WO2021013244A1 - 一种构建捕获文库的方法和试剂盒 - Google Patents

一种构建捕获文库的方法和试剂盒 Download PDF

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WO2021013244A1
WO2021013244A1 PCT/CN2020/104351 CN2020104351W WO2021013244A1 WO 2021013244 A1 WO2021013244 A1 WO 2021013244A1 CN 2020104351 W CN2020104351 W CN 2020104351W WO 2021013244 A1 WO2021013244 A1 WO 2021013244A1
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sequence
dna
library
kit
hybridization
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French (fr)
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桂丹
陈迪
王光园
张建光
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北京贝瑞和康生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6844—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 belongs to the field of molecular biology, and specifically relates to a method and a kit for constructing a hybrid capture library.
  • Exon capture is a technology that uses probes to capture and enrich DNA sequences in exon regions, and is widely used in scientific research and clinical testing. Compared with whole genome sequencing, its cost is lower, the cycle is shorter, the coverage is better, and it is more economical and efficient.
  • the construction of a traditional exon capture library generally includes the following steps: fragment genomic DNA, perform end repair and add A at the end, connect the adapter and tag sequence, and obtain the pre-library through the first round of PCR amplification; The pre-library is hybridized with the hybridization probe in the presence of the blocking sequence, and after purification, the final capture library is obtained by the second round of PCR amplification (see Figure 1).
  • linker and tag sequence are of great significance for on-machine sequencing, sample identification and tracing the source of the original DNA molecule.
  • these linker and tag sequences often form a long-length (about 60 bp at each end) and reverse complementary sequence structure.
  • This sequence structure is very easy to anneal to each other during the hybrid capture process, so that non-target sequences are captured together when the probe binds to the specific sequence, thereby reducing the specificity of the overall capture. Therefore, it is necessary to effectively block non-target sequences other than these inserts during the hybrid capture process to prevent non-specific binding.
  • the blocking sequence adopts a reverse complementary sequence to the linker sequence, and the linker is sealed by complementary base pairing with the linker sequence.
  • the closed sequence is divided into two parts, one part is reverse complementary to the sequence of amplification primer P5 and sequencing primer 1 (also called Read 1 sequencing primer), and the other part is complementary to sequencing primer 2 (also called Read 2 sequencing primer),
  • the index tag and the amplification primer P7 sequence are reverse complementary, and the adapter is blocked by complementary pairing with the corresponding part.
  • the binding of the linker blocking sequence is easily affected by temperature during the hybridization process, and dimers are easily formed between the blocking sequences, which leads to a reduction in the blocking efficiency and further reduces the capture efficiency of the target region.
  • hypoxanthine In order to control costs, some people have proposed a strategy of using a corresponding number of hypoxanthines to block the tag sequence, that is, using hypoxanthine to modify the end of the tag sequence instead of adding additional blocking sequences.
  • hypoxanthine has a certain preference for blocked bases, resulting in poor blocking effect on some tag sequences, which in turn affects the capture efficiency.
  • the cost of synthetic hypoxanthine is relatively expensive.
  • Some people also proposed a bridge closure design strategy, that is, design corresponding closure sequences for the linker sequences at both ends of the target fragment, and use 6-8 C3 inter-arms for bridge connection for the tag sequence in the middle part.
  • CN108456713A also proposes blocking modification to the end of the linker, such as reverse dT modification, infra-arm modification, amino modification, ddNTP modification, so as to realize the blocking of the linker sequence. Regardless of the strategy, additional blocking sequences or special blocking modifications to the linker are required, which is of limited help in controlling hybridization costs.
  • the amount of DNA used for hybridization capture (ie, the content of the prelibrary) be 500 ng or more.
  • the commonly used kit in hybrid capture Twist Human Core Exome EF Singleplex Complete Kit, 96Samples (Twist Bioscience, catalog number 100790) and Exome Research Panel v1.0 (IDT, catalog number 1056115) requires that the initial volume of the pre-library used for hybridization is at least 500ng, and SureSelect XT HS Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library (Agilent Technologies, catalog number G9704N) The initial amount of the pre-library required for hybridization is 500-1000ng.
  • the traditional capture library construction method requires PCR amplification to amplify the amount of DNA extracted from the genome and compensate for the aforementioned purification bands.
  • PCR amplification to amplify the amount of DNA extracted from the genome and compensate for the aforementioned purification bands.
  • the inventors proposed a method for constructing a pre-library without PCR pre-amplification, and without adding blocking sequences or end-modification of the adaptor The capture library construction method.
  • the present invention is based on the following two facts discovered by the inventors: (1) With an initial amount of 5-50ng DNA, a pre-library obtained without PCR amplification can achieve good coverage and coverage uniformity. Therefore, a large number of pre-libraries (500ng-1000ng) are not necessary for hybrid capture, and PCR pre-amplification is not a necessary step to construct a pre-library; (2) By connecting fragmented DNA to a Y-linker, blocking can be omitted during hybrid capture The closed sequence of the linker and the tag sequence will not have any impact on the capture efficiency, coverage and coverage uniformity, thereby saving hybrid capture costs.
  • the present invention provides a method for constructing a capture library, including the following steps:
  • the hybridization product is amplified by PCR to obtain a capture library.
  • the fragmented DNA refers to naturally occurring short fragments of DNA or short fragments of DNA obtained by artificially interrupting genomic DNA.
  • the fragmented DNA may be derived from blood, serum, plasma, joint fluid, semen, urine, sweat, saliva, feces, cerebrospinal fluid, ascites, pleural fluid, bile, or pancreatic fluid.
  • the natural short fragment DNA is free peripheral blood DNA, free tumor DNA or naturally degraded genomic DNA.
  • genomic DNA can have various sources, such as from peripheral blood, dried blood spots, buccal swabs, and the like.
  • methods for interrupting genomic DNA for example, ultrasonic treatment, mechanical interruption, or restriction enzyme digestion. Since sonication and mechanical interruption will relatively lose more DNA, when the initial DNA content is small (for example, as low as 50 ng), it is preferable to use enzyme digestion to fragment the DNA.
  • the length of the fragmented DNA is 150-400 bp, preferably 180-230 bp.
  • the method of the present invention further includes a step of performing end repair and/or adding A to the end of the fragmented DNA before connecting with the Y-type linker (ie, step 2).
  • any enzyme known to those skilled in the art that is suitable for end repair can be used to perform end repair on DNA, such as T4 DNA polymerase, Klenow enzyme and mixtures thereof.
  • any enzyme known to those skilled in the art that is suitable for end-addition of A can be used to end-add DNA. Examples of such enzymes include, but are not limited to, Taq enzyme, klenowex-enzyme and mixtures thereof.
  • the end repair and the end addition of A can be performed in two reaction systems, that is, after the end repair, the end is purified and then the end is added.
  • the end repair and the end addition of A are performed in one reaction system, that is, the end repair and the end addition of A are completed at the same time, and then the nucleic acid is purified.
  • DNA fragmentation, end repair, and end addition are performed in one reaction system, and then the linker is connected. This not only simplifies the operation steps and saves costs, but also reduces the contamination between samples.
  • the incubation time and temperature for end-filling and end-adding A can be determined by those skilled in the art according to conventional techniques according to specific needs.
  • step (2) can be performed with any enzyme known to those skilled in the art that is suitable for linking linkers.
  • enzymes include, but are not limited to, T4 DNA ligase, T7 DNA ligase or their mixtures.
  • the conditions for carrying out the ligation reaction are well known to those skilled in the art.
  • Y-linker refers to a linker formed by two strands that are not completely complementary. One end of the linker forms a double strand due to the base complementarity of the two strands, and the other end is due to the two strands. The bases are not completely complementary, and no double strands are formed.
  • the commonly used Y-joints mainly include long Y-joints ( Figure 3a) and truncated Y-joints ( Figure 3b).
  • the conventional long Y-joint mainly includes amplification primer sequence (P5/P7), index tag sequence, read 1/read 2 sequencing primer sequence and index read sequencing primer sequence, among which read 1/read 2 sequencing The primer sequence and the sequence of the index read sequencing primer are not completely complementary to form a part of the double strand.
  • the conventional truncated Y-joint mainly includes read 1/read 2 sequencing primer sequence and index sequencing primer sequence, or partial read 1/read 2 sequencing primer sequence and partial index sequencing primer sequence, where read 1/ The sequence of the read 2 sequencing primer and the index read sequencing primer are not completely complementary to form a part of the double strand.
  • This truncated Y-linker usually needs to be used with another linker containing P5/P7 primers and index tag sequence.
  • sequence of the two chains included in the Y-linker that can be used in the present invention is as follows:
  • the underline shows the part where the bases in the two chains are complementary.
  • the 5'end of the oligonucleotide can be phosphorylated by polynucleotide kinase, or a phosphate group can be directly added to the 5'end when the primer is synthesized.
  • step (3) of the method of the present invention is performed in a liquid phase hybridization system.
  • blocking sequence refers to a sequence used to block the linker and tag sequence, including sequences designed to be complementary to the linker and/or tag sequence.
  • special modifications are added to the end of the blocked sequence, such as reverse dT modification, amino modification, ddNTP modification (including ddCTP, ddATP, ddGTP and ddTTP), spacer Modification, hypoxanthine modification, random base modification, etc.
  • PCR amplification is often performed after linking the adapter and the tag sequence to amplify the content of the target DNA, thereby ensuring the efficiency of the subsequent hybridization step and meeting the requirements of on-machine sequencing.
  • a blocking sequence to the hybridization system. Its function is to block the amplified linker and tag sequence through base complementation so that they do not interfere with the target sequence and the target sequence during the hybridization process. Combination of hybridization probes.
  • the blocking sequence is base complementary to the linker and tag sequence, they can not only bind to the linker and tag sequence during the hybridization process, but they can also bind to each other.
  • the inventors found that, without the need for PCR pre-amplification to prepare a pre-library and the use of Y-type adapters, the hybridization system does not need to add any blocking sequence to achieve better capture efficiency.
  • the system used for hybridization includes hybridization buffer, Cot-1 DNA and hybridization probes, but does not include blocking sequences.
  • hybridization buffer such as hybridization temperature and hybridization time
  • hybridization probes are also well known to those skilled in the art.
  • the present invention provides a kit for constructing a capture library, which includes:
  • the reagents used for hybridization include hybridization buffer, Cot-1 DNA and hybridization probes, but do not include blocking sequences.
  • the reagents used for PCR amplification include buffer, PCR polymerase, and amplification primers.
  • the capture library prepared according to the method of the present invention can be used in a variety of second-generation sequencing platforms, including but not limited to such as Roche/454 FLX, Illumina/Hiseq, Miseq, NextSeq and Life Technologies/SOLID system, PGM, Proton And other sequencing platforms.
  • second-generation sequencing platforms including but not limited to such as Roche/454 FLX, Illumina/Hiseq, Miseq, NextSeq and Life Technologies/SOLID system, PGM, Proton And other sequencing platforms.
  • the excellent technical effects of the present invention are: (1) The content of starting DNA is low, even as low as 5ng, which greatly improves the utilization rate of rare samples and expands the scope of application of the present invention, such as the method and The kit can be applied to dried blood spots, buccal swabs, cfDNA and other sample types that cannot meet the common exon capture process due to the small amount of DNA extraction; (2) The library construction process is simple, and the method of the present invention is before obtaining the pre-library library No PCR reaction is required, so it only takes about 2 hours to complete the pre-library construction, while it takes about 6 hours to construct the pre-library in the traditional capture library construction method; (3) because the method of the present invention does not include the hybridization system The closed sequence can greatly save the cost of building a database while ensuring that the capture efficiency and coverage are not affected.
  • Figure 1 Schematic diagram of the conventional capture library construction method.
  • Figure 2 A schematic flow chart of an embodiment of the capture library construction method of the present invention.
  • Figures 3a and 3b schematic diagram of the Y-joint structure.
  • Figure 4 Schematic diagram of the closed sequence.
  • Example 1 Construction of a capture library according to the method of the present invention
  • Step 1 Obtain fragmented DNA, end repair and end add A
  • sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 are annealed under the following conditions to form a long Y-joint: 95°C, 2min; 95°C, 2min, cooling to 90°C at a rate of 0.1°C/s , And keep for 2min; decrease the temperature to 85°C at a rate of 0.1°C/s and keep it for 2min; decrease the temperature to 80°C at a rate of 0.1°C/s and keep it for 2min; and so on, until the temperature drops at a rate of 0.1°C/s To 25°C, and keep for 2min; finally keep at 4°C.
  • step 1 Using the WGS Ligase kit (Enzymatics, article number L6030-WL), the reaction system of step 1 was used to prepare the connection system shown in Table 2, and the connection system was incubated at 20°C for 15 minutes, and then kept at 4°C.
  • the ligation product is purified with the Beckman Agencourt AMPure XP kit (Beckman, catalog number A63882).
  • xGen Lockdown Reagents kit IDT, Catalog No. 1072281
  • 14.5 ⁇ l hybridization reagent 9.5 ⁇ l xGen 2 ⁇ hybridization buffer, 3 ⁇ l xGen hybridization buffer enhancer and 2 ⁇ l Cot-1DNA
  • Step 4 PCR amplification
  • the sequence of the amplification primer is as follows:
  • P5 primer 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 3);
  • P7 primer 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 4).
  • the Beckman Agencourt AMPure XP kit (Beckman, catalog number A63882) is used for purification to obtain the final capture library.
  • the library construction method of this embodiment is basically the same as that of embodiment 1, except that the hybridization reagent in step 3 also includes 2 ⁇ l of blocking sequence, and the blocking sequence is xGen Universal Blockers-TS Mix (IDT, catalog number 1075475).
  • the library construction method of this embodiment is the same as that of embodiment 1, except that after step 2 is completed, the purified product is pre-amplified by PCR to prepare a pre-library, and 2 ⁇ l of blocking sequence is added to the hybridization reagent in step 3.
  • 2 ⁇ KAPA HiFiHotStartReadyMix kit KAPA, article number KK2602
  • PCR was performed according to the following procedure: 95°C for 45s; 98°C for 15s, 65°C for 30s, 72°C 30s, 7 cycles; 72°C for 1 minute; keep at 4°C.
  • the sequence of the pre-amplification primer is as follows:
  • P5 primer 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 3);
  • P7 primer 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 4).
  • the Beckman Agencourt AMPure XP kit (Beckman, catalog number A63882) is used for purification, and then the capture hybridization is performed.
  • the blocking sequence added to the hybridization reagent in step 3 is xGen Universal Blockers-TS Mix (IDT, catalog number 1075475).
  • the library construction method of this embodiment is the same as that of Comparative Example 2, except that in step 3, no blocking sequence is added to the hybridization system.
  • Example 2 Construction of a capture library according to the method of the present invention
  • a capture library was prepared with peripheral blood gDNA, dried blood spot gDNA, and buccal swab gDNA.
  • the captured library was quantified by qPCR, and then the library was sequenced (150bp paired-end sequencing) using the Illumina NovaSeq 6000 sequencing platform according to the standard operating procedure of the sequencer, and 10G data was measured for each sample.
  • the sequencing results are shown in Table 6.
  • the method for constructing a sequencing library of the present invention is applicable to a variety of sample types, especially samples of peripheral blood, dried blood spots, buccal swabs and the like with less DNA content.
  • Example 3 The effect of the initial amount of DNA on the capture library.
  • the capture library constructed according to the method of the present invention has no significant difference in capture efficiency, coverage, and comparison rate. This shows that the method according to the present invention can use samples with an initial DNA content as low as 5 ng, and the prepared capture library fully meets the requirements of on-machine sequencing and subsequent data analysis.

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Abstract

本发明提供一种构建捕获文库的方法，包括以下步骤：(1)获得片段化的DNA；(2)将片段化的DNA与Y型接头连接，获得预文库；(3)在不存在封闭序列的情况下将预文库与杂交探针进行杂交，获得杂交产物；和(4)将杂交产物进行PCR扩增，获得捕获文库。本发明还提供用于实施该方法的试剂盒。

Description

一种构建捕获文库的方法和试剂盒 技术领域
本发明属于分子生物学领域，具体涉及构建杂交捕获文库的方法和试剂盒。
背景技术
外显子捕获是利用探针捕获并富集外显子区域的DNA序列的技术，在科研和临床检测领域被广泛应用。与全基因组测序相比，其费用更低、周期更短、覆盖度更好、更加经济、高效。传统的外显子捕获文库的构建一般包括以下步骤：将基因组DNA片段化，进行末端修复和末端加A，然后连接接头和标签序列，并通过第一轮PCR扩增获得预文库；之后再在封闭序列存在下将预文库与杂交探针进行杂交，纯化后通过第二轮PCR扩增以获得最终的捕获文库(参见图1)。接头和标签序列对上机测序、样本区分和追溯原始DNA分子的来源有着重要的意义。然而，在构建预文库的第一轮PCR扩增过程中，这些接头和标签序列往往会形成长度较长(每端60bp左右)、反向互补的序列结构。这种序列结构在杂交捕获过程中非常容易相互退火，使得非目标序列在探针与特异性序列结合时被一同捕获下来，从而降低整体捕获的特异性。因此，在杂交捕获过程中需要将这些插入片段以外的非目标序列进行有效封闭，以防止非特异性结合的发生。目前，封闭序列采用的是与接头序列反向互补的序列，通过与接头序列的碱基互补配对完成对接头的封闭。具体而言，封闭序列分两部分，一部分与扩增引物P5及测序引物1(也称为Read 1测序引物)序列反向互补，另一部分与测序引物2(也称为Read 2测序引物)、index标签及扩增引物P7序列反向互补，通过与其对应部分的互补配对进行接头封闭。然而，这种接头封闭序列的结合在杂交过程中容易受到温度的影响，封闭序列之间也容易形成二聚 体，从而导致封闭效率降低，进一步使得目标区域的捕获效率也降低。此外，在高通量测序时，通常涉及大量样本，需要多种标签序列进行区分。上述封闭策略意味着需要对每一种标签序列单独设计封闭序列，这无疑增加了实验操作的繁琐性，同时也增加了建库成本。
为了控制成本，有人提出用相应数目的次黄嘌呤来封闭标签序列的策略，即用次黄嘌呤修饰标签序列的末端以代替额外加入封闭序列的做法。然而，次黄嘌呤对封闭的碱基具有一定的偏好性，造成对有些标签序列封闭效果差，进而影响捕获效率。同时，合成次黄嘌呤成本比较昂贵。也有人提出桥式封闭设计策略，即，分别对目标片段两端的接头序列设计对应的封闭序列，对于中间部分的标签序列则采取用6-8个C3间臂进行桥式连接。CN108456713A还提出对接头末端进行封闭修饰，例如反向dT修饰、间臂修饰、氨基修饰、ddNTP修饰，从而实现对接头序列的封闭。但无论哪种策略，均需要额外加入封闭序列或对接头进行特殊的封闭修饰，对控制杂交成本的帮助比较有限。
此外，为了增加测序文库多样性并保证文库的丰度，一般要求用于进行杂交捕获的DNA量(即，预文库的含量)为500ng甚至更高。例如，在杂交捕获中常用的试剂盒Twist Human Core Exome EF Singleplex Complete Kit,96Samples(Twist Bioscience，货号100790)和
Figure PCTCN2020104351-appb-000001
Exome Research Panel v1.0(IDT，货号1056115)均要求用于杂交的预文库的起始量为至少500ng，SureSelect XT HS Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library(Agilent Technologies，货号G9704N)则要求用于杂交的预文库的起始量为500-1000ng。正是由于预文库对DNA含量的这种要求，以及因为纯化步骤造成的DNA量损失，传统的捕获文库构建方法中才需要通过PCR扩增来放大从基因组提取的DNA量并补偿上述因纯化带来的损失，通过提供足量的预文库来满足杂交捕获反应的要求。因此，需要建立一种简单且经济的捕获文库的构建方法， 其能够有效降低杂交过程中的非特异性结合，提高捕获效率。
发明内容
鉴于捕获文库构建中存在的以上问题，为了节约成本和简化文库构建过程中的繁琐性，发明人提出了一种无需PCR预扩增构建预文库，并且不需要加入封闭序列或对接头进行末端修饰的捕获文库构建方法。
本发明是基于发明人发现的以下两个事实：(1)在5-50ng DNA的起始量下，无需PCR扩增获得的预文库也可以达到很好的覆盖度和覆盖均一性。因而大量预文库(500ng-1000ng)并非杂交捕获所必需，PCR预扩增并非构建预文库的必要步骤；(2)通过将片段化DNA连接Y型接头，可以在杂交捕获时省去用于封闭接头和标签序列的封闭序列，并且不会对捕获效率、覆盖度和覆盖均一性产生任何影响，从而节约杂交捕获成本。
因此，在第一个方面，本发明提供一种构建捕获文库的方法，包括以下步骤：
(1)获得片段化的DNA；
(2)将片段化的DNA与Y型接头连接，获得预文库；
(3)在不存在封闭序列的情况下将预文库与杂交探针进行杂交，获得杂交产物；
(4)将杂交产物进行PCR扩增，获得捕获文库。
在一个实施方案中，所述片段化的DNA是指天然存在的短片段DNA或通过人工打断基因组DNA获得的短片段DNA。在一个实施方案中，片段化的DNA可以源自血液、血清、血浆、关节液、精液、尿液、汗液、唾液、粪便、脑脊液、腹水、胸水、胆汁或胰腺液等。在一个优选的实施方案中，天然短片段DNA是外周血游离DNA、肿瘤游离DNA或自然降解的基因组DNA。在另一个实施方案中，基因组DNA可以有各种来源，例如来自外周血、干血斑、口腔拭子等。本领域技术人员知晓打断基因组DNA的方法，例如通过超声处 理、机械打断或通过酶切等。由于超声处理和机械打断相对而言会损失较多的DNA，因此在起始DNA含量较少(例如，低至50ng)的情况下，优选用酶切的方法使DNA片段化。
在一个实施方案中，所述片段化的DNA长度为150-400bp，优选180-230bp。
在一个实施方案中，本发明的方法还包括在与Y型接头连接(即，步骤2)之前，将片段化的DNA进行末端修复和/或末端加A的步骤。在该实施方案中，可以用本领域技术人员已知的任何适用于末端修复的酶对DNA进行末端修复，例如T4DNA聚合酶、Klenow酶及其混合物。在该实施方案中，可以用本领域技术人员已知的任何适用于末端加A的酶对DNA进行末端加A。这种酶的实例包括但不限于Taq酶、klenow ex-酶及其混合物。在该实施方案中，末端修复和末端加A可以在两个反应体系中进行，即，在末端修复后，经过纯化再进行末端加A。可替换地且为优选地，末端修复和末端加A在一个反应体系中进行，即，末端修复和末端加A同时完成，之后再对核酸进行纯化。或者，更加优选地，将DNA片段化、末端修复和末端加A三者在一个反应体系中进行，之后再连接接头。这样不仅简化了操作步骤、节约成本，同时也降低了样本间的污染。
在一个实施方案中，末端补平和末端加A所用的温育时间和温度可以根据具体需要由本领域技术人员根据常规技术确定。
在一个实施方案中，步骤(2)可以用本领域技术人员已知的任何适用于连接接头的酶进行。这种酶的实例包括但不限于T4 DNA连接酶、T7 DNA连接酶或它们的混合物。进行连接反应的条件是本领域技术人员熟知的。
在本发明的上下文中，“Y型接头”是指不完全互补的两条链形成的接头，所述接头的一端由于两条链的碱基互补形成双链，而另一端由于两条链的碱基之间不完全互补，没有形成双链。目前常用的Y型接头主要包括长Y型接头(图3a)和截短Y型接头(图3b)。如图3a所示，常规的长Y型接头主要包括扩增引物序列 (P5/P7)、index标签序列、read 1/read 2测序引物序列和index read测序引物序列，其中read 1/read 2测序引物序列和index read测序引物的序列不完全互补形成一部分双链。如图3b所示，常规的截短Y型接头主要包括read 1/read 2测序引物序列和index测序引物序列，或部分read 1/read 2测序引物序列和部分index测序引物序列，其中read 1/read 2测序引物和index read测序引物的序列不完全互补形成一部分双链。这种截短Y型接头通常需要搭配另外的包含P5/P7引物和index标签序列的接头一起使用。
举例而言，可用于本发明的Y型接头包含的两条链的序列如下：
SEQ ID NO：1
5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA CGCTCTTCCGATCT-3’
SEQ ID NO：2(5’端具有磷酸化修饰)
5’- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC{index}ATCTCGTATGCCGTCTTCTGCTTG-3’
其中，下划线示出了两条链中碱基互补的部分。
对寡核苷酸进行磷酸化修饰的方法是本领域技术人员熟知的。例如，可以通过多核苷酸激酶使寡核苷酸5’端磷酸化，或在合成引物时直接在5’端加上磷酸基团。
在一个实施方案中，本发明方法的步骤(3)在液相杂交系统中进行。
在本发明的上下文中，“封闭序列”是指用于封闭接头和标签序列的序列，包括设计为与接头和/或标签序列互补的序列。在一些实施方案中，为增加封闭序列的封闭效果，在封闭序列的末端添加特殊修饰，如反向dT修饰、氨基修饰、ddNTP修饰(包括ddCTP、ddATP、ddGTP和ddTTP)、间臂(spacer)修饰、次黄嘌呤修饰、随机碱基修饰等。
在传统的捕获文库构建中，往往在连接接头和标签序列后进行PCR扩增，以放大目标DNA的含量，从而保证后续杂交步骤的效率， 满足上机测序的要求。为了减少特异性结合，提高中靶率，通常需要在杂交体系中加入封闭序列，其作用在于通过碱基互补来封闭被扩增的接头和标签序列，使它们在杂交过程中不干扰目标序列与杂交探针的结合。然而，由于封闭序列与接头和标签序列是碱基互补的，因此在杂交过程中它们不仅能与接头和标签序列结合，其彼此之间也能互相结合。这种封闭序列之间的结合会导致封闭效果不够理想，进而降低捕获效率。此外，鉴于多个样品同时测序时会需要多种标签序列(有时多达96种)，而针对每种标签序列都需要单独设计封闭序列，这会加大后续测序数据分析的难度，并且增加实验成本。
出乎意料地，本发明人发现，在无需PCR预扩增制备预文库并且采用Y型接头的情况下，杂交体系中不需要加入任何封闭序列也能实现较好的捕获效率。
因此，在一个实施方案中，用于杂交的体系包括杂交缓冲液、Cot-1DNA和杂交探针，但不包括封闭序列。本领域技术人员根据实际需要可以调节杂交的条件，例如杂交温度、杂交时间等。设计和制备杂交探针的一般原理也是本领域技术人员熟知的。
进行步骤(3)PCR扩增的方法
在第二个方面，本发明提供一种用于构建捕获文库的试剂盒，其包括：
(1)用于连接接头的试剂，包括Y型接头；
(2)用于杂交的试剂，不包括封闭序列；
(3)用于PCR扩增的试剂。
在一个实施方案中，用于杂交的试剂包括杂交缓冲液、Cot-1 DNA和杂交探针，但不包括封闭序列。
在一个实施方案中，用于PCR扩增的试剂包括缓冲液、PCR聚合酶和扩增引物。
在一个实施方案中，根据本发明方法制备的捕获文库可用于多种二代测序平台，包括但不限于如Roche/454 FLX、Illumina/Hiseq、 Miseq、NextSeq和Life Technologies/SOLID system、PGM、Proton等测序平台。
本发明的优异技术效果在于：(1)起始DNA的含量要求较低，甚至可以低至5ng，这大大提高了罕见样本的利用率，扩展了本发明的应用范围，例如本发明的方法和试剂盒可以应用于干血斑、口腔拭子、cfDNA等因DNA提取量少而无法满足普通外显子捕获流程的样本类型；(2)建库流程简单，本发明的方法在获得预文库之前无需经过PCR反应，因此仅需约2小时即可完成预文库构建，而传统的捕获文库构建方法中构建预文库则需要大约6小时，；(3)由于本发明的方法在杂交体系中不包括封闭序列，因此能够在保证捕获效率和覆盖度不受影响的前提下大大节约建库成本。
下面将参考附图并结合实例来详细说明本发明。需要说明的是，本领域的技术人员应该理解本发明的附图及其实施例仅仅是为了例举的目的，并不能对本发明构成任何限制。在不矛盾的情况下，本申请中的实施例及实施例中的特征可以相互组合。
附图说明
图1：常规的捕获文库构建方法的流程示意图。
图2：本发明的捕获文库构建方法的一个实施例的流程示意图。
图3a和3b：Y型接头结构示意图。
图4：封闭序列的结构示意图。
具体实施方式
实施例1：根据本发明的方法构建捕获文库
步骤1.获得片段化的DNA、末端修复和末端加A
根据制造商的说明，用5X WGS Fragmentation Mix试剂盒(Enzymatics，货号Y9410L)制备如表1所示的反应体系以一步完成片段化、末端修复和末端加A，并将该反应体系按以下程序进行反应：4℃,1分钟；32℃，16分钟；65℃，30分钟，然后保持在4 ℃。
表1
Figure PCTCN2020104351-appb-000002
步骤2.连接接头
(1)制备接头
合成如SEQ ID NO：1和SEQ ID NO：2所示的序列，其中SEQ ID NO：2的5’端具有磷酸化修饰。
SEQ ID NO：1
5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’
SEQ ID NO：2
5’-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC{index}ATCTCGTATGCCGTCTTCTGCTTG-3’
将SEQ ID NO：1和SEQ ID NO：2所示的序列在以下条件下退火，以形成长Y型接头：95℃，2min；95℃，2min，以0.1℃/s的速率降温到90℃，并保持2min；以0.1℃/s的速率降温到85℃，并保持2min；以0.1℃/s的速率降温到80℃，并保持2min；以此类推，直到以0.1℃/s的速率降温到25℃，并保持2min；最后保持在4℃。
(2)连接接头
使用WGS Ligase试剂盒(Enzymatics，货号L6030-WL)，用步骤1的反应体系制备如表2所示的连接体系，并将该连接体系在20℃温育15分钟，然后保持在4℃。
表2
Figure PCTCN2020104351-appb-000003
连接反应结束后，用Beckman Agencourt AMPure XP试剂盒(Beckman，货号A63882)对连接产物进行纯化。
步骤3：捕获杂交
使用xGen Lockdown Reagents试剂盒(IDT，货号1072281)，在步骤2的纯化产物中加入14.5μl杂交试剂(9.5μl xGen 2×杂交缓冲液、3μl xGen杂交缓冲液增强剂和2μl Cot-1DNA)，充分混匀后于室温孵育10分钟。孵育结束后，取12.75μl上清液到新的低吸附的0.2mL离心管中，然后加入4.25μl杂交探针。孵育结束后，充分混匀后瞬离，并运行如下程序：95℃30s；65℃，1分钟，37℃3s，60个循环；65℃16小时；然后保持在65℃。
杂交结束后，根据制造商的说明，用xGen Lockdown Reagents试剂盒(IDT，货号1072281)清洗并纯化杂交产物(即，与目标序列结合的磁珠)
步骤4：PCR扩增
根据制造商的说明，用2×KAPA HiFiHotStartReadyMix试剂盒(KAPA，货号KK2602)，制备如表3所示的扩增体系，并按照以下程序进行PCR：95℃45s；98℃15s，65℃30s，72℃30s，12个循环；72℃1分钟；保持在4℃。
扩增引物的序列如下：
P5引物：5'-AATGATACGGCGACCACCGA-3'(SEQ ID NO：3)；
P7引物：5'-CAAGCAGAAGACGGCATACGA-3'(SEQ ID NO：4)。
表3
Figure PCTCN2020104351-appb-000004
PCR程序完成之后，用Beckman Agencourt AMPure XP试剂盒(Beckman，货号A63882)对进行纯化，即获得最终的捕获文库。
对照实施例1
本实施例的文库构建方法与实施例1基本相同，区别仅在于在步骤3的杂交试剂中还包括2μl封闭序列，所述封闭序列是xGen Universal Blockers-TS Mix(IDT，货号1075475)。
对照实施例2
本实施例的文库构建方法与实施例1相同，区别仅在于在步骤2结束后，对纯化产物进行PCR预扩增制备预文库，并在步骤3的杂交试剂中加入2μl封闭序列。具体地，用2×KAPA HiFiHotStartReadyMix试剂盒(KAPA，货号KK2602)，制备如表4所示的预扩增体系，并按照以下程序进行PCR：95℃45s；98℃15s，65℃30s，72℃30s，7个循环；72℃1分钟；保持在4℃。
预扩增引物的序列如下：
P5引物：5'-AATGATACGGCGACCACCGA-3'(SEQ ID NO：3)；
P7引物：5'-CAAGCAGAAGACGGCATACGA-3'(SEQ ID NO：4)。
表4
Figure PCTCN2020104351-appb-000005
Figure PCTCN2020104351-appb-000006
PCR程序完成之后，用Beckman Agencourt AMPure XP试剂盒(Beckman，货号A63882)对进行纯化，然后进行捕获杂交。步骤3的杂交试剂中加入的封闭序列是xGen Universal Blockers-TS Mix(IDT，货号1075475)。
对照实施例3
本实施例的文库构建方法与对照实施例2相同，区别仅在于在步骤3中，杂交体系中未加入封闭序列。
将以上实施例1和对照实施例1-3制备的捕获文库进行qPCR定量，然后按照测序仪标准操作流程，用Illumina NovaSeq 6000测序平台对文库进行测序(150bp双端测序)，每个样本测得10G数据。测序结果如表5所示。
表5
Figure PCTCN2020104351-appb-000007
从表5可以看出，在没有PCR预扩增的情况下，是否添加封闭序列对最终的捕获效率、覆盖度和比对率没有显著影响，所制备的捕获文库均满足测序的质量要求(实施例1vs对照实施例1)。
此外，比较对照实施例1与对照实施例2的测序结果发现，在添加封闭序列的情况下，捕获效率、覆盖度和比对率没有显著差异，说明PCR预扩增可以省略而不会影响最终文库的质量。而比较实施例1与对照实施例3的测序结果发现，在不添加封闭序列的情况下，PCR预扩增反而会造成捕获效率和20×覆盖度等质控参数显著降 低。
最后，将对照实施例2和对照实施例3进行比较可以发现，在进行PCR预扩增的情况下，不添加封闭序列会导致捕获效率和20×覆盖度等质控参数显著降低。这表明，当连接接头后的DNA通过PCR预扩增形成预文库后，在与杂交探针的杂交过程中必须加入封闭序列，否则将严重影响最终捕获文库的质量，使其无法满足上机测序和后续数据分析的要求。
实施例2：根据本发明的方法构建捕获文库
根据实施例1所述的方法，分别用外周血gDNA、干血斑gDNA、口腔拭子gDNA制备捕获文库。将捕获文库进行qPCR定量，然后按照测序仪标准操作流程，用Illumina NovaSeq 6000测序平台对文库进行测序(150bp双端测序)，每个样本测得10G数据。测序结果如表6所示。
表6
样本类型 捕获效率 4x覆盖度 20x覆盖度 比对率 重复率
外周血gDNA 65.91％ 99.28％ 98.73％ 92.64％ 17.89％
干血斑gDNA 67.56％ 99.33％ 98.73％ 91.25％ 17.98％
口腔拭gDNA 65.08％ 99.29％ 98.75％ 91.32％ 14.58％
由上表可见，本发明的测序文库的构建方法可适用于多种样本类型，尤其是DNA含量较少的外周血、干血斑、口腔拭子等样本。
实施例3：DNA起始量对捕获文库的影响。
根据实施例1所述的方法，用不同起始量的基因组DNA样本构建捕获文库。将捕获文库进行qPCR定量，然后按照测序仪标准操作流程，用Illumina NovaSeq 6000测序平台对文库进行测序(150bp双端测序)，每个样本测得10G数据。测序结果如表7所示。
表7
起始量 捕获效率 4x覆盖度 20x覆盖度 比对率
5ng 66.04％ 99.22％ 97.46％ 91.50％
10ng 66.70％ 99.28％ 98.60％ 91.55％
20ng 64.56％ 99.24％ 98.72％ 92.12％
30ng 64.18％ 99.25％ 98.72％ 92.00％
40ng 64.57％ 99.23％ 98.77％ 92.04％
50ng 67.19％ 99.16％ 98.61％ 92.00％
80ng 67.16％ 99.23％ 98.63％ 91.39％
100ng 66.99％ 99.27％ 98.80％ 91.50％10
200ng 64.34％ 99.27％ 98.74％ 91.98％
由上表可见，在5ng-200ng的范围内，根据本发明方法构建的捕获文库在捕获效率、覆盖度和比对率方面没有显著差异。这表明，根据本发明的方法可以使用起始DNA含量低至5ng的样本，所制备的捕获文库完全满足上机测序和后续数据分析的要求。
需要说明的是，以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。本领域技术人员理解的是，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (24)

  1. 一种构建捕获文库的方法，包括以下步骤：
    (1)获得片段化的DNA；
    (2)将片段化的DNA与Y型接头连接，获得预文库；
    (3)在不存在封闭序列的情况下将预文库与杂交探针进行杂交，获得杂交产物；
    (4)将杂交产物进行PCR扩增，获得捕获文库。
  2. 权利要求1所述的方法，其中所述片段化的DNA是指天然存在的短片段DNA或通过人工打断基因组DNA获得的短片段DNA。
  3. 权利要求2所述的方法，其中天然存在的短片段DNA是外周血游离DNA、肿瘤游离DNA或自然降解的基因组DNA。
  4. 权利要求2所述的方法，其中人工打断基因组DNA通过超声处理、机械打断或通过酶切实现。
  5. 权利要求1所述的方法，其中所述片段化的DNA源自血液、血清、血浆、关节液、精液、尿液、汗液、唾液、粪便、脑脊液、腹水、胸水、胆汁或胰腺液。
  6. 权利要求1所述的方法，其中所述片段化的DNA长度为150-400bp。
  7. 权利要求6所述的方法，其中所述片段化的DNA长度为180-230bp。
  8. 权利要求1所述的方法，还包括在步骤(2)之前，将片段化的DNA进行末端修复和/或末端加A的步骤。
  9. 权利要求8所述的方法，其中所述末端修复和末端加A在一个反应体系中进行。
  10. 权利要求8所述的方法，其中将DNA片段化、末端修复和末端加A在一个反应体系中进行。
  11. 权利要求1所述的方法，其中所述Y型接头是长Y型接头 或截短Y型接头。
  12. 权利要求11所述的方法，其中所述长Y型接头包括扩增引物、index标签序列、read 1/read 2测序引物和index read测序引物。
  13. 权利要求11所述的方法，其中所述截短Y型接头包括read1/read 2测序引物和index测序引物，或部分read 1/read 2测序引物和部分index测序引物。
  14. 权利要求1所述的方法，其中所述封闭序列包括设计为与接头和/或标签序列反向互补的序列。
  15. 权利要求1所述的方法，其中步骤(3)在液相系统中进行。
  16. 一种用于构建捕获文库的试剂盒，其包括：
    (1)用于连接接头的试剂，包括Y型接头；
    (2)用于杂交的试剂，不包括封闭序列；
    (3)用于PCR扩增的试剂。
  17. 权利要求16所述的试剂盒，其中所述Y型接头是长Y型接头或截短Y型接头。
  18. 权利要求17所述的试剂盒，其中所述长Y型接头包括扩增引物序列、index标签序列、read 1/read 2测序引物序列和index read测序引物序列。
  19. 权利要求17所述的试剂盒，其中所述截短Y型接头包括read1/read 2测序引物序列和index测序引物序列，或部分read 1/read 2测序引物序列和部分index测序引物序列。
  20. 权利要求16所述的试剂盒，还包括用于进行末端修复和/或末端加A的试剂。
  21. 权利要求16所述的试剂盒，其中所述用于杂交的试剂包括杂交缓冲液、Cot-1DNA和杂交探针，但不包括封闭序列。
  22. 权利要求16所述的试剂盒，其中所述封闭序列包括设计为与接头和/或标签序列反向互补的序列。
  23. 权利要求16所述的试剂盒，其中所述用于PCR扩增的试剂包括缓冲液、PCR聚合酶和扩增引物。
  24. 根据权利要求1-15任一项构建的捕获文库，或用权利要求16-23任一项的试剂盒构建的捕获文库，其中所述捕获文库用于二代测序平台。
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