WO2020258084A1 - 巢式多重pcr高通量测序文库制备方法及试剂盒 - Google Patents

巢式多重pcr高通量测序文库制备方法及试剂盒 Download PDF

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WO2020258084A1
WO2020258084A1 PCT/CN2019/093066 CN2019093066W WO2020258084A1 WO 2020258084 A1 WO2020258084 A1 WO 2020258084A1 CN 2019093066 W CN2019093066 W CN 2019093066W WO 2020258084 A1 WO2020258084 A1 WO 2020258084A1
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primer
sequence
tag
nested
universal
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PCT/CN2019/093066
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French (fr)
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WO2020258084A9 (zh
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杨林
张艳艳
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 PCT/CN2019/093066 priority Critical patent/WO2020258084A1/zh
Priority to JP2021574288A priority patent/JP7281565B2/ja
Priority to US17/617,854 priority patent/US20220235414A1/en
Priority to EP19935242.8A priority patent/EP3992303A4/en
Priority to CA3140403A priority patent/CA3140403A1/en
Priority to AU2019453690A priority patent/AU2019453690B2/en
Priority to CN201980095420.3A priority patent/CN113811620B/zh
Priority to KR1020217042321A priority patent/KR20220011725A/ko
Publication of WO2020258084A1 publication Critical patent/WO2020258084A1/zh
Publication of WO2020258084A9 publication Critical patent/WO2020258084A9/zh

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    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the invention relates to the technical field of library preparation, in particular to a method and kit for preparing a nested multiple PCR high-throughput sequencing library.
  • the target area capture technology can be roughly divided into two types: one is the capture sequencing technology based on hybridization, and the other is the capture technology based on multiplex PCR.
  • the two use multiple probes or primers to capture the gene region of interest at one time, combined with high-throughput sequencing technology, multiple samples are sequenced at the same time, to obtain the sequence information of the target region.
  • target capture sequencing technology greatly reduces costs while screening large sample sizes.
  • the former has cumbersome experimental procedures and high probe costs, which limits its clinical application.
  • the latter has simple experimental operations and strong flexibility, and is suitable for the screening and diagnosis of Mendelian genetic diseases, and GWAS candidate segments. Sequencing, QTL localization segment resequencing, precision medicine research and application, etc.
  • the high-throughput SNP detection service combines multiplex PCR and high-throughput sequencing technologies to design specific primers for the sites to be detected, and perform multiplex PCR amplification in a single tube. Different samples are distinguished by different barcode primers. After the samples are mixed, the amplicons are sequenced on the sequencing platform, and the sequencing results use bioinformatics methods to distinguish different samples, and finally obtain SNP information for each site.
  • This method is suitable for genetic research for different purposes, such as disease genome research, tumor genome research, disease and gene association research, clinical molecular diagnosis, etc. In plant genome research, it can be used for QTL mapping and molecular breeding, and is very suitable for large-scale research. SNP analysis of the sample.
  • the multiplex PCR experiment is simple to operate and the cost of a single detection is very low, it needs to repeatedly test and optimize multiple pairs of primers in the early stage of the experiment, which is time-consuming and labor-intensive.
  • the complexity of primer sequences makes it easy for primers to form primer dimers.
  • the formation of primer dimers will drastically consume the raw materials in the PCR reaction system, causing PCR to quickly reach a plateau; the formed primer dimers will also be sequenced in subsequent sequencing, resulting in invalid data and affecting data utilization efficiency.
  • primers that easily form primer dimers which will seriously affect the amplification efficiency of the target amplification region corresponding to the primer, resulting in low sequencing depth of the target, and ultimately affecting the uniformity of the entire amplification system.
  • specificity of primers greatly affects the performance of multiplex amplification.
  • primer-dimers In multiple amplification, as the number of primer pairs increases, the formation of primer-dimers is inevitable. Many companies use additional enzyme treatments to eliminate excess primer-dimers. For example, Ampliseq uses one-step specific amplification first. Then, the specific primer sequence in the primer dimer and the amplicon is digested by enzyme, and the library is prepared for the product finally, the whole process is cumbersome. Later, Paragon Company improved this method and invented the Clean Plex dimer elimination technology. It uses specific enzymes to eliminate only the primer dimers in the multiplex PCR process, and then uses universal primers to universally amplify the digested products. There is no better solution for non-specific amplification of primers.
  • the shortcomings of the prior art are as follows: (1) The existing method itself cannot reduce the formation of primer dimers. The method is to use enzymatic digestion after the primer dimer is produced, and remove the primers afterwards. In the dimer mode, the primer dimer produced in the multiplex PCR process will greatly affect the efficiency and uniformity of PCR amplification; (2) The existing method requires an additional digestion step, which is cumbersome; (3) Adopt In conventional two-primer amplification, it is difficult to design specific primers for some regions with poor specificity; (4) Existing methods need to separately perform amplification of continuous regions in multiple PCR tubes.
  • the present application provides a method and kit for preparing a nested multiplex PCR high-throughput sequencing library, which effectively avoids dimers generated in the multiplex PCR amplification process, and adopts a nested amplification method to greatly increase the specificity of primers and improve data
  • the utilization rate can also effectively inhibit mutual amplification between overlapping amplicon primers.
  • an embodiment provides a method for preparing a nested multiplex PCR high-throughput sequencing library, the method comprising:
  • the first round of PCR amplification use forward primers and reverse primers to amplify the target region, wherein the forward primer is a forward specific sequence that binds to the target region, and the reverse primer includes the 3'end and the above The reverse specific sequence bound to the target region and the first universal sequencing sequence at the 5'end;
  • Purification of the amplified product Purify the product of the first round of PCR amplification
  • the second round of PCR amplification Amplify the purified product using nested primers, first tag primers and second universal primers, where the nested primers are located downstream of the forward primers, and the nested primers include 5' The second universal sequencing sequence at the end and the specific sequence at the 3′ end, the 3′ end sequence of the first tag primer and the first universal sequencing sequence at the 5′ end of the reverse primer are partially or completely the same, and the first tag primer further includes The first tag sequence, the 3'end sequence of the second universal primer, and the second universal sequencing sequence at the 5'end of the nested primer are partially or completely identical.
  • the second universal primer is a second tag primer
  • the 3'end sequence of the second tag primer is partially or the same as the second universal sequencing sequence at the 5'end of the nested primer
  • the second tag also includes a second tag sequence.
  • another embodiment provides a method for preparing a nested multiplex PCR high-throughput sequencing library, the method comprising:
  • the first round of PCR amplification use forward primer, reverse primer and first tag primer to amplify a target region, wherein the forward primer is a forward specific sequence that binds to the target region, and the reverse primer includes The 3'end of the reverse specific sequence that binds to the target region and the 5'end of the first universal sequencing sequence, the 3'end sequence of the first tag primer and the 5'end of the reverse primer part or all of the first universal sequencing sequence Same, and the above-mentioned first tag primer also includes a first tag sequence;
  • Purification of the amplified product Purify the product of the first round of PCR amplification
  • the second round of PCR amplification Amplify the purified product using a nested primer, a first universal primer and a second universal primer.
  • the nested primer is located downstream of the forward primer, and the nested primer includes 5' The second universal sequencing sequence and the specific sequence of the 3'end, the 3'end sequence of the first universal primer and the 5'end of the first tag primer are partially or completely the same, and the 3'end sequence of the second universal primer is the same as the nest Part or all of the second universal sequencing sequence at the 5'end of the formula primer is identical.
  • the second universal primer is a second tag primer
  • the 3'end sequence of the second tag primer is partially or the same as the second universal sequencing sequence at the 5'end of the nested primer
  • the second tag also includes a second tag sequence.
  • the above reverse primer further includes a molecular tag sequence.
  • the aforementioned molecular tag sequence is a random sequence of 8-24 bp.
  • the above method amplifies multiple consecutive target regions, and each target region has a corresponding forward primer, reverse primer, and nested primer, and an amplification is formed between adjacent amplicon regions. Increase overlap.
  • the above-mentioned forward primer, reverse primer and nested primer are all primer pools composed of multiple primers respectively targeting different target regions.
  • the forward primer of each target region and the forward primer, nested primer of the adjacent region and the nested primer, reverse primer of the adjacent region and the reverse primer of the adjacent region have opposite amplification directions. .
  • the number of cycles of the first round of PCR amplification and/or the second round of PCR amplification is 2-30 cycles.
  • the length of the first tag sequence and/or the second tag sequence is 8-15 bp.
  • an embodiment provides a nested multiplex PCR high-throughput sequencing library preparation kit, which includes:
  • the first round of PCR amplification primers which include a forward primer and a reverse primer, wherein the forward primer is a forward specific sequence that binds to the target region, and the reverse primer includes a 3'end that binds to the target region.
  • the above-mentioned first round of PCR amplification primers are used for the first round of PCR amplification of the target region;
  • the second round of PCR amplification primers which include a nested primer, a first tag primer and a second universal primer, wherein the nested primer is located downstream of the forward primer, and the nested primer includes a second universal sequencing sequence at the 5'end And the specific sequence at the 3'end, the 3'end sequence of the first tag primer and the first universal sequencing sequence at the 5'end of the reverse primer are partially or completely the same, and the first tag primer also includes a first tag sequence, The 3'end sequence of the second universal primer is partly or completely the same as the second universal sequencing sequence at the 5'end of the nested primer.
  • the second round of PCR amplification primers are used for the purified products of the first round of PCR amplification. Perform the second round of PCR amplification.
  • the second universal primer is a second tag primer
  • the 3'end sequence of the second tag primer is partially or the same as the second universal sequencing sequence at the 5'end of the nested primer
  • the second tag also includes a second tag sequence.
  • another embodiment provides a nested multiplex PCR high-throughput sequencing library preparation kit, which includes:
  • the first round of PCR amplification primers which include a forward primer, a reverse primer and a first label primer, wherein the forward primer is a forward specific sequence that binds to the target region, and the reverse primer includes a 3'end and The reverse specific sequence bound to the target region and the first universal sequencing sequence at the 5'end, the 3'end sequence of the first tag primer and the first universal sequencing sequence at the 5'end of the reverse primer are partially or completely identical, and
  • the first tag primer also includes a first tag sequence, and the above-mentioned first round of PCR amplification primers are used to perform the first round of PCR amplification of the target region;
  • the second round of PCR amplification primers which include a nested primer, a first universal primer and a second universal primer, wherein the nested primer is located downstream of the forward primer, and the nested primer includes a second universal sequencing sequence at the 5'end And the specific sequence of the 3'end, the 3'end sequence of the first universal primer and the 5'end of the first tag primer are partially or completely the same, the 3'end sequence of the second universal primer and the 5'end of the nested primer
  • the two universal sequencing sequences are partly or completely the same, and the second round of PCR amplification primers are used to perform the second round of PCR amplification on the purified product of the first round of PCR amplification.
  • the second universal primer is a second tag primer
  • the 3'end sequence of the second tag primer is partially or the same as the second universal sequencing sequence at the 5'end of the nested primer
  • the second tag also includes a second tag sequence.
  • the above reverse primer further includes a molecular tag sequence.
  • the aforementioned molecular tag sequence is a random sequence of 8-24 bp.
  • the above-mentioned forward primer, reverse primer and nested primer are all primer pools composed of a plurality of primers respectively targeting a plurality of consecutive target regions, and an expansion is formed between adjacent amplicon regions. Increase overlap.
  • the forward primer of each target region and the forward primer, nested primer of the adjacent region and the nested primer, reverse primer of the adjacent region and the reverse primer of the adjacent region have opposite amplification directions. .
  • the length of the first tag sequence and/or the second tag sequence is 8-15 bp.
  • the method and kit for preparing a nested multiplex PCR high-throughput sequencing library of the present invention can effectively reduce the primer dimers generated in the multiplex PCR amplification process, the amplification mode of three primers, and effectively increase the specificity of the product.
  • the preferred embodiment can effectively suppress invalid amplified fragments generated during continuous region amplification; using molecular tags to label specific primers can uniquely label the original template, and can trace and correct the original template.
  • Figure 1 is a schematic diagram of a two-step PCR amplification process in the prior art
  • FIG. 2 is a schematic diagram of a nested multiplex PCR amplification process in an embodiment of the present invention
  • Fig. 3 is a schematic diagram of continuous region amplification of nested multiplex PCR in an embodiment of the present invention
  • Figure 4 is a schematic diagram of conventional nested PCR primer design in an embodiment of the present invention (a), a schematic diagram of nested PCR primer design with molecular tags introduced (b), and a schematic diagram of continuous region nested PCR primer design (c);
  • Figure 5 is a schematic diagram of different sample label introduction methods in an embodiment of the present invention.
  • Figure 6 is a schematic diagram of different sample label introduction methods in an embodiment of the present invention.
  • FIG. 7 is a diagram showing the results of experiments of uniformity of different amplicons in an embodiment of the invention.
  • Fig. 8 is a library electrophoresis diagram obtained by nested multiplex PCR in an embodiment of the present invention.
  • the two-step PCR amplification process in the prior art shows that in the process of specific amplification, two specific primers (specific primer 1 and specific primer 2 in FIG. 1) respectively carry two The partial sequence of a sequencing adapter. These two primers form a primer dimer in the first round of PCR amplification. This primer dimer will be amplified in the second round of PCR universal amplification to obtain a large amount of The dimer product affects the sequencing data.
  • the method of the present invention solves the problem that the primer dimer product produced in the specific PCR amplification process is amplified in the subsequent general PCR amplification process.
  • one specific primer (hereinafter referred to as reverse primer) carries the first universal sequencing sequence
  • the other specific primer (hereinafter referred to as forward primer) does not With sequencing sequence.
  • Reverse primers and reverse primers form primer dimers (primer dimer 3 in Figure 2). Due to the existence of universal sequencing sequences, such primers will form a neck loop structure during subsequent amplification without being amplified Increase the amplification, the primer dimer formed between the forward primer and the forward primer, the forward primer and the reverse primer (primer dimer 1 and primer dimer 2 in Figure 2) cannot be used by the subsequent universal primers. Amplification can effectively reduce the ratio of primer-dimer in the final product in the first round of PCR amplification.
  • the use of semi-nested amplification can improve the specificity of target region amplification.
  • a nested primer is designed downstream of the forward primer, and the nested primer has a second universal sequencing sequence. Use this primer and the first tag primer to perform the first step on the product obtained.
  • Two rounds of PCR amplification the final sequencing library is obtained.
  • This method adopts nested amplification, and a step of specific selection is performed on the basis of the first round of PCR amplification products to improve the specificity of the final product.
  • This step includes adding a nested primer and a second universal primer for PCR amplification.
  • primer dimer 4 in Figure 2
  • the primer-dimer of the following amplification will form a neck loop structure, but will not be amplified, so it can significantly inhibit the formation of primer-dimer.
  • each region consists of three primers, forward primer, nested primer, and reverse primer.
  • the design of the forward primer and the next adjacent forward primer forms an amplification overlap
  • the nested primer and the next An amplification overlap is formed between adjacent nested primers
  • an amplification overlap is formed between the reverse primer and the next reverse primer ( Figure 4).
  • the forward primer and the forward primer will produce a product (product 4 in Figure 3), which will be used by the nested primer in the second step of nested amplification for amplification, but because the nested primers all have a second universal
  • the sequencing sequence will form a neck loop structure during subsequent amplification and will be inhibited; the reverse primer and reverse primer will produce products (product 2 in Figure 3). Since the reverse primers all carry the first universal sequencing sequence, In the subsequent amplification, the product will form a neck-loop structure to inhibit amplification ( Figure 3), so these overlapping products will not be amplified.
  • the nested primer is located at the downstream primer of the forward primer.
  • the two can be overlapped or separated.
  • the 5'end of the nested primer has a universal sequence for sequencing, the 3'end is a specific sequence, and the 3'end sequence is designed in the target region to be amplified Upstream;
  • the forward primer is designed upstream of the nested primer, and its 5'end can be added or not (preferably not) a general sequencing sequence;
  • the 5'end of the reverse primer has a general sequencing sequence, and the 3'end is a specific sequence
  • the 3'end sequence is designed downstream of the target region to be amplified.
  • the molecular tag can be a random sequence of 8-24 bp, which can generate 4 8 -24 kinds of random tag molecules, used to tag the original DNA molecules.
  • each template will be equipped with a unique molecular index (Unique molecular index), through which the original DNA template can be traced back to remove duplicate amplification And sequencing errors.
  • the introduction of molecular tags to primers will dramatically increase the possibility of dimer formation between primers, but the primer dimers formed by our method will be effectively inhibited in the subsequent process.
  • each region is amplified by three primers, forward primer, nested primer, and reverse primer.
  • the forward primer of each region and the forward primer of the adjacent region, the nested primer and the nested primer of the adjacent region, the reverse primer and the reverse primer of the adjacent region are designed in opposite directions (the amplification direction is opposite).
  • the first amplicon from 5'to 3'is forward primer 1, nested primer 1, and reverse primer 1 then the second amplicon is reverse from 5'to 3' Primer 2, nested primer 2, and forward primer 2, the third amplicon from 5'to 3'is forward primer 3, nested primer 3, and direction primer 3, and so on, until all amplicons It can cover the entire continuous region.
  • the 3'end of reverse primer 2 of the second amplicon is upstream of the 3'end of reverse primer 1, and the 3'end of nested primer 3 of the third amplicon is nested. Upstream of the 3'end of primer 2, all the sequences in the continuous region can be amplified into the corresponding amplicon by this design.
  • forward primers and reverse primers are used to perform 2-30 rounds of PCR amplification.
  • the obtained product is purified, excess primers and other ions are removed, and then the second round of PCR amplification is performed.
  • the nested primer, the first tag primer and the second universal primer are used for amplification for 2-30 cycles, where the 3'end sequence of the first tag primer and the 5'end of the reverse primer are either All sequences are the same.
  • the tag sequence has different fixed sequences on different sequencing platforms, which are used to distinguish different samples for subsequent mixed sequencing of multiple samples; the 3'end sequence of the second universal primer and Part or all of the sequence at the 5'end of the nested primer is the same.
  • the nested primer and the first tag primer are amplified.
  • the nested primer, the first tag primer and the second universal primer are used together to amplify the target library, and finally the amplification is obtained. Additive library.
  • nested primers, first tag primers, and second tag primers are used in the second round of PCR amplification for 2-30 cycles, where the 3'end of the first tag primer The sequence is the same as part or all of the 5'end of the reverse primer.
  • the 3'end of the second tag primer is the same as the 5'end of the nested primer.
  • the tag sequence There are different fixed sequences on different sequencing platforms, which are used to distinguish different samples for subsequent mixed sequencing of multiple samples.
  • the nested primer and the first tag primer are amplified.
  • the nested primer, the first tag primer and the second tag primer are used together to amplify the target library, and finally a double Amplicon library of sample tags.
  • the first tag primer has a tag sequence of 8-15 nt.
  • the first round of PCR Amplification can distinguish samples for different samples for subsequent mixed sequencing of multiple samples.
  • the 3'end of the first tag primer and the 5'end of the reverse primer partially or completely overlap.
  • the forward primer and reverse primer are amplified, and in the subsequent cycles, the forward primer, reverse primer and the first tag primer are amplified together.
  • the second round of PCR amplification the nested primer, the first universal primer and the second universal primer are used for amplification for 2-30 cycles.
  • the 3'end of the first universal primer and the 5'end of the first tag primer are either All are the same.
  • the first universal primer and the nested primer are amplified first, and in the subsequent cycles, the first universal primer, the second universal primer and the nested primer are amplified together to obtain Sequencing library.
  • nested primers, second tag primers and first universal primers are used in the second round of PCR amplification for 2-30 cycles, where the 3'end of the second tag
  • the sequence is the same as part or all of the 5'end of the nested primer.
  • the tag sequence has different fixed sequences on different sequencing platforms, which are used to distinguish different samples for subsequent multi-sample mixed sequencing ;
  • the 3'end sequence of the first universal primer is the same as part or all of the 5'end sequence of the first tag primer.
  • the nested primer and the first universal primer are amplified.
  • the nested primer, the second tag primer and the first universal primer are used together to amplify the target library, and finally a double Amplicon library of sample tags.
  • Primer design Design nested multiplex PCR primers for tumor drug-related sites. Each amplicon consists of forward primer, nested primer and reverse primer. A 15nt random sequence is introduced into the reverse primer for template For traceability and error correction, these primers are used to prepare a multiplex PCR library of the standard HD701 (horizon company) and complete high-throughput sequencing. The data obtained is analyzed and then the mutation at each target site is detected.
  • the first round of PCR amplification uses QIAGEN Multiplex PCR Kit (Cat No./ID: 206143) for PCR amplification.
  • the forward primer pool is formed by mixing the above primers at an equimolar ratio of 10 ⁇ M, and Y is a degenerate base.
  • the reverse primer pool is formed by mixing the above primers at 10 ⁇ M equimolar.
  • XP magnetic beads 80 ⁇ L of XP magnetic beads (beads) were added to the obtained PCR product for purification (Agencourt AMPure XP magnetic beads from Beckman, catalog number A63881), and the obtained product was dissolved in 20 ⁇ L TE solution.
  • the second round of PCR amplification uses QIAGEN Multiplex PCR Kit (Cat No./ID: 206143) for PCR amplification.
  • #Nested primer pool is shown in Table 6.
  • the nested primer pool is formed by mixing the above primers at 10 ⁇ M equimolar.
  • the second universal primer is: /5Phos/#GAACGACATGGCTACGATCCGACTT (SEQ ID NO: 41).
  • the 5'end of the primer is phosphorylated and used for the circularization of the MGI platform.
  • XP magnetic beads 80 ⁇ L of XP magnetic beads (beads) were added to the obtained PCR product for purification (Agencourt AMPure XP magnetic beads from Beckman, catalog number A63881), and the obtained product was dissolved in 20 ⁇ L TE solution.
  • the analysis step includes basic steps such as filtering the adapter primer sequence and comparison.
  • the basic information obtained is shown in Table 8, and then GATK is used to detect mutations at the target site (Table 9).

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Abstract

一种巢式多重PCR高通量测序文库制备方法及试剂盒，巢式多重PCR高通量测序文库制备方法包括：使用正向引物和反向引物对目标区域进行扩增，正向引物是正向特异性序列，反向引物包括3'端的反向特异性序列和5'端的第一通用测序序列；对第一轮PCR扩增的产物进行纯化；使用巢式引物、第一标签引物和第二通用引物对纯化后的产物进行扩增，其中巢式引物位于正向引物的下游，巢式引物包括5'端的第二通用测序序列和3'端的特异性序列，第一标签引物3'端序列和反向引物5'端的部分或全部序列相同，且第一标签引物还包括第一标签序列，第二通用引物3'端序列和巢式引物5'端的部分或者全部序列相同。巢式多重PCR高通量测序文库制备方法能够有效避免多重PCR扩增过程中产生的二聚体，并且采用巢式扩增方法大大增加PCR扩增的特异性。

Description

巢式多重PCR高通量测序文库制备方法及试剂盒 技术领域
本发明涉及文库制备技术领域，具体涉及一种巢式多重PCR高通量测序文库制备方法及试剂盒。
背景技术
随着测序技术的发展，对基因组候选区段的重测序需求日益增加，人们对序列的关注超过了少数的SNP，候选区段的范围可能在5kb-10M之间。使用传统sanger法或目标区域杂交捕获测序价格昂贵，使用目标区域捕获测序很好地解决了这一难题。目标区域捕获技术可大致分为两种：一种是基于杂交的捕获测序技术，另一种是基于多重PCR的捕获技术。两者通过多重探针或者引物对感兴趣的基因区域一次性捕获，结合高通量测序技术，多样本同时测序，得到目标区域的序列信息。相对于全基因组测序，目标捕获测序技术在大样本量筛查的同时极大地降低成本。但前者的实验流程繁琐，探针成本较高，限制了其在临床上的应用，后者实验操作简单，灵活性强，适用于孟德尔遗传性疾病的筛查和诊断、GWAS候选区段重测序、QTL定位区段重测序、精准医疗研究与应用等领域。
高通量SNP检测服务结合多重PCR和高通量测序技术，对需要检测的位点设计特异性引物，在单管内进行多重PCR扩增，不同的样本以不同的标签(barcode)引物区分。混合样本后，在测序平台上，对扩增子进行测序，测序结果使用生物信息学方法，区分不同样本，最终获得每个位点的SNP信息。这种方法适用于不同目的遗传学研究，例如疾病基因组研究、肿瘤基因组研究、疾病与基因的关联研究、临床分子诊断等，在植物基因组研究中，可用于QTL定位及分子育种，非常适合大规模样本的SNP分析。
虽然多重PCR实验操作简单、单个检测成本很低，但其在实验前期需要对多对引物进行反复测试优化，费时费力。特别是在超高重的PCR中，引物序列的复杂性使得引物很容易形成引物二聚体。引物二聚体的形成会急剧消耗PCR反应体系中的原料，导致PCR很快达到平台期；形成的引物二聚体在后续的测序中也会被测序，形成无效数据，影响数据的利用效率。最严重的是那些容易形成引物二聚体的引物，会严重影响该引物对应的目标扩增区域的扩增效率，导致该目标测序深度低，最终影响整个扩增体系的均一性。此外，引物的特异性也极大地影响了多重扩增的性能。
在多重扩增中随着引物对数的增加，引物二聚体的形成不可避免，很多公司通过额外的酶处理对过多的引物二聚体进行消除，如Ampliseq公司，先采用一步特异性扩增，然后通过酶消化掉引物二聚体和扩增子中的特异性引物序列，最后对产物进行文库制备，整个过程繁琐。后来Paragon公司改良了这个方法，发明了Clean Plex二聚体消除技术，采用特定的酶只消除多重PCR过程中的引物二聚体，然后采用通用引物对消化后的产物进行通用扩增，但是对于引物的非特异性扩增没有更好的解决方法。
概而言之，现有技术的缺陷体现在：(1)现有方法本身无法减少引物二聚体的形成，其方法是在引物二聚体产生之后采用酶消化的方式进行，采用事后去除引物二聚体的方式，其多重PCR过程中产生的引物二聚体会极大地影响PCR扩增的效率和均一性；(2)现有方法需额外采用一步消化步骤，操作比较繁琐；(3)采用常规的两条引物扩增，对于一些特异性不好的区域很难设计特异的引物；(4)现有方法对于连续区域扩增需要在多个PCR管中分别进行。
发明内容
本申请提供一种巢式多重PCR高通量测序文库制备方法及试剂盒，有效避免多重PCR扩增过程中产生的二聚体，并且采用巢式扩增方法大大增加引物的特异性，提高数据的利用率，还能有效抑制重叠扩增子引物之间的互相扩增。
根据第一方面，一种实施例中提供一种巢式多重PCR高通量测序文库制备方法，该方法包括：
第一轮PCR扩增：使用正向引物和反向引物对目标区域进行扩增，其中上述正向引物是与上述目标区域结合的正向特异性序列，上述反向引物包括3’端的与上述目标区域结合的反向特异性序列和5’端的第一通用测序序列；
扩增产物的纯化：对上述第一轮PCR扩增的产物进行纯化；
第二轮PCR扩增：使用巢式引物、第一标签引物和第二通用引物对纯化后的产物进行扩增，其中上述巢式引物位于上述正向引物的下游，上述巢式引物包括5’端的第二通用测序序列和3’端的特异性序列，上述第一标签引物3’端序列和上述反向引物5’端的第一通用测序序列部分或全部序列相同，且上述第一标签引物还包括第一标签序列，上述第二通用引物3’端序列和上述巢式引物5’端的第二通用测序序列部分或者全部序列相同。
在优选实施例中，上述第二通用引物是第二标签引物，该第二标签引物3’端序 列和上述巢式引物5’端的第二通用测序序列部分或者全部序列相同，且上述第二标签引物还包括第二标签序列。
根据第一方面，另一种实施例中提供一种巢式多重PCR高通量测序文库制备方法，该方法包括：
第一轮PCR扩增：使用正向引物、反向引物和第一标签引物对目标区域进行扩增，其中上述正向引物是与上述目标区域结合的正向特异性序列，上述反向引物包括3’端的与上述目标区域结合的反向特异性序列和5’端的第一通用测序序列，上述第一标签引物3’端序列和上述反向引物5’端的第一通用测序序列部分或全部序列相同，且上述第一标签引物还包括第一标签序列；
扩增产物的纯化：对上述第一轮PCR扩增的产物进行纯化；
第二轮PCR扩增：使用巢式引物、第一通用引物和第二通用引物对纯化后的产物进行扩增，其中上述巢式引物位于上述正向引物的下游，上述巢式引物包括5’端的第二通用测序序列和3’端的特异性序列，上述第一通用引物3’端序列和上述第一标签引物5’端部分或全部序列相同，上述第二通用引物3’端序列和上述巢式引物5’端的第二通用测序序列部分或者全部序列相同。
在优选实施例中，上述第二通用引物是第二标签引物，该第二标签引物3’端序列和上述巢式引物5’端的第二通用测序序列部分或者全部序列相同，且上述第二标签引物还包括第二标签序列。
在优选实施例中，上述反向引物上还包括分子标签序列。
在优选实施例中，上述分子标签序列是8-24bp的随机序列。
在优选实施例中，上述方法对多个连续的目标区域进行扩增，每个目标区域都有对应的正向引物、反向引物和巢式引物，并且相邻扩增子区域之间形成扩增重叠。
在优选实施例中，上述正向引物、反向引物和巢式引物均是由多条分别靶向不同的目标区域的引物组成的引物池。
在优选实施例中，每个目标区域的正向引物和相邻区域的正向引物、巢式引物和相邻区域的巢式引物、反向引物和相邻区域的反向引物扩增方向相反。
在优选实施例中，上述第一轮PCR扩增和/或第二轮PCR扩增的循环数均是2-30个循环。
在优选实施例中，上述第一标签序列和/或第二标签序列的长度均是8-15bp。
根据第二方面，一种实施例中提供一种巢式多重PCR高通量测序文库制备试剂 盒，该试剂盒包括：
第一轮PCR扩增引物，其包括正向引物和反向引物，其中上述正向引物是与目标区域结合的正向特异性序列，上述反向引物包括3’端的与上述目标区域结合的反向特异性序列和5’端的第一通用测序序列，上述第一轮PCR扩增引物用于对目标区域进行第一轮PCR扩增；
第二轮PCR扩增引物，其包括巢式引物、第一标签引物和第二通用引物，其中上述巢式引物位于上述正向引物的下游，上述巢式引物包括5’端的第二通用测序序列和3’端的特异性序列，上述第一标签引物3’端序列和上述反向引物5’端的第一通用测序序列部分或全部序列相同，且上述第一标签引物还包括第一标签序列，上述第二通用引物3’端序列和上述巢式引物5’端的第二通用测序序列部分或者全部序列相同，上述第二轮PCR扩增引物用于对纯化后的上述第一轮PCR扩增的产物进行第二轮PCR扩增。
在优选实施例中，上述第二通用引物是第二标签引物，该第二标签引物3’端序列和上述巢式引物5’端的第二通用测序序列部分或者全部序列相同，且上述第二标签引物还包括第二标签序列。
根据第二方面，另一种实施例中提供一种巢式多重PCR高通量测序文库制备试剂盒，该试剂盒包括：
第一轮PCR扩增引物，其包括正向引物、反向引物和第一标签引物，其中上述正向引物是与上述目标区域结合的正向特异性序列，上述反向引物包括3’端的与上述目标区域结合的反向特异性序列和5’端的第一通用测序序列，上述第一标签引物3’端序列和上述反向引物5’端的第一通用测序序列部分或全部序列相同，且上述第一标签引物还包括第一标签序列，上述第一轮PCR扩增引物用于对目标区域进行第一轮PCR扩增；
第二轮PCR扩增引物，其包括巢式引物、第一通用引物和第二通用引物，其中上述巢式引物位于上述正向引物的下游，上述巢式引物包括5’端的第二通用测序序列和3’端的特异性序列，上述第一通用引物3’端序列和上述第一标签引物5’端部分或全部序列相同，上述第二通用引物3’端序列和上述巢式引物5’端的第二通用测序序列部分或者全部序列相同，上述第二轮PCR扩增引物用于对纯化后的上述第一轮PCR扩增的产物进行第二轮PCR扩增。
在优选实施例中，上述第二通用引物是第二标签引物，该第二标签引物3’端序 列和上述巢式引物5’端的第二通用测序序列部分或者全部序列相同，且上述第二标签引物还包括第二标签序列。
在优选实施例中，上述反向引物上还包括分子标签序列。
在优选实施例中，上述分子标签序列是8-24bp的随机序列。
在优选实施例中，上述正向引物、反向引物和巢式引物均是由多条分别靶向多个连续的目标区域的引物组成的引物池，并且相邻扩增子区域之间形成扩增重叠。
在优选实施例中，每个目标区域的正向引物和相邻区域的正向引物、巢式引物和相邻区域的巢式引物、反向引物和相邻区域的反向引物扩增方向相反。
在优选实施例中，上述第一标签序列和/或第二标签序列的长度均是8-15bp。
本发明的巢式多重PCR高通量测序文库制备方法及试剂盒，能够有效降低多重PCR扩增过程中产生的引物二聚体，三条引物的扩增方式，有效增加产物的特异性。此外，优选实施例能够有效抑制连续区域扩增中产生的无效扩增片段；采用分子标签标记特异性引物，能够对原始模板进行唯一标记，可以对原始模板进行溯源和错误矫正。
附图说明
图1为现有技术中两步PCR扩增流程示意图；
图2为本发明实施例中巢式多重PCR扩增流程示意图；
图3为本发明实施例中巢式多重PCR连续区域扩增示意图；
图4为本发明实施例中常规巢式PCR引物设计示意图(a)，引入分子标签的巢式PCR引物设计示意图(b)，连续区域巢式PCR引物设计示意图(c)；
图5为本发明实施例中不同样本标签引入方式示意图；
图6为本发明实施例中不同样本标签引入方式示意图；
图7为本发明实施例中不同扩增子均一性实验结果图；
图8为本发明实施例中巢式多重PCR得到的文库电泳图。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。在以下的实施方式中，很多细节描述是为了使得本申请能被更好的理解。然而，本领域技术人员可以毫不费力的认识到，其中部分特征在不同情况下是可以省略的，或者可以由其他材料、方法所替代。
另外，说明书中所描述的特点、操作或者特征可以以任意适当的方式结合形成 各种实施方式。同时，方法描述中的各步骤或者动作也可以按照本领域技术人员所能显而易见的方式进行顺序调换或调整。因此，说明书和附图中的各种顺序只是为了清楚描述某一个实施例，并不意味着是必须的顺序，除非另有说明其中某个顺序是必须遵循的。
本文中为特征所编序号本身，例如“第一”、“第二”等，仅用于区分所描述的对象，不具有任何顺序或技术含义。
如图1所示，现有技术中两步PCR扩增流程显示，在特异性扩增的过程中，两条特异性引物(图1中特异性引物1和特异性引物2)分别带上两个测序接头的部分序列，这两种引物在第一轮PCR扩增过程中形成引物二聚体，这种引物二聚体在后续的第二轮PCR通用扩增中会被放大，得到大量的二聚体产物，影响测序数据。
本发明的方法，解决了特异性PCR扩增过程中产生的引物二聚体产物在随后的通用PCR扩增过程中放大的问题。
如图2所示，本发明的方法，在特异性扩增中，一条特异性引物(下称反向引物)带有第一通用测序序列，另一条特异性引物(下称正向引物)不带测序序列。反向引物和反向引物形成引物二聚体(图2中引物二聚体3)，由于通用测序序列的存在，这类引物在后续的扩增中会形成颈环结构，而不会被扩增放大，正向引物和正向引物、正向引物和反向引物之间形成的引物二聚体(图2中引物二聚体1和引物二聚体2)无法被后续的通用引物利用而无法进行扩增，可以有效降低第一轮PCR扩增中引物二聚体在最终产物中的比例。
如图2所示，采用半巢式扩增，能够提高目标区域扩增的特异性。具体而言，在第二轮PCR扩增中，在正向引物下游设计巢式引物，该巢式引物带有第二通用测序序列，用这条引物和第一标签引物对得到的产物进行第二轮PCR扩增，得到最终的测序文库。该方法采用巢式扩增，在第一轮PCR扩增产物的基础上再进行一步特异性选择，提高最终产物的特异性。该步包括加入巢式引物和第二通用引物进行PCR扩增，扩增过程中只有巢式引物之间容易形成引物二聚体(图2中引物二聚体4)，巢式引物之间形成的引物二聚体在后续的扩增中会形成颈环结构，而不会被扩增放大，因此也能明显抑制引物二聚体的形成。
如图3和图4所示，对于连续区域，特殊的引物扩增策略，避免重叠扩增子之间重叠引物互相扩增。具体而言，每个区域有三条引物组成，正向引物、巢式引物、反向引物，设计正向引物和下一个相邻的正向引物之间形成扩增重叠，巢式引物和 下一个相邻的巢式引物之间形成扩增重叠，反向引物和下一个反向引物之间形成扩增重叠(图4)。正向引物和正向引物会产生产物(图3中产物4)，该产物会被第二步巢式扩增中的巢式引物利用，进行扩增，但是由于巢式引物都带有第二通用测序序列，在后续的扩增中会形成颈环结构而被抑制；反向引物和反向引物会产生产物(图3中产物2)，由于反向引物都带有第一通用测序序列，该产物在后续扩增中会形成颈环结构而被抑制扩增(图3)，因此这些重叠的产物都不会被扩增放大。
如图4a所示，对于每一个扩增子，设计三条引物：正向引物、巢式引物、反向引物进行两步PCR扩增。巢式引物位于正向引物的下游引物，两者可以重叠或相隔，巢式引物5’端有一段测序通用序列，3’端为特异性序列，3’端的序列设计在需要扩增的目标区域上游；正向引物设计在巢式引物的上游，其5’端可以加或者不加(优选不加)测序通用序列；反向引物5’端有一段测序通用序列，3’端为特异性序列，3’端的序列设计在需要扩增的目标区域的下游。
如图4b所示，如需要对低频突变进行检测，需要在反向引物中的通用序列和特异序列之间加上分子标签序列，该分子标签可以是8-24bp的随机序列，可以产生4 8-24种随机标签分子，用来标记原始的DNA分子。当分子标签的数量远远大于模板的数量时，每一个模板都会带上一个唯一的分子标签(Unique molecular index)，通过这个分子标签可以对原始的DNA模板进行追溯，用来去掉扩增的重复和测序错误。引入分子标签到引物上会急剧增加引物之间二聚体生成的可能性，但是我们的方法形成的引物二聚体在后续的过程中会被有效抑制。
如图4c所示，对于连续区域的扩增，设计多对引物对连续区域进行覆盖，相邻扩增子区域有重叠(除去扩增子中引物序列外，上一个扩增的3’端在下一个扩增子的5’端的下游)。每一个区域由三条引物扩增，正向引物、巢式引物、反向引物。每个区域的正向引物和相邻区域的正向引物、巢式引物和相邻区域的巢式引物、反向引物和相邻区域的反向引物相向设计(扩增方向相反)。例如，第一个扩增子从5’至3’方向依次为正向引物1、巢式引物1和反向引物1，那么第二个扩增子从5’至3’方向依次为反向引物2、巢式引物2和正向引物2，第三个扩增子从5’至3’方向依次为正向引物3、巢式引物3和方向引物3，以此类推，直到所有扩增子能够覆盖整个连续区域，第二个扩增子的反向引物2的3’端在反向引物1的3’端的上游，第三个扩增子的巢式引物3的3’端在巢式引物2的3’端的上游，通过该种设计将连续区域的所有序列都能够扩增到相应的扩增子中。
如图5a所示，在第一轮PCR扩增中，采用正向引物和反向引物进行2-30轮PCR扩增。得到的产物进行纯化，去掉多余的引物和其他离子，然后进行第二轮PCR扩增。第二轮PCR扩增中用巢式引物、第一标签引物和第二通用引物进行进行扩增2-30个循环，其中第一标签引物的3’端序列和反向引物5’端的部分或全部序列相同，标签引物中间有8-15bp的标签序列，该标签序列在不同测序平台有不同的固定序列，用于区分不同样本以便后续的多样本混合测序；第二通用引物3’端序列和巢式引物5’端的部分或者全部序列相同。在扩增的第一个循环，巢式引物和第一标签引物进行扩增，在后续的循环中巢式引物、第一标签引物和第二通用引物一起对目标文库进行扩增，最终得到扩增子文库。
如图5b所示，对于双样本标签序列测序，第二轮PCR扩增中用巢式引物、第一标签引物和第二标签引物扩增2-30个循环，其中第一标签引物3’端的序列和反向引物5’端的部分或全部序列相同，第二标签引物3’端的序列和巢式引物的5’端部分或者全部序列相同，标签引物中间有8-15bp的标签序列，该标签序列在不同测序平台有不同的固定序列，用于区分不同样本以便后续的多样本混合测序。在扩增的第一个循环，巢式引物和第一标签引物进行扩增，在后续的循环中巢式引物、第一标签引物和第二标签引物一起对目标文库进行扩增，最终得到双样本标签的扩增子文库。
如图6a所示，对于第一轮PCR扩增，除了加正向引物和反向引物，还可以加入第一标签引物，第一标签引物中间有8-15nt的标签序列，在第一轮PCR扩增就可以对样本进行区分，用于区分不同样本以便后续的多样本混合测序，该第一标签引物的3’端和反向引物的5’端部分或者全部重叠。在扩增的第一个循环，正向引物和反向引物进行扩增，在后续的循环正向引物、反向引物和第一标签引物一起进行扩增。第二轮PCR扩增中用巢式引物、第一通用引物和第二通用引物进行进行扩增2-30个循环，第一通用引物的3’端和第一标签引物的5’端部分或者全部相同，在扩增的第一个循环，第一通用引物和巢式引物先进行扩增，在后续的循环中，第一通用引物、第二通用引物和巢式引物一起进行扩增，得到测序文库。
如图6b所示，对于双样本标签序列测序，第二轮PCR扩增中用巢式引物、第二标签引物和第一通用引物扩增2-30个循环，其中第二标签的3’端序列和巢式引物5’端的部分或全部序列相同，标签引物中间有8-15bp的标签序列，该标签序列在不同测序平台有不同的固定序列，用于区分不同样本以便后续的多样本混合测序； 第一通用引物的3’端序列和第一标签引物的5’端部分或者全部序列相同。在扩增的第一个循环，巢式引物和第一通用引物进行扩增，在后续的循环中巢式引物、第二标签引物和第一通用引物一起对目标文库进行扩增，最终得到双样本标签的扩增子文库。
以下通过具体实施例详细说明本发明的技术方案，应当理解，实施例仅是示例性的，不能理解为对本发明保护范围的限制。
实施例1
引物设计：针对肿瘤用药相关位点设计巢式多重PCR引物，每个扩增子由正向引物、巢式引物和反向引物组成，在反向引物上引入15nt的随机序列，用于模板的溯源和错误矫正，采用这些引物对标准品HD701(horizon公司)进行多重PCR文库制备并完成高通量测序，得到的数据进行分析后检测每个目标位点的突变。
1、第一轮PCR扩增，采用QIAGEN Multiplex PCR Kit(货号Cat No./ID：206143)，进行PCR扩增。
在200μL PCR管子配置如下表1所示的试剂体系：
表1
组分 用量
上一步反应物(标准品HD701) 20μL
2X PCR反应酶 25μL
正向引物池(10μM) 2.5μL
反向引物池(10μM) 2.5μL
总量 50μL
#正向引物池、反向引物池如表2和表3所示。
表2正向引物池
Figure PCTCN2019093066-appb-000001
Figure PCTCN2019093066-appb-000002
正向引物池由上述引物10μM等摩尔混合形成，Y为兼并碱基。
表3反向引物池
Figure PCTCN2019093066-appb-000003
Figure PCTCN2019093066-appb-000004
反向引物池由上述引物10μM等摩尔混合形成。
按照以下表4所示的程序进行第一轮PCR扩增：
表4
Figure PCTCN2019093066-appb-000005
得到的PCR产物中加入80μL的XP磁珠(beads)进行纯化(beckman公司Agencourt AMPure XP磁珠，货号A63881)，得到的产物溶解于20μL TE溶液中。
2、第二轮PCR扩增，采用QIAGEN Multiplex PCR Kit(货号Cat No./ID：206143)，进行PCR扩增。
在200μL PCR管子配置如下表5所示的试剂体系：
表5
组分 用量
上一步反应物 20μL
2X PCR反应酶 25μL
巢式引物池 2.5μL
第二通用引物 2.5μL
第一标签引物 2.5μL
总量 50μL
#巢式引物池如表6。
表6巢式引物池
Figure PCTCN2019093066-appb-000006
Figure PCTCN2019093066-appb-000007
巢式引物池由上述引物10μM等摩尔混合形成。
第一标签引物为：
TGTGAGCCAAGGAGTTNNNNNNNNNN #TTGTCTTCCTAAGACCGCTTGGCCTCCGACTT(SEQ ID NO：40)，#其中N为标签序列，是随机碱基。
第二通用引物为：/5Phos/#GAACGACATGGCTACGATCCGACTT(SEQ ID NO：41)，引物5’端磷酸化，用于MGI平台的环化。
按照以下表7的程序进行第一轮PCR扩增。
表7
Figure PCTCN2019093066-appb-000008
得到的PCR产物中加入80μL的XP磁珠(beads)进行纯化(beckman公司Agencourt AMPure XP磁珠，货号A63881)，得到的产物溶解于20μL TE溶液中。
3、文库质检
对文库进行检测，条带范围在150-200bp之间，结果如图8所示。
4、上机测序
得到的所有产物进行标准化，进行等量混合，混合得到的文库进行平行测序，测序平台MGISEQ-2000，测序类型PE100。
5、数据分析
分析步骤包括过滤接头引物序列、比对等基本的步骤，得到的基本信息如表8，然后采用GATK对目标位点进行突变检测(表9)。
表8下机数据统计
Figure PCTCN2019093066-appb-000009
表9突变位点检测
Figure PCTCN2019093066-appb-000010
Figure PCTCN2019093066-appb-000011
由图8可以看到，采用巢式多重PCR得到的产物没有引物二聚体和非特异性产物。表8显示下机数据中比对率、捕获率都能达到99％，0.2X平均深度的均一性可以达到100％，其中各个扩增子深度在7倍以内(图7)，表明扩增性能优良。对目标位点进行突变检测，所有样本实际检测到的突变频率和标准品突变检测频率接近，实际值大约等于1±0.1倍的理论值(表9)。
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (20)

  1. 一种巢式多重PCR高通量测序文库制备方法，其特征在于，所述方法包括：
    第一轮PCR扩增：使用正向引物和反向引物对目标区域进行扩增，其中所述正向引物是与所述目标区域结合的正向特异性序列，所述反向引物包括3’端的与所述目标区域结合的反向特异性序列和5’端的第一通用测序序列；
    扩增产物的纯化：对所述第一轮PCR扩增的产物进行纯化；
    第二轮PCR扩增：使用巢式引物、第一标签引物和第二通用引物对纯化后的产物进行扩增，其中所述巢式引物位于所述正向引物的下游，所述巢式引物包括5’端的第二通用测序序列和3’端的特异性序列，所述第一标签引物3’端序列和所述反向引物5’端的第一通用测序序列部分或全部序列相同，且所述第一标签引物还包括第一标签序列，所述第二通用引物3’端序列和所述巢式引物5’端的第二通用测序序列部分或者全部序列相同。
  2. 根据权利要求1所述的方法，其特征在于，所述第二通用引物是第二标签引物，该第二标签引物3’端序列和所述巢式引物5’端的第二通用测序序列部分或者全部序列相同，且所述第二标签引物还包括第二标签序列。
  3. 一种巢式多重PCR高通量测序文库制备方法，其特征在于，所述方法包括：
    第一轮PCR扩增：使用正向引物、反向引物和第一标签引物对目标区域进行扩增，其中所述正向引物是与所述目标区域结合的正向特异性序列，所述反向引物包括3’端的与所述目标区域结合的反向特异性序列和5’端的第一通用测序序列，所述第一标签引物3’端序列和所述反向引物5’端的第一通用测序序列部分或全部序列相同，且所述第一标签引物还包括第一标签序列；
    扩增产物的纯化：对所述第一轮PCR扩增的产物进行纯化；
    第二轮PCR扩增：使用巢式引物、第一通用引物和第二通用引物对纯化后的产物进行扩增，其中所述巢式引物位于所述正向引物的下游，所述巢式引物包括5’端的第二通用测序序列和3’端的特异性序列，所述第一通用引物3’端序列和所述第一标签引物5’端部分或全部序列相同，所述第二通用引物3’端序列和所述巢式引物5’端的第二通用测序序列部分或者全部序列相同。
  4. 根据权利要求3所述的方法，其特征在于，所述第二通用引物是第二标签引物，该第二标签引物3’端序列和所述巢式引物5’端的第二通用测序序列部分或者全 部序列相同，且所述第二标签引物还包括第二标签序列。
  5. 根据权利要求1至4任一项所述的方法，其特征在于，所述反向引物上还包括分子标签序列。
  6. 根据权利要求5所述的方法，其特征在于，所述分子标签序列是8-24bp的随机序列。
  7. 根据权利要求1至4任一项所述的方法，其特征在于，所述方法对多个连续的目标区域进行扩增，每个目标区域都有对应的正向引物、反向引物和巢式引物，并且相邻扩增子区域之间形成扩增重叠。
  8. 根据权利要求7所述的方法，其特征在于，所述正向引物、反向引物和巢式引物均是由多条分别靶向不同的目标区域的引物组成的引物池。
  9. 根据权利要求7所述的方法，其特征在于，每个目标区域的正向引物和相邻区域的正向引物、巢式引物和相邻区域的巢式引物、反向引物和相邻区域的反向引物扩增方向相反。
  10. 根据权利要求1至4任一项所述的方法，其特征在于，所述第一轮PCR扩增和/或第二轮PCR扩增的循环数均是2-30个循环。
  11. 根据权利要求1至4任一项所述的方法，其特征在于，所述第一标签序列和/或第二标签序列的长度均是8-15bp。
  12. 一种巢式多重PCR高通量测序文库制备试剂盒，其特征在于，所述试剂盒包括：
    第一轮PCR扩增引物，其包括正向引物和反向引物，其中所述正向引物是与目标区域结合的正向特异性序列，所述反向引物包括3’端的与所述目标区域结合的反向特异性序列和5’端的第一通用测序序列，所述第一轮PCR扩增引物用于对目标区域进行第一轮PCR扩增；
    第二轮PCR扩增引物，其包括巢式引物、第一标签引物和第二通用引物，其中所述巢式引物位于所述正向引物的下游，所述巢式引物包括5’端的第二通用测序序列和3’端的特异性序列，所述第一标签引物3’端序列和所述反向引物5’端的第一通用测序序列部分或全部序列相同，且所述第一标签引物还包括第一标签序列，所述第二通用引物3’端序列和所述巢式引物5’端的第二通用测序序列部分或者全部序列相同，所述第二轮PCR扩增引物用于对纯化后的所述第一轮PCR扩增的产物进行第二轮PCR扩增。
  13. 根据权利要求12所述的试剂盒，其特征在于，所述第二通用引物是第二标签引物，该第二标签引物3’端序列和所述巢式引物5’端的第二通用测序序列部分或者全部序列相同，且所述第二标签引物还包括第二标签序列。
  14. 一种巢式多重PCR高通量测序文库制备试剂盒，其特征在于，所述试剂盒包括：
    第一轮PCR扩增引物，其包括正向引物、反向引物和第一标签引物，其中所述正向引物是与所述目标区域结合的正向特异性序列，所述反向引物包括3’端的与所述目标区域结合的反向特异性序列和5’端的第一通用测序序列，所述第一标签引物3’端序列和所述反向引物5’端的第一通用测序序列部分或全部序列相同，且所述第一标签引物还包括第一标签序列，所述第一轮PCR扩增引物用于对目标区域进行第一轮PCR扩增；
    第二轮PCR扩增引物，其包括巢式引物、第一通用引物和第二通用引物，其中所述巢式引物位于所述正向引物的下游，所述巢式引物包括5’端的第二通用测序序列和3’端的特异性序列，所述第一通用引物3’端序列和所述第一标签引物5’端部分或全部序列相同，所述第二通用引物3’端序列和所述巢式引物5’端的第二通用测序序列部分或者全部序列相同，所述第二轮PCR扩增引物用于对纯化后的所述第一轮PCR扩增的产物进行第二轮PCR扩增。
  15. 根据权利要求14所述的试剂盒，其特征在于，所述第二通用引物是第二标签引物，该第二标签引物3’端序列和所述巢式引物5’端的第二通用测序序列部分或者全部序列相同，且所述第二标签引物还包括第二标签序列。
  16. 根据权利要求12至15任一项所述的试剂盒，其特征在于，所述反向引物上还包括分子标签序列。
  17. 根据权利要求16所述的试剂盒，其特征在于，所述分子标签序列是8-24bp的随机序列。
  18. 根据权利要求12至15任一项所述的试剂盒，其特征在于，所述正向引物、反向引物和巢式引物均是由多条分别靶向多个连续的目标区域的引物组成的引物池，并且相邻扩增子区域之间形成扩增重叠。
  19. 根据权利要求18所述的试剂盒，其特征在于，每个目标区域的正向引物和相邻区域的正向引物、巢式引物和相邻区域的巢式引物、反向引物和相邻区域的反向引物扩增方向相反。
  20. 根据权利要求12至15任一项所述的试剂盒，其特征在于，所述第一标签序列和/或第二标签序列的长度均是8-15bp。
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