Free technology has strict sample requirements, cumbersome steps and high cost, which compresses people's desire for it.
  • the current library construction technology based on the DNBseq platform is classified according to whether it is amplified or not.
  • the technical problem to be solved by the present invention is to provide a linker and its application, especially the application in rapid construction of a single-stranded circular library.
  • the present invention develops a novel linker and a corresponding method for library construction and sequencing.
  • the library construction and sequencing method is simpler, can more truly reflect the original information of samples, and can meet the needs of this field with higher cost performance.
  • the present invention mainly solves the above-mentioned technical problems through the following technical solutions.
  • the present invention provides a linker, which comprises linker 1, linker 2 and anchor primers; wherein the nucleic acid sequences at both ends of the anchor primers are complementary to part of the nucleic acid sequences of linker 1 and linker 2, respectively.
  • the adapter 1 and the adapter 2 preferably both contain one or more tag sequences, reverse complementary sequences and sequencing primer binding sequences; and the 5' end of the adapter has phosphorylation modification, and the 3' end locks and exposes at least one T base after annealing with the reverse complementary sequence at the 5' end;
  • the two ends of the anchor primer respectively contain capture sequences complementary to adapter 1 and adapter 2, and a single-stranded nucleic acid structural sequence.
  • the adapter 1 also contains 1 or 2 index primer binding sequences.
  • the linker 2 also contains 1 or 2 index primer binding sequences.
  • both the adapter 1 and the adapter 2 further contain 1 or 2 index primer binding sequences.
  • the binding sequence of the index primer and the binding sequence of the sequencing primer are limiting factors of the sequencing platform, and can be adjusted accordingly according to the sequencing platform or bioinformatics analysis.
  • the reverse complementary sequences inside the adapter 1 and the adapter 2 are 5-10 bp locked.
  • the linker 1 and the linker 2 can respectively anneal to form a hairpin structure through the reverse complementary sequence under appropriate conditions.
  • both the length of the linker and the length range of the anchor primer can be adjusted to meet the needs of the actual library construction scenario.
  • the length of the anchor primer is determined according to the length of the inserted library.
  • the anchor primer is a single-stranded nucleic acid sequence longer than 35 bp, for example, the length can be 35-200 bp, such as 75 bp; the capture sequence length of the anchor primer is 15-25 bp, and the single-stranded structural sequence length of the anchor primer is designed according to the length of the library fragment to be tested.
  • the single-stranded structural sequence of the anchor primer is 35-55 bp.
  • the present invention also provides a kit for constructing a sequencing library, which comprises adapter 1, adapter 2 and anchor primers as defined above.
  • the kit further comprises a polymerase.
  • the molar ratio of the adapter 1, the adapter 2 and the anchor primer is 1:1:1.
  • the kit also includes other solutions routinely used in this field, such as 5 ⁇ STE Buffer and NF Water, etc.
  • the kit is usually prepared in the form of serial linker complex dilutions during use.
  • the preparation method of the adapter complex dilution comprises the following steps:
  • the conditions of the reaction described in step (1) are preferably: 95°C for 5min, 70°C for 5min, 45°C for 5min, and 25°C for 5min.
  • the condition of reaction described in step (2) is preferably: 25 °C 15min.
  • the present invention also provides a method for constructing a sequencing library, the method comprising:
  • Adapter complex preparation mix the adapter 1 and adapter 2 defined above with the anchor primer and incubate to obtain the adapter complex;
  • step (1) (2) performing a ligation reaction between the nucleic acid fragment to be detected and the linker complex in step (1), the ligation reaction further comprising a ligase;
  • the nucleic acid fragment is selected from: nucleic acid fragments obtained by genome disruption, PCR products, free nucleic acid fragments and reverse transcription products.
  • the present invention also provides a method for simultaneously detecting the sense strand and the antisense strand of nucleic acid, which comprises:
  • a new adapter complex structure ( Figure 1), which is composed of tag adapter 1, tag adapter 2 and anchor primers, can capture DNA fragments with phosphorylated 5-terminals and cohesive ends with A bases at the 3-terminal for library construction;
  • Index adapter 1 (adapter 1, Figure 2): This adapter is composed of one or more tags, index primer binding sequence, a complementary structure and sequencing primer binding sequence. Its 5' end has phosphorylation modification, and its 3' end is locked after refolding and a T base is exposed for pairing connection;
  • Adapter 2 (adapter 2, Figure 3): This adapter is composed of one or more tags, tag primer binding sequence, a complementary structure and sequencing primer binding sequence. Its 5' end has phosphorylation modification, and its 3' end is locked after refolding and a T base is exposed for pairing connection;
  • An anchor primer contains two nucleic acid sequences complementary to adapter 1 and adapter 2 respectively, and a single-stranded nucleic acid sequence, which can simultaneously capture and anchor two adapters correspondingly designed to form an arched structure, and can be used as a primer for the subsequent preparation of DNA nanoballs;
  • PCR Free format can be selected for library building, which has good compatibility and independent selectivity; PCR links can be exempted for better quality samples, and PCR amplification steps can be reduced for poor quality and trace samples; low sample size requirements, good compatibility, and high accuracy;
  • the double-strand combined library construction and sequencing method can completely retain the original double-strand information of the sample, maintain the authenticity of the sample information to the greatest extent, and expand new applications for special needs.
  • Figure 1 is a schematic diagram of the connection of the novel linker complex.
  • Figure 2 is the connector adapter 1.
  • Figure 3 is the connector adapter2.
  • Figure 4 is the connector Link.
  • Figure 5 shows the database building process
  • Figure 6 is a graph showing the size distribution of fragmented DNA products.
  • Fig. 7 is an electrophoresis diagram of the product after verification and amplification of DNB.
  • Figure 8 is a 6 ⁇ GAPDH Primer mix gel electrophoresis image.
  • Step 1 Pre-synthesize three primers of adapter 1, adapter 2 and Link and dilute them into 100 ⁇ L dilution using TE (Sangon, Cat. No. B548106-0500). Anneal the Adapter 1 primer dilution and Adapter 2 primer dilution. The annealing method is "take Adapter 1 (100 ⁇ M)/12.5 ⁇ L+Adapter 2 (100 ⁇ M)/12.5 ⁇ L, 5 ⁇ STE Buffer (BGI)/3 ⁇ L, and place it on a PCR instrument for reaction.
  • the program is “25 °C for 15 min, 4 °C storage’” to obtain the linker dilution (25 ⁇ M) required for this method.
  • linker dilution solution 25 ⁇ M/8 ⁇ L, 10 ⁇ Phi29Buffer (Thermo, product number B62)/40 ⁇ L, NF Water (Invitrogen, product number AM9932)/152 ⁇ L and mix to prepare Make DNB buffer for later use.
  • Step 2 Fragment the DNA sample using Hieff NGS OnePot DNA Fragmentation Reagent ( ⁇ , Cat. No. 13474ES96).
  • the processing method is "take sample DNA/150ng, OnePot mix/7.5 ⁇ L, NF water UP to 30 ⁇ L; 30°C for 10min, 65°C for 20min, and store at 4°C (heat lid set at 70°C)".
  • the processed sample DNA was taken out and centrifuged for a simple purification.
  • the operation was "take the reaction product of the previous step and add 27 ⁇ L (0.9 ⁇ ) magnetic beads (VAZYME, product number N411-03), mix well and place at room temperature for 5 minutes; then place it on a magnetic stand for adsorption for 2 minutes, and remove the supernatant; add 80% ethanol to rinse once and dry until the surface of the magnetic beads is rough; add 22 ⁇ L TE (Sanko, product number B548106-0500) Mix and elute, and keep the supernatant; take 1 ⁇ L and use Qubit dsDNA HS Assay Kit (Invitrogen, product number Q32854) to quantify the concentration, and the total yield is required to be greater than 30ng for future use.
  • Step 3 DNB preparation before sequencing, the operation is "take 20 ⁇ L of the Make DNB buffer prepared in step 1 and mix with 30 ng of the product obtained in step 2, then add DNB polymerase I (MGI, 1000004803)/40 ⁇ L, T4 DNA ligase (repaid) (enzymatic, product number L6030-HC-L) 1 ⁇ L, make up to 80 ⁇ L with NF Water, 2 5°C for 10 minutes, stored at 4°C (heated lid set at 35°C); after the reaction, the reaction solution was taken out and centrifuged, and DNB polymerase II (MGI, 1000017261)/4 ⁇ L was added, 30°C for 25 minutes, stored at 4°C (total volume 84 ⁇ L, heated lid set at 35°C)".
  • DNB polymerase I MMI, 1000004803
  • T4 DNA ligase repaid
  • Step 1 Prepare Make DNB buffer according to the method of step 1 in Experimental Method 1.
  • Step 2 Perform PCR amplicon library preparation.
  • the first combination is 5-terminal phosphorylated F primer and R primer, template and Taq enzyme (TaKaRa, Cat. No. R001A), and the second combination is conventional F primer and R primer, template, Taq enzyme, and T4 polynucleotide kinase (Thermo, Cat. No. EK0031).
  • the preparation method is "dilute each F primer and R primer to 25 ⁇ M, and mix them in equimolar amounts at a ratio of 1:1 for later use; take the mixed primer/4 ⁇ L, 2 ⁇ M ⁇ Ltiplex PCR Readymix (BGI, catalog number 1000014386)/25 ⁇ L, sample/1ng, NF Water/UP to 50 ⁇ L; the amplification program is 95°C/3min, 1 cycle; 95°C/ 20s, 60°C/20s, 72°C/30s, 30 cycles; 72°C/10min, one cycle; 12°C storage".
  • This process can be optimized according to its own situation, and no specific requirements are required, but it is necessary to ensure that the product is fully extended so that A is added to the end to facilitate ligation.
  • the processed sample DNA was taken out and centrifuged and purified once.
  • the operation was as follows: take the reaction product from the previous step and add 40 ⁇ L (0.8 ⁇ ) magnetic beads, mix well and place at room temperature for 5 minutes; then place it on a magnetic stand for 2 minutes and remove the supernatant; add 80% ethanol to rinse once and discard the supernatant; add 80% ethanol to rinse once and dry until the surface of the magnetic beads is rough; Assay Kit quantitative concentration, the total output must be greater than 30ng for use.
  • Step 3 DNB preparation before sequencing, according to the situation described in step 2, we need to prepare in two ways.
  • the amplicon library prepared by the first combination we can directly prepare it according to step 4 in method 1.
  • the second combination we need to change the steps to "take 20 ⁇ L of the Make DNB buffer prepared in step 1 and mix it with 30 ng of the product obtained in step 3, then add DNB polymerase I/40 ⁇ L, T4 DNA ligase (repaid) 1 ⁇ L, T4 polynucleotide kinase 1 ⁇ L, make up to 80 ⁇ L with NF Water, 30°C for 15 minutes, and store at 4°C (heat lid set at 35 degrees); Centrifuge, add DNB polymerase II/4 ⁇ L, store at 30°C for 25 minutes, and store at 4°C (total volume 84 ⁇ L, heat lid set at 35°C)”. After the reaction, take 2 ⁇ L and use the Qubit ssDNA Assay Kit for quantification. If the concentration is greater than
  • F1 CAGAACGACATGGCTACGATCCGACGT (SEQ ID NO: 16)
  • R1 TGCATGGCGACCTTATCAGAGTTA (SEQ ID NO: 17)
  • GAPDH-1F CCCCGGTTTCTATAAATTGAGC (SEQ ID NO: 4)
  • GAPDH-1R CGCTTGGCCTCCGACTTGAACTCACCCGTTGACTCCG (SEQ ID NO:5)
  • GAPDH-2F CTCCTCTGCGACACGTGA (SEQ ID NO: 6)
  • GAPDH-2R CGCTTGGCCTCCGACTTGTTCTCTCCCCTCCGCGCA (SEQ ID NO: 7)
  • GAPDH-3F CAGGAGGTCCCTACTCCCG (SEQ ID NO: 8)
  • GAPDH-3R CGCTTGGCCTCCGACTTGAGAATAATCTAGGAAAAGCATCACCCG (SEQ ID NO:9)
  • GAPDH-4F CCCAATCCTCCCGGTGACAT (SEQ ID NO: 10)
  • GAPDH-4R CGCTTGGCCTCCGACTTATGGGTGGAGTCGCGTGT (SEQ ID NO: 11)
  • GAPDH-5F ATCAATGACCCCTTCATTGA (SEQ ID NO: 12)
  • GAPDH-5R CGCTTGGCCTCCGACTTGATCTCGCTCCTGGAAGAT (SEQ ID NO: 13)
  • GAPDH-6F TAAGCAGTTGGTGGTGCA (SEQ ID NO: 14)
  • GAPDH-6R CGCTTGGCCTCCGACTTCTTCACCACCATGGAGAAG (SEQ ID NO: 15)
  • GAPDH-1F 5phos-CCCCGGTTTCTATAAATTGAGC (SEQ ID NO: 4)
  • GAPDH-1R 5phos-CGCTTGGCCTCCGACTTGAACTCACCCGTTGACTCCG (SEQ ID NO:5)
  • GAPDH-2F 5phos-CTCCTCTGCGACACGTGA (SEQ ID NO: 6)
  • GAPDH-2R 5phos-CGCTTGGCCTCCGACTTGTTCTCTCCCCTCCGCGCA (SEQ ID NO: 7)
  • GAPDH-3F 5phos-CAGGAGGTCCCTACTCCCG (SEQ ID NO:8)
  • GAPDH-3R 5phos-CGCTTGGCCTCCGACTTGAGAATAATCTAGGAAAAGCATCACCCG (SEQ ID NO:9)
  • GAPDH-4F 5phos-CCCAATCCTCCCGGTGACAT (SEQ ID NO: 10)
  • GAPDH-4R 5phos-CGCTTGGCCTCCGACTTATGGGTGGAGTCGCGTGT (SEQ ID NO: 11)
  • GAPDH-5F 5phos-ATCAATGACCCCTTCATTGA (SEQ ID NO: 12)
  • GAPDH-5R 5phos-CGCTTGGCCTCCGACTTGATCTCGCTCCTGGAAGAT (SEQ ID NO: 13)
  • GAPDH-6F 5phos-TAAGCAGTTGGTGGTGCA (SEQ ID NO: 14)
  • GAPDH-6R 5phos-CGCTTGGCCTCCGACTTCTTTCACCACCATGGAGAAG (SEQ ID NO: 15)
  • the primers used in this protocol were all synthesized by Jinweizhi (Suzhou).
  • step 2 Carry out the experiment according to the experimental method one.
  • step 2 three 150ng DNA samples were processed respectively, and the concentration and total amount of the obtained products were shown in Table 1, which met the requirements.
  • Agilent 2100 high sensitivity
  • the interrupted product obtained in the previous step was prepared as DNB according to step 3.
  • the concentration is shown in Table 2, which meets the requirements of the machine.
  • a pair of quality inspection primers F1/R1 was used to amplify the DNB, and electrophoresis was performed on a 1% agarose gel to check that the size of the amplified product was in line with expectations ( FIG. 7 ).
  • the library was sequenced using the MGI-2000 platform and supplemented with the data of a routine library construction (MGIEasy Universal DNA Library Preparation Kit) for comparison, and the data GC_Content and Mapping_Rate were analyzed using BWA; Raw_Q30, Raw_Reads, Clean_Reads and Clean_Rate were analyzed using Soapnuke, and the results are shown in Table 3.
  • the test was carried out according to the second experimental method. Six pairs of amplification primers were mixed and then amplified. Three parallel replicates were performed for samples with different concentrations. The PCR products were electrophoresed on 1% agarose gel and compared with Maker 100 (TIANGEN, Cat. No. MD109).
  • MGI-2000 platform uses the MGI-2000 platform to sequence the library and supplement the data of a conventional process library (MGIEasy Universal DNA Library Preparation Kit) for comparison, use BWA to analyze the data GC_Content and Mapping_Rate; use Soapnuke to analyze Raw_Q30, Raw_Reads, Clean_Reads and Clean_Rate, and the results are shown in Table 6.

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Abstract

一种接头及其在构建DNB文库中的应用，所述接头包含接头1、接头2以及锚定引物；其中锚定引物两端的核酸序列分别与接头1和接头2部分核酸序列互补。利用所述接头进行DNB文库的构建可以完成一步式文库制备，相较于其他方法具有极高的性价比；且能够适配多种载量的样本，适配形式多样化；还可以用于捕获双链DNA并进行双链测序。

Description

一种接头及其在构建DNB文库中的应用 技术领域
本发明属于生物工程领域，具体涉及一种接头及其在构建DNB文库中的应用。
背景技术
近些年来建库测序技术飞速发展，基于测序阐释的生命科学愈发彰显出它无穷的魅力，在分子育种、病原防控或是精准健康等领域都显示了不可替代的优势。现如今市场上有多个测序平台，华大的DNBSeq平台以其高性价比、高准确率独树一帜。建库技术方面已有众多新颖实用的技术发明，而其大多可归为两类。一是含PCR扩增过程，这种技术具有高富集性，对于微量样本及热点SNP具有极高的适应性，能够以极低的成本获得高质量的数据。但是PCR技术本身可能会引入错误碱基并在后续过度放大这种错误，使得数据准确性下降。二是无PCR扩增(PCR Free)，这种技术避免了扩增引入的错误，可获得真实的样本信息。完全的PCR Free技术具有严格的样本需求、繁琐的步骤和高昂的成本代价，压缩了人们对于它的需求欲望。
现基于DNBseq平台的建库技术按扩增与否来进行分类。
对于需要扩增的建库技术，例如常规WGS文库构建，其需要对样本核酸进行质量检测、打断、打断后片段质检、末端修复、片段双选纯化、片段质检、接头连接及纯化，扩增纯化及定量，最后完成环化、消化及DNB制备的上机准备。如果涉及到探针捕获流程，则需要更久的时间以及更昂贵的成本；再例如扩增子建库技术，它优先进行一次模板的PCR富集和纯化，再进行第二次的PCR富集、接头添加及纯化、文库质量鉴定以及环化、消化和上机前准备。
对于不需要扩增的建库技术，例如PCR Free建库，其需要对样本核酸 进行质量检测定量、打断、打断后片段质检、片段双选纯化、双选片段质检、末端修复、接头连接及纯化，连接产物质检，最后完成环化、消化及DNB制备的上机准备。
现有的建库技术存在一系列缺陷。例如，建库技术流程十分冗长，扩增子建库、WGS建库或PCR Free建库可能8-12小时能完成建库，而涉及到探针捕获的则需要更久才能完成建库。加上后续的不同类型的测序，一个周期的时间过久，大大影响效率。WGS和PCR Free所需要的成本代价远远大于扩增子建库。一部分科研人员因扩增会引入不可控错误不愿意选择扩增子建库，但是又受制于过高的成本，折中的办法就是选择低程度的扩增并结合其他的方法进行建库。但这种方式依旧不能解决准确度、成本以及时间上存在的问题。
发明内容
本发明所要解决的技术问题是提供一种接头及其应用，特别是在快速构建单链环状文库中的应用。本发明开发一种新型的接头以及相应的建库测序方法，该建库测序方法更为简捷、能更真实反应样本原始信息，能够以更高的性价比实现本领域的需求。
本发明主要通过以下技术方案解决上述技术问题。
本发明提供一种接头，其包含接头1、接头2以及锚定引物；其中锚定引物两端的核酸序列分别与接头1和接头2部分核酸序列互补。
其中，所述接头1和所述接头2优选均含有一个或多个标签序列、反向互补序列以及测序引物结合序列；且接头的5’端具有磷酸化修饰，3’端在与5’端的反向互补序列退火结合后闭锁并裸露至少一个T碱基；
所述锚定引物的两端分别含有与接头1和接头2互补的捕获序列，以及一段单链核酸结构序列。
在本发明一较佳实施方案中，所述接头1还含有1个或2个标签引物结 合序列。
在本发明一较佳实施方案中，所述接头2还含有1个或2个标签引物结合序列。
在本发明另一较佳实施方案中，所述接头1和所述接头2均还含有1个或2个标签引物结合序列。
其中，所述标签引物结合序列以及测序引物结合序列为测序平台限制性因素，可以根据测序平台或者生信分析做相应调整。
在本发明一较佳实施方案中，所述接头1和所述接头2内部的反向互补序列为5-10bp闭锁。
本发明中所述接头1和所述接头2在条件合适的情况下，可分别通过所述反向互补序列退火形成发卡结构。
在本发明中接头长度以及锚定引物的长度范围均可以作调整以适配实际建库场景需要。其中，锚定引物的长度是根据插入文库的长度来决定的。
在本发明一较佳实施方案中，所述锚定引物为长度大于35bp的单链核酸序列，例如长度可为35-200bp，如75bp；所述锚定引物的捕获序列长度为15-25bp，所述锚定引物的单链结构序列长度根据待测文库片段长度设计。
在本发明一较佳实施方案中，所述锚定引物的单链结构序列为35-55bp。
本发明还提供一种用于构建测序文库的试剂盒，其包含如上所定义的接头1、接头2和锚定引物。
较佳地，所述试剂盒还包含聚合酶。
在本发明一较佳实施方案中，所述接头1、所述接头2和所述锚定引物的摩尔比为1:1:1。
在本发明一更佳实施方案中，所述试剂盒还包含本领域常规使用的其他溶液，例如5×STE Buffer以及NF Water等。
所述试剂盒在使用过程中通常制备成串连接头复合物稀释液的形式。
在本发明一优选实施方案中，所述接头复合物稀释液的制备方法包括如 下步骤：
(1)将12.5μL浓度为100μM的所述接头1、12.5μL浓度为100μM的所述接头2以及3μL 5×STE Buffer混合，于PCR仪中进行反应；
(2)加入12.5μL浓度为100μM的所述锚定引物，以及2μL 5×STE Buffer和7.5μL NF Water，于PCR仪中进行反应。
其中，步骤(1)中所述反应的条件较佳地为：95℃ 5min,70℃ 5min，45℃ 5min，25℃ 5min。
步骤(2)中所述反应的条件较佳地为：25℃ 15min。
本发明还提供一种构建测序文库的方法，所述方法包括：
(1)接头复合物制备：将如上所定义的接头1和接头2与锚定引物混合后孵育，获得接头复合物；
(2)将待检测核酸片段与步骤(1)中的接头复合物进行连接反应，所述连接反应进一步包括连接酶；
(3)获得单链环状文库。
较佳地，所述核酸片段选自：基因组打断获得的核酸片段、PCR产物、游离核酸片段以及反转录产物。
本发明还提供同时检测核酸正义链与反义链的方法，其包括：
(1)使用如上所述的方法构建单链环状文库；
(2)扩增单链环状文库并进行核酸序列测定，根据测序数据中标签序列的信息，鉴定互补的正义链和反义链。
本发明的技术方案具体如下：
1.一种新的型接头复合物结构(图1)，由标签接头1、标签接头2以及锚定引物组合而成，可对5端磷酸化、3端为A碱基的粘性末端的DNA片段进行捕获建库；
2.标签接头1(adapter 1，图2)：该接头由一个或多个标签、标签引物结合序列、一段互补结构以及测序引物结合序列组合而成，其5’端具有磷酸 化修饰，3’端在复性结合后闭锁并裸露一个T碱基用于配对连接；
3.标签接头2(adapter 2，图3)：该接头由一个或多个标签、标签引物结合序列、一段互补结构以及测序引物结合序列组合而成，其5’端具有磷酸化修饰，3’端在复性结合后闭锁并裸露一个T碱基用于配对连接；
4.一种锚定引物(Link，图4)：含有与接头1、接头2分别互补的两段核酸序列，一段单链核酸序列，能够同时捕获锚定相应设计的两个接头，形成拱形结构，并可以作为后续制备DNA纳米球的引物；
5.一种两步退火反应制备接头复合物的方法及DNA纳米球制备缓冲液的配制方法；
6.一种可在扩增子产物3端加A碱基的扩增方法；
7.一种使用多5端磷酸化引物的多重PCR引物捕获方法；
8.一种将无5端磷酸化双链DNA制备成有5端磷酸化双链DNA的方法；
9.一种基于接头复合物，将片段化并末端修复加A的DNA或者是PCR产物与该接头复合物及反应缓冲液混合进行反应，快速得到可直接测序的DNB文库的方法(图5)。
本发明的积极进步效果为：
1、缩短冗长的流程，免去中间环节多项操作，完成一步式文库制备；本方法极大的压缩了流程和成本，操作起来简单高效，耗时极短，相较于其他方法有极高的性价比；
2、可以适配多种载量的样本，适配形式多样化。在极少量样本时可结合扩增子建库进行使用，在样本量充足时可选择PCR Free形式进行建库，具有良好的兼容性和自主选择性；对于质量较好的样本可免除PCR环节，对于质量较差以及微量的样本可减少PCR扩增的步骤；对样本量需求量低，兼容性好，准确度高；
3、可以捕获双链DNA并进行双链测序，双链结合的建库测序方式可以 完整保留样本的原始双链信息，最大限度的保持了样本信息的真实性，针对特殊的需求可以拓展新的应用。
附图说明
图1为新型接头复合物连接示意图。
图2为接头adapter 1。
图3为接头adapter2。
图4为接头Link。
图5为建库流程。
图6为打断DNA产物的大小分布图。
图7为对DNB进行验证扩增后的产物的电泳图。
图8为6×GAPDH Primer mix凝胶电泳图。
具体实施方式
实施例1
步骤1：预先合成adapter 1、adapter2以及Link三条引物并使用TE(生工，货号B548106-0500)稀释成100μL稀释液。将Adapter 1引物稀释液以及Adapter 2引物稀释液进行退火处理，退火方法为“取Adapter 1(100μM)/12.5μL+Adapter 2(100μM)/12.5μL，5×STE Buffer(BGI)/3μL，置于PCR仪上进行反应，其程序设置为‘95℃5min,70℃5min，45℃5min,25℃5min,4℃保存’反应结束后取出并离心；再加入Link(100μM)/12.5μL，5×STE Buffer/2μL，NF Water/7.5μL，置于PCR仪上进行反应，其程序为‘25℃15min,4℃保存’”，即可得到本方法所需要用的接头稀释液(25μM)。取接头稀释液(25μM)/8μL，10×Phi29Buffer(Thermo，货号B62)/40μL，NF Water(Invitrogen,货号AM9932)/152μL混合，制备成Make DNB buffer备用。
步骤2：对DNA样本使用Hieff NGS OnePot DNA Fragmentation Reagent (翊圣，货号13474ES96)进行片段化处理，处理方法为“取样本DNA/150ng，OnePot mix/7.5μL，NF water UP to 30μL；30℃ 10min，65℃ 20min，4℃保存(热盖设置70℃)”。将处理后的样本DNA取出离心并进行一次简单纯化，其操作为“取上一步反应产物加入27μL(0.9×)磁珠(VAZYME，货号N411-03)，充分混匀并室温放置5min；然后置于磁力架上吸附2min，并去除上清；加入80％乙醇漂洗一次并晾干至磁珠表面粗糙即可；加入22μL TE(生工，货号B548106-0500)混匀洗脱，保留上清；取1μL使用Qubit dsDNA HS Assay Kit(Invitrogen，货号Q32854)定量浓度，总产量要求大于30ng备用。
步骤3：测序前DNB制备，其操作为“取20μL步骤一配制的Make DNB buffer与步骤2所得产物30ng混合，再加入DNB聚合酶I(MGI,1000004803)/40μL,T4 DNA ligase(repaid)(enzymatic，货号L6030-HC-L)1μL，用NF Water补足至80μL，25℃10min，4℃保存(热盖设置35度)；反应结束后取出反应液并离心，加入DNB聚合酶II(MGI,1000017261)/4μL，30℃ 25min，4℃保存(总体积84μL，热盖设置35度)”。反应结束后取2μL使用Qubit ssDNA Assay Kit(Invitrogen，货号Q10212)进行定量，浓度大于8ng即为合格文库，可用于MGI测序平台测序。
实施例2
步骤1：按照实验方法一中步骤1的方法配制Make DNB buffer备用。
步骤2：进行PCR扩增子文库制备，这里根据不同的实验室条件推荐两种制备方式。第一种组合为5端磷酸化的F引物和R引物、模板以及Taq酶(TaKaRa，货号R001A)，第二种组合为常规的F引物和R引物、模板以及Taq酶、T4多聚核苷酸激酶(Thermo，货号EK0031)。制备方法为“将每条F引物与R引物分别稀释至25μM，按1:1的比例进行等摩尔量混合备用；取混合引物/4μL，2×MμLtiplex PCR Readymix(BGI,货号1000014386)/25μL， 样本/1ng，NF Water/UP to 50μL；扩增程序为95℃/3min，1个循环；95℃/20s，60℃/20s,72℃/30s,30个循环；72℃/10min，一个循环；12℃保存”。该过程可根据自身情况进行优化，不作具体要求，但需要保证产物得到充分延伸使其末端加A，便于连接。将处理后的样本DNA取出离心并进行一次纯化，其操作为：取上一步反应产物加入40μL(0.8×)磁珠，充分混匀并室温放置5min；然后置于磁力架上吸附2min，并去除上清；加入80％乙醇漂洗一次并弃上清；再加入80％乙醇漂洗一次并晾干至磁珠表面粗糙即可；加入22μL TE混匀洗脱，保留上清；取1μL使用Qubit dsDNA HS Assay Kit定量浓度，总产量需大于30ng备用。
步骤3：测序前DNB制备，根据步骤二中所述情况，我们需要分为两种方式制备。对于第一种组合制备的扩增子文库，我们直接按照方法一中的步骤四制备即可。在第二种组合中，我们需要变更步骤为“取20μL步骤一配制的Make DNB buffer与步骤三所得产物30ng混合，再加入DNB聚合酶I/40μL,T4 DNA ligase(repaid)1μL，T4多聚核苷酸激酶1μL，用NF Water补足至80μL，30℃15min，4℃保存(热盖设置35度)；反应结束后取出反应液并离心，加入DNB聚合酶II/4μL，30℃25min，4℃保存(总体积84μL，热盖设置35度)”。反应结束后取2μL使用Qubit ssDNA Assay Kit进行定量，浓度大于8ng即为合格文库，可用于MGI测序平台测序。
三个结构部分的引物序列举其中一例用于描述试验：
Adapter 1：
5phos·TCCCCCTAAGTCAGCTCAGTACGTCAGCAGTTCGTGATACACGCTCACAGAACGACATGGCTACGATCCGACGTAAGGGGGAT(SEQ ID NO:1)
Adapter 2：
5phos·GAAAAAGTTAACTCTGATAAGGTCGCCATGCATCAAGTGCCTAAGTCGGAGGCCAAGCGGTCTTAGGAAGACAATCTTTTTCT(SEQ ID  NO:2)
Link：
Figure PCTCN2022073026-appb-000001
设计一对捕获产物验证引物，可对连接后产物进行检测：
F1:CAGAACGACATGGCTACGATCCGACGT(SEQ ID NO:16)
R1:TGCATGGCGACCTTATCAGAGTTA(SEQ ID NO:17)
设计六对多重PCR扩增引物，以人DNA(NA12878，BGI)为模板，捕获GAPDH基因的6个片段：
GAPDH-1F：CCCCGGTTTCTATAAATTGAGC(SEQ ID NO:4)
GAPDH-1R：CGCTTGGCCTCCGACTTGAACTCACCCGTTGACTCCG(SEQ ID NO:5)
GAPDH-2F：CTCCTCTGCGACACGTGA(SEQ ID NO:6)
GAPDH-2R：CGCTTGGCCTCCGACTTGTTCTCTCCCTCCGCGCA(SEQ ID NO:7)
GAPDH-3F：CAGGAGGTCCCTACTCCCG(SEQ ID NO:8)
GAPDH-3R：CGCTTGGCCTCCGACTTGAGAATAATCTAGGAAAAGCATCACCCG(SEQ ID NO:9)
GAPDH-4F：CCCAATCCTCCCGGTGACAT(SEQ ID NO:10)
GAPDH-4R：CGCTTGGCCTCCGACTTATGGGTGGAGTCGCGTGT(SEQ ID NO:11)
GAPDH-5F：ATCAATGACCCCTTCATTGA(SEQ ID NO:12)
GAPDH-5R：CGCTTGGCCTCCGACTTGATCTCGCTCCTGGAAGAT(SEQ ID NO:13)
GAPDH-6F：TAAGCAGTTGGTGGTGCA(SEQ ID NO:14)
GAPDH-6R：CGCTTGGCCTCCGACTTCTTCACCACCATGGAGAAG (SEQ ID NO:15)
另外合成一份分别磷酸化修饰的六对引物：
GAPDH-1F：5phos-CCCCGGTTTCTATAAATTGAGC(SEQ ID NO:4)
GAPDH-1R：5phos-CGCTTGGCCTCCGACTTGAACTCACCCGTTGACTCCG(SEQ ID NO:5)
GAPDH-2F：5phos-CTCCTCTGCGACACGTGA(SEQ ID NO:6)
GAPDH-2R：5phos-CGCTTGGCCTCCGACTTGTTCTCTCCCTCCGCGCA(SEQ ID NO:7)
GAPDH-3F：5phos-CAGGAGGTCCCTACTCCCG(SEQ ID NO:8)
GAPDH-3R：5phos-CGCTTGGCCTCCGACTTGAGAATAATCTAGGAAAAGCATCACCCG(SEQ ID NO:9)
GAPDH-4F：5phos-CCCAATCCTCCCGGTGACAT(SEQ ID NO:10)
GAPDH-4R：5phos-CGCTTGGCCTCCGACTTATGGGTGGAGTCGCGTGT(SEQ ID NO:11)
GAPDH-5F：5phos-ATCAATGACCCCTTCATTGA(SEQ ID NO:12)
GAPDH-5R：5phos-CGCTTGGCCTCCGACTTGATCTCGCTCCTGGAAGAT(SEQ ID NO:13)
GAPDH-6F：5phos-TAAGCAGTTGGTGGTGCA(SEQ ID NO:14)
GAPDH-6R：5phos-CGCTTGGCCTCCGACTTCTTCACCACCATGGAGAAG(SEQ ID NO:15)
本方案使用的引物均由金唯智(苏州)合成。
按照实验方法一进行实验。按步骤2分别将三份150ng DNA样本进行处理，得到产物的浓度及总量如表1所示，符合要求。为了便于后续验证，我们使用安捷伦2100(高敏)对打断产物进行了一次片段大小的检测(图6)。
表1
150ng样本 1 2 3
打断回收浓度ng/μL 4.42 4.38 4.51
回收总量ng 88.4 87.6 90.2
将上一步得到的打断产物按步骤3进行DNB制备，浓度如表2所示，符合上机要求。为了检验DNB是否正确，使用一对质检引物(F1/R1)对DNB进行扩增，并用1％琼脂糖凝胶进行电泳检查，扩增产物大小符合预期(图7)。
表2
样本 1 2 3
DNB浓度ng/μL 22.4 19.7 23.3
使用MGI-2000平台对文库进行测序并补充一个常规流程建库(MGIEasy通用DNA文库制备试剂盒)的数据作为比较，使用BWA对数据GC_Content和Mapping_Rate进行分析；使用Soapnuke对Raw_Q30、Raw_Reads、Clean_Reads以及Clean_Rate进行分析，得到结果如表3所示。
表3
样本 Raw_Q30 GC_Content Raw_Reads Clean_Reads Clean_Rate Mapping_Rate
1 95.04％ 43.34％ 34309037 34248909 99.72％ 99.35％
2 95.09％ 43.36％ 34326210 34266052 99.77％ 99.40％
3 95.16％ 43.39％ 34350207 34290007 99.84％ 99.47％
MGIEasy 95.14％ 43.38％ 34343337 34283149 99.82％ 99.45％
下机数据显示，以本方法测序得到的结果与常规方法测得的结果具有良好的一致性。
按照实验方法二进行试验，将六对扩增引物混合后扩增，对于不同浓度的样本做三个平行重复，对PCR产物使用1％琼脂糖凝胶电泳，Maker 100做对比(TIANGEN,货号MD109)，电泳图(图8)显示，PCR产物条带明亮，大小符合预期，阴性对照无条带，表明实验结果正常，产物浓度及总量如表 4所示：
表4
Figure PCTCN2022073026-appb-000002
分别取1ng、2.5ng、5ng、7.5ng、10ng样本投入量的常规引物PCR产物和磷酸化引物PCR产物各一份进行DNB制备，浓度如表5所示，均符合要求。
表5
样本组别 1ng常规组 2.5ng常规组 5ng常规组 7.5ng常规组 10ng常规组
DNB浓度ng/μL 18.9 21.7 20.6 21.3 23.6
样本组别 1ng-phos 2.5ng-phos 5ng-phos 7.5ng-phos 10ng-phos
DNB浓度ng/μL 19.8 23.2 25.7 20.4 22.8
使用MGI-2000平台对文库进行测序并补充一个常规流程建库(MGIEasy通用DNA文库制备试剂盒)的数据作为比较，使用BWA对数据GC_Content和Mapping_Rate进行分析；使用Soapnuke对Raw_Q30、Raw_Reads、Clean_Reads以及Clean_Rate进行分析，得到结果如表6所示。
表6
Figure PCTCN2022073026-appb-000003
Figure PCTCN2022073026-appb-000004
下机数据显示，以本方法测序得到的结果与常规方法测得的结果具有良好的一致性。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (10)

  1. 一种接头，其特征在于，其包含接头1、接头2以及锚定引物；其中锚定引物两端的核酸序列分别与接头1和接头2部分核酸序列互补。
  2. 如权利要求1所述的接头，其特征在于，所述接头1和接头2均含有一个或多个标签序列、反向互补序列以及测序引物结合序列；其中，5’端具有磷酸化修饰，3’端与5’端的反向互补序列退火结合后闭锁并裸露至少一个T碱基；
    所述锚定引物的两端分别含有与接头1和接头2互补的捕获序列，以及一段单链核酸结构序列。
  3. 如权利要求2所述的接头，其特征在于，所述接头1还含有1个或2个标签引物结合序列；
    和/或，所述接头2还含有1个或2个标签引物结合序列。
  4. 如权利要求2所述的接头，其特征在于，所述接头1和所述接头2内部的反向互补序列为5-10bp闭锁。
  5. 如权利要求1～4任一项所述的接头，其特征在于，所述接头1和所述接头2在条件合适的情况下，分别通过所述反向互补序列退火形成发卡结构。
  6. 如权利要求1～4任一项所述的接头，其特征在于，所述锚定引物为长度大于35bp的单链核酸序列，所述锚定引物的捕获序列长度为15-25bp，所述锚定引物的单链结构序列长度根据待测文库片段长度设计；
    所述锚定引物的单链结构序列长度优选35-55bp。
  7. 一种用于构建测序文库的试剂盒，其特征在于，其包含如权利要求1～6中任一项所定义的接头1、接头2和锚定引物；
    较佳地，所述试剂盒还包含聚合酶。
  8. 如权利要求7所述的试剂盒，其特征在于，所述接头1、所述接头2和所述锚定引物的摩尔比为1:1:1。
  9. 一种构建测序文库的方法，其特征在于，所述方法包括：
    (1)接头复合物制备：将权利要求1～6任一项中所定义的接头1和接头2与锚定引物混合后孵育，获得接头复合物；
    (2)将待检测核酸片段与步骤(1)中的接头复合物进行连接反应，所述连接反应进一步包括连接酶；较佳地，所述待检测核酸片段选自：基因组打断获得的核酸片段、PCR产物、反转录产物以及游离核酸片段；
    (3)获得单链环状文库。
  10. 一种同时检测核酸正义链与反义链的方法，其包括：
    (1)使用如权利要求9所述的方法构建单链环状文库；
    (2)扩增单链环状文库并进行核酸序列测定，根据测序数据中标签序列的信息，鉴定互补的正义链和反义链。
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