WO2016045105A1 - Pf快速建库方法及其应用 - Google Patents

Pf快速建库方法及其应用 Download PDF

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WO2016045105A1
WO2016045105A1 PCT/CN2014/087603 CN2014087603W WO2016045105A1 WO 2016045105 A1 WO2016045105 A1 WO 2016045105A1 CN 2014087603 W CN2014087603 W CN 2014087603W WO 2016045105 A1 WO2016045105 A1 WO 2016045105A1
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sequencing
library
cnv
unit
rapid
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PCT/CN2014/087603
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English (en)
French (fr)
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李剑
甄贺富
张彩芬
张爱萍
夏滢颖
陈大洋
张现东
刘赛军
李尉
黄奕乐
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深圳华大基因股份有限公司
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Priority to CN201480082249.XA priority Critical patent/CN106795650B/zh
Priority to PCT/CN2014/087603 priority patent/WO2016045105A1/zh
Publication of WO2016045105A1 publication Critical patent/WO2016045105A1/zh

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    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00Libraries per se, e.g. arrays, mixtures
    • C40B40/04Libraries containing only organic compounds
    • C40B40/06Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06Biochemical methods, e.g. using enzymes or whole viable microorganisms

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  • the invention relates to a PF rapid database construction method and application thereof, and more particularly to a PF rapid database construction method for DNA sequencing, a CNV detection method and device for embryo chromosomes.
  • Preimplantation Genetic Screening refers to the process of culturing blastocysts in vitro after in vitro fertilization (IVF), using a new generation of high-throughput sequencing ( Next Generation Sequencing (NGS) method, which sequenced and analyzed the chromosomes of the Whole Genome Amplification (WGA) product on the third day of blastomere single cell or fifth day blastocyst trophoblast cells Whether the number is abnormal and whether fragments are missing or repeated to screen for embryos with normal karyotype for implantation. Avoid abortion due to chromosomal abnormalities, repeated IVF failure, and pregnancy and birth in chromosomal-affected children.
  • NGS Next Generation Sequencing
  • high-throughput sequencing has become more and more widely used in the medical field; for example, non-invasive prenatal Down syndrome screening based on high-throughput sequencing, based on high-throughput Sequencing HPV screening and genetic diagnosis of various complex genetic diseases based on high-throughput sequencing.
  • the common features of high-throughput sequencing-based detection technology are high degree of automation, good stability, high sensitivity, large throughput and accurate results. With the further reduction of sequencing costs, high-throughput sequencing technology will be widely used in the medical field.
  • Inventors of the present invention is based on the new high-throughput sequencing platform semiconductor (e.g. Ion Proton TM, Ion Torrent TM, etc.), a self-developed single chromosome copy number variation Rapid PCR-Free and building a database technology (non-equal length sequences) ( Copy Number Variation, abbreviated CNV) analysis technique, achieves the goal of reducing the time and cost of sequencing library preparation by reducing unnecessary steps while achieving aneuploidy and CNV detection of embryonic single-cell whole-genome amplification products.
  • the new high-throughput sequencing platform semiconductor e.g. Ion Proton TM, Ion Torrent TM, etc.
  • CNV Copy Number Variation, abbreviated CNV
  • the object of the present invention is to provide a method for performing pre-implantation genetic testing using high-throughput sequencing technology, aiming at solving the problem of analyzing aneuploid or CNV in a single embryonic cell sample or other single-cell micro-sample.
  • the sample used in the present invention is an embryonic cell sample, a single blastomere cell or a blastocyst trophoblast cell cluster (generally 3-8).
  • the basic operation procedure is that after the whole sample of the cell sample is amplified, the sequencing library is constructed, and the sequencing data is analyzed after the library is sequenced, and the detection result is obtained.
  • the whole genome amplification refers to genome-wide amplification of single cells, several cells or micronucleic acid samples
  • the method may be partial random primer amplification (Degenerate Oligonucleotide Primer PCR, abbreviated as DOP- PCR), Primer Extension Preamplification PCR (abbreviated PEP-PCR), Multiple Strand Displacement Amplification (MDA), OmniPlex WGA, and the like.
  • DOP-PCR Degenerate Oligonucleotide Primer PCR
  • PEP-PCR Primer Extension Preamplification PCR
  • MDA Multiple Strand Displacement Amplification
  • OmniPlex WGA and the like.
  • Commercial kits such as REPLI-g from QIAgen, GenomePlex WGA from Sigma Aldrich, Sureplex from New England Biolabs, PicoPlex WGA from Rubicon Genomics, and illustra Genomiphi V2 from GE Healthcare can also be used. .
  • a first aspect of the present invention provides a PF rapid database construction method comprising the steps of: step 1 of disrupting genomic DNA to obtain a DNA fragment; and second step of performing end repair on the DNA fragment; a third step of adding a P1 and a BarcodeX linker at the end of the end-repaired DNA fragment; a fourth step of library mixing according to the yield required for each library; a fifth step of nick translation of the mixed library; Step 6 of quality testing of products that have undergone nick translation.
  • the genomic DNA is disrupted by a Covaris LE220 interrupter.
  • end-repair is performed using a polynucleotide kinase buffer, a dNTP mixed solution, a T4 DNA polymerase, and a large Klenow fragment.
  • magnetic bead purification is performed before the third step after the second step, before the fourth step after the third step, and before the sixth step after the fifth step.
  • a second aspect of the present invention provides a CNV detection method for an embryo chromosome, comprising the steps of: constructing a library according to the PF rapid database construction method of the first aspect of the present invention; performing sequencing on the constructed library to obtain a sequencing result; And performing information analysis on the sequencing results.
  • the sequencing is performed using high throughput sequencing technology.
  • the on-machine sequencing is performed using an Ion Proton sequencer.
  • a third aspect of the present invention provides a CNV detecting device for an embryo chromosome, comprising: a building unit that constructs a library according to the PF rapid database building method of the first aspect of the present invention and outputs the same; the sequencing unit, the sequencing unit The library is connected to the database unit and the library outputted by the database unit is sequenced to output the sequencing result; and an analysis unit is connected to the sequencing unit and performs information analysis on the sequencing result output by the sequencing unit.
  • the sequencing is performed using high throughput sequencing technology.
  • the on-machine sequencing is performed using an Ion Proton sequencer.
  • the invention focuses on the development of PCR-Free rapid library construction technology (referred to as PF database) and (non-equal length sequence) single cell CNV analysis technology.
  • PF database PCR-Free rapid library construction technology
  • non-equal length sequence single cell CNV analysis technology
  • PF builds down and optimizes the steps and reaction systems for constructing libraries, reduces the time and cost of sequencing library preparation, and on the other hand eliminates the possibility that library PCR enrichment may introduce biased amplification to influence CNV judgment.
  • the newly developed CNV analysis technology for non-equal length sequences can maximize the utilization of the original sequence, and can perform accurate CNV detection analysis on single cell samples such as embryos.
  • FIG. 1 is a flow chart showing a method of building a database in the prior art.
  • FIG. 2 is a flow chart showing the PF rapid database building method of the present invention.
  • Figure 3 is a schematic diagram showing the results of comparison of WGA product and gDNA on two interrupters, respectively.
  • FIG. 4 is a flow chart showing a CNV detecting method of an embryo chromosome of the present invention.
  • Fig. 5 is a configuration diagram showing a CNV detecting device of an embryonic chromosome of the present invention.
  • FIG. It is to be understood that the following examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
  • genomic DNA as a sample. Seven embryonic samples were selected and the whole genome amplification was performed according to the kit instructions using New Zealand Biolabs' Sureplex Single Cell WGA Kit. The amplified products were quantified by DNA, and 100 ng of DNA was taken from each sample to complete the PF library construction. .
  • 8 cell line samples of known karyotype were selected, including aneuploid, fragments with different fragment/repetition sizes (with a minimum of about 1.5 Mb).
  • single cells were picked to simulate embryonic single cells, and whole genome amplification was performed and DNA quantification was performed. 100 ng of DNA was taken from each sample to complete PF library construction.
  • FIG. 2 is a flow chart showing the PF rapid database building method of the present invention.
  • the advanced sample was interrupted.
  • the official routine library manual recommends the use of a Bioruptor interrupter.
  • the interrupter was unable to interrupt the single-cell whole-gene amplification product (hereinafter referred to as WGA product) to the requirement of 150-200 bp insert for sequencing. Therefore, if the insertion requirement required for sequencing is 150-200 bp, the inventors of the present invention have found that if the Covaris LE220 interrupter is used instead, the insert size requirement can be satisfied, and the interrupt throughput and the saving can be greatly improved. time consuming.
  • Table 1 below shows the relevant values for the two types of interrupters.
  • Figure 3 is a schematic diagram showing the results of comparison of WGA product and gDNA on two interrupters, respectively.
  • lanes 1 and 10 are DNA molecular markers
  • lane 2 is uninterrupted WGA product
  • lane 3 is uninterrupted gDNA
  • lanes 4 and 5 are two WGA samples interrupted by Bioruptor interrupter
  • Lane 6 is a gDNA sample interrupted by a Bioruptor interrupter
  • lanes 7 and 8 are two WGA samples interrupted by a Covaris LE220 interrupter
  • lane 9 is a gDNA sample interrupted by a Covaris LE220 interrupter.
  • the sample is interrupted. After the interruption was completed, the interrupted sample was slowly aspirated and stored in a 1.5 ml centrifuge tube.
  • the DNA fragment obtained after the disruption was subjected to end repair.
  • Klenow large fragment, T4 polynucleotide kinase was placed on an ice box, and the reagent was allowed to melt and thoroughly mixed and centrifuged (the enzyme was not shaken).
  • the end-repair reaction system was prepared in a 2 ml centrifuge tube according to the following usage:
  • the total volume configured is 100 microliters.
  • thermomixer Thermomixer
  • a volume of 46 ⁇ L of EB is dissolved using 1 volume (100 ⁇ L) of Ampure XP magnetic beads.
  • the repaired DNA fragment is ligated end-to-end.
  • the ligation reaction system was prepared in a 1.5 ml centrifuge tube according to the following usage:
  • Ion Xpress TM Barcode X (1.25 ⁇ M) (single plus) 1 microliter.
  • the total volume configured is 100 microliters.
  • the library is mixed, and the amount of the substance such as a single library is mixed according to the yield required for each library.
  • the final volume can be made 37.2 microliters.
  • the notch translation can be performed. 10 ⁇ Pfx buffer, dNTP mixed solution (10 mM), MgSO 4 (50 mM) and Platinum Pfx DNA polymerase (2.5 U/ ⁇ L) were preliminarily removed from the kit stored at -20 ° C and placed on an ice box. And mix thoroughly and centrifuge (the enzyme is not oscillating).
  • reaction system was prepared in a 1.5 ml centrifuge tube according to the following amounts:
  • the total volume configured is 100 microliters.
  • the used reagents were returned to the original kit and stored at -20 ° C.
  • the prepared mixture was shaken and mixed, and 12.8 ⁇ l of the enzyme reaction mixture was added to each reaction. After shaking and centrifugation, it was placed in a PCR machine or a thermomixer (Thermomixer) to react at 72 ° C for 20 minutes.
  • PCR amplification of the library can further increase the concentration of the effective library, and the purification of a certain size of the fragment by subsequent gel-removing can improve the quality of the preparation before the machine.
  • the biased amplification accompanying PCR may seriously affect the true situation of chromosome copy number variation, and the reproducibility and stability of gel purification are poor, and the flux is low. Therefore, the conventional database construction scheme is not suitable for large sample detection.
  • the PF rapid database construction method optimizes the library construction step under the premise that the sequencing output data is substantially consistent, removes the PCR enrichment and the gelation purification step, and mixes the library before performing the notch translation. It greatly saves the investment of material reagents and saves time and time. The advantages of simultaneous detection of a large number of samples are more obvious.
  • Table 2 below compares the sequencing output data for conventional database construction (0, 2, 5, and 8 cycles of PCR).
  • the 0 cycle is a step of extending without denaturation annealing, and only nick translation is performed;
  • GC content is the ratio of guanine and cytosine among the four bases of DNA.
  • data utilization is the quotient of the unique sequence and the number of sequences after filtration. The higher the value, the higher the quality of the library; the “alignment rate” is the ratio of the number of sequences that can be aligned to the number of reference sequences to the total number of sequences.
  • the "repetition rate” is the ratio of the number of identical or identical sequences to the total number of sequences. The lower the value, the more uniform the WGA amplification, and the fewer amplicon introduced artificially during the library construction process.
  • Table 3 compares the data obtained from the conventional building (0, 8 cycles of PCR) and the non-cutting recovery sequencing.
  • the 8 cycle construction step in the “cutting glue” is the conventional database construction step
  • the 0 cycle construction step in the “not cutting glue” is the construction step of the invention.
  • a CNV detecting method for an embryo chromosome comprising the steps of: constructing a library according to the PF rapid database building method of the first aspect of the present invention; The library is subjected to sequencing on the machine to obtain a sequencing result; and information analysis is performed on the sequencing result.
  • sequencing can be performed by any high throughput sequencing technique known in the art, preferably using an Ion Proton sequencer, in accordance with a specific example of the invention.
  • the inventors of the present invention found that the Ion Proton sequencer can effectively obtain the sequencing result, and the sequencing time is small, the efficiency is high, the sequencing result is accurate, and the repeatability is good.
  • a CNV detecting device for an embryonic chromosome As shown in FIG. 5, the apparatus includes a database building unit 100, a sequencing unit 200, and an analysis unit 300.
  • the database building unit 100 constructs a library and outputs the same using the PF rapid database construction technique described above.
  • the sequencing unit 200 is connected to the database building unit 100 and performs sequencing on the library outputted by the database unit 100 to output the sequencing result.
  • the analysis unit 300 is connected to the sequencing unit 200 and performs information analysis on the sequencing result output by the sequencing unit 200.

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Abstract

一种PF快速建库方法及其应用。PF快速建库方法包括以下步骤:对基因组DNA进行打断以获得DNA片段的第1步骤;对所述DNA片段进行末端修复的第2步骤;在经过末端修复的所述DNA片段的末端添加P1和BarcodeX接头的第3步骤;根据每个文库所需要的产量,进行文库混合的第4步骤;对混合后的文库进行缺口平移的第5步骤;以及对经过缺口平移的产物进行质量检测的第6步骤。

Description

PF快速建库方法及其应用 技术领域
本发明涉及一种PF快速建库方法及其应用,更具体地,涉及用于DNA测序的PF快速建库方法、胚胎染色体的CNV检测方法和装置。
背景技术
植入前筛查检测(Preimplantation Genetic Screening,缩写PGS)是指经过体外人工受精(In vitro fertilization,缩写IVF)后的受精卵在体外培养成囊胚的过程中,利用新一代高通量测序(Next Generation Sequencing,缩写NGS)的方法,对其第三天卵裂球单细胞或第五天囊胚滋养层细胞的全基因组扩增(Whole Genome Amplification,缩写WGA)产物进行测序,并分析其染色体数目是否异常和是否存在片段缺失或重复,以筛选出核型正常的胚胎进行植入。避免因染色体异常而引起流产、反复IVF失败以及染色体患病儿的妊娠和出生。
世界上首个试管婴儿诞生于1978年,截止目前全球已有超过500万试管婴儿;辅助生殖技术的安全性得到了充分验证。随着不孕不育发生率的不断上升和遗传学知识的普及,越来越多的夫妇正在寻求通过辅助生殖技术和PGS获得正常的小孩。以荧光原位杂交(Fluorescent In Situ Hybridization,缩写FISH)、多重PCR和微阵列比较基因组杂交(array CGH)为主的传统植入前遗传学诊断技术,因操作流程复杂,人为干扰因素大,在检测稳定性、准确性和通量方面各有所欠缺,不利于PGS的推广。近年来,随着高通量测序技术的快速发展,高通量测序被越来越广泛地应用于医学领域;例如基于高通量测序的无创产前唐氏综合症筛查、基于高通量测序的HPV筛查以及基于高通量测序的各种复杂遗传病的基因诊断等。基于高通量测序的检测技术的共同特点是自动化程度高、稳定性好、灵敏度高、通量大和结果准确,随着测序成本的进一步降低,高通量测序技术将会普遍应用于医学领域。
发明内容
本发明的发明人们基于新一代高通量半导体测序平台(如Ion ProtonTM、Ion TorrentTM等),自主研发了PCR-Free快速建库技术和(非等长序列)单细胞染色体拷贝数变异(Copy Number Variation,缩写CNV)分析技术,通过缩减不必要的步骤以达到降低测序文库制备的时间与成本的目的,同时实现对胚胎单细胞全基因组扩增产物的非整倍体和CNV检测。
本发明的目的在于提供一种利用高通量测序技术完成胚胎植入前遗传检测的方法,旨在解决对单个胚胎细胞样本或者其他单细胞级微量样本分析其非整倍体或者CNV的问题。
本发明所用样本为胚胎细胞样本,单个卵裂球细胞或者囊胚期滋养层细胞团(一般为3-8个)。基本操作流程为细胞样本经全基因组扩增后,进行测序文库构建,文库上机测序后对测序数据进行分析,得出检测结果。
本发明中,所述的全基因组扩增是指对单个细胞、几个细胞或微量核酸样本进行全基因组范围的扩增,其方法可以是部分随机引物扩增(Degenerate Oligonucleotide Primer PCR,缩写DOP-PCR),完全随机引物扩增(Primer Extension Preamplification PCR,缩写PEP-PCR),多重链置换扩增(Multiple Displacement Amplification,缩写MDA),OmniPlex WGA等方法中的任一种。也可采用商业试剂盒如QIAgen公司的REPLI-g,Sigma Aldrich公司的GenomePlex WGA,New England Biolabs公司的Sureplex,Rubicon Genomics公司的PicoPlex WGA,GE Healthcare公司的illustra Genomiphi V2等试剂盒中的任一种。
本发明的第一方面提供了一种PF快速建库方法,包括以下步骤:对基因组DNA进行打断以获得DNA片段的第1步骤;对所述DNA片段进行末端修复的第2步骤;在经过末端修复的所述DNA片段的末端添加P1和BarcodeX接头的第3步骤;根据每个文库所需要的产量,进行文库混合的第4步骤;对混合后的文库进行缺口平移的第5步骤;以及对经过缺口平移的产物进行质量检测的第6步骤。
优选为,在第1步骤中以Covaris LE220打断仪对基因组DNA进行打断。
优选为,在第2步骤中利用多聚核苷酸激酶缓冲液、dNTP混合溶液、T4DNA聚合酶、Klenow大片段所进行末端修复。
优选为,在第2步骤之后第3步骤之前,第3步骤之后第4步骤之前,第5步骤之后第6步骤之前,分别进行磁珠纯化。
本发明的第二方面提供了一种胚胎染色体的CNV检测方法,包括以下步骤:根据本发明第一方面的PF快速建库方法构建文库;对所构建的文库进行上机测序,得到测序结果;以及对所述测序结果进行信息分析。
优选为,所述上机测序是用高通量测序技术进行的。
优选为,所述上机测序是利用Ion Proton测序仪进行的。
本发明的第三方面提供了一种胚胎染色体的CNV检测装置,具备:建库单元,该建库单元根据本发明第一方面的PF快速建库方法构建文库且输出;测序单元,该测序单元连接于建库单元且对建库单元输出的文库进行上机测序以输出测序结果;以及分析单元,该分析单元连接于测序单元且对测序单元输出的测序结果进行信息分析。
优选为,所述上机测序是用高通量测序技术进行的。
优选为,所述上机测序是利用Ion Proton测序仪进行的。
本发明重点开发了PCR-Free快速文库构建技术(简称PF建库)和(非等长序列)单细胞CNV分析技术。PF建库一方面缩减和优化构建文库的步骤和反应体系,降低测序文库制备的时间与成本,另一方面消除文库PCR富集中可能引入偏向性扩增影响CNV判断的可能性。而针对非等长序列新开发的CNV分析技术,可最大限度地提高原始序列的利用率,可对胚胎等单细胞样本进行快递精确的CNV检测分析。
附图说明
图1是示出现有技术中的建库方法的流程图。
图2是示出本发明的PF快速建库方法的流程图。
图3是示出WGA产物和gDNA分别在两种打断仪上进行打断的对比结果的示意图。
图4是示出本发明的胚胎染色体的CNV检测方法的流程图。
图5是示出本发明的胚胎染色体的CNV检测装置的结构图。
具体实施方式
以下参照附图,结合具体实施方式,进一步阐述本发明。应理解,以下实施方式仅用于说明本发明而不用于限制本发明的范围。
PF文库构建
首选,准备作为样本的基因组DNA。选择胚胎样本7例,利用New England Biolabs公司的Sureplex Single Cell WGA Kit,严格按照试剂盒说明完成全基因组扩增,将扩增的产物做DNA定量,每例样本取100ng的DNA来完成PF文库构建。
另外,选择已知核型的细胞系样本8例,包括非整倍体,片段缺失/重复大小不同的样本(其中最小为1.5Mb左右)。将其培养至最佳状态时,挑取单个细胞,以模拟胚胎单细胞,同样完成全基因组扩增并做DNA定量,每例样本取100ng的DNA来完成PF文库构建。
图2是示出本发明的PF快速建库方法的流程图。
如图2所示,先进性样品打断。如图1所示,在现有技术中,官方常规建库手册推荐使用Bioruptor打断仪。但是经测试,在指引的条件下,该打断仪无法将单细胞全基因扩增产物(下称WGA产物)打断至测序需要150-200bp插入片段要求。因此,如果测序需要的插入片段要求是150-200bp的话,本发明的发明人们发现,如果利用Covaris LE220打断仪代替,就可以满足插入片段大小的要求,并提高打断的通量和大大节省耗时。当然,如果插入片段要求不是上述范围的话,也可以使用Bioruptor打断仪或者其他打断仪。
下表1 为两种打断仪的相关数值。
Figure PCTCN2014087603-appb-000001
Figure PCTCN2014087603-appb-000002
图3是示出WGA产物和gDNA分别在两种打断仪上进行打断的对比结果的示意图。其中,泳道1和10为DNA分子标记;泳道2为未经打断的WGA产物;泳道3为未经打断的gDNA;泳道4和5为两例经Bioruptor打断仪打断的WGA样本;泳道6为经Bioruptor打断仪打断的gDNA样本;泳道7和8为两例经Covaris LE220打断仪打断的WGA样本;泳道9为经Covaris LE220打断仪打断的gDNA样本。
在利用Covaris LE220-96 well plate打断仪进行样品打断时,先打开变压器电源(输入/输出电压为220/110V),然后打开恒温水浴锅开关,将温度设为4℃,再打开Covaris LE仪器开关和计算机;平稳地取出Covaris LE水槽,往水槽中加入去离子水至相应位置,小心地将水槽放入仪器内对应位置;打开计算机和桌面上的Covaris LE,转换器架自动向下移动至相应位置;最后按下上排气按钮(DEGAS PUMP)进行排气,排气至少45分钟。
取一块96孔板,用来打断的每个孔放入2根专用的打断棒,用TE缓冲液将50至300ng经全基因组扩增后的样品稀释至80μL后加入相应的96孔PCR板中,然后用封膜机封膜,短暂离心。按住打断仪绿色按钮(DOOR)开门,将96孔PCR板放在架上,进入Covaris LE的设置选择打断顺序和打断程序,再进行打断条件的设置。
确认所有设置的内容无误后开始打断样品。在打断完成之后,将打断后的样品缓慢吸出,放入1.5毫升离心管中保存。
接下来,对打断后得到的DNA片段进行末端修复。预先从-20℃保存的试剂盒中取出10×多聚核苷酸激酶缓冲液、10mM的dNTP混合溶液、T4 DNA聚合酶、 Klenow大片段、T4多聚核苷酸激酶将其置于冰盒上,待试剂融解充分混匀离心(酶不可震荡)。
在2毫升的离心管中按照以下的使用量配制末端修复反应体系:
上一步DNA  80微升
10×多聚核苷酸激酶缓冲液  10微升
dNTP混合溶液(10mM)  2.5微升
T4 DNA聚合酶  1微升
Klenow大片段  0.1微升
T4多聚核苷酸激酶  1微升
超纯水  5.4微升。
配置出的总体积是100微升。
把使用后的试剂放回原试剂盒中并-20℃保存。然后将配置好的混合液震荡离心后,每个反应加入20微升酶反应混合液。震荡离心后,置于恒温混匀仪(Thermomixer)中在20℃下温浴30分钟,以完成整个修复过程。
温浴完成后,优选地,使用1倍体积(100μL)Ampure XP磁珠纯化,溶46μL的EB。
接下来,对修复后的DNA片段进行末端连接。预先从-20℃保存的试剂盒中取出2×快速连接缓冲液、Ion P1 Adapter(1.25μM)、Ion XpressTM Barcode X(1.25μM)和T4 DNA连接酶将其置于冰盒上融解并充分混匀离心(酶不可震荡)。在1.5毫升的离心管中按照以下的使用量配制连接反应体系:
上一步DNA  44微升
2×快速连接缓冲液  50微升
Ion P1 Adapter(1.25μM)  1微升
T4 DNA连接酶(Rapid,L603-HC-L)  4微升
Ion XpressTM Barcode X(1.25μM)(单加)  1微升。
配置出的总体积是100微升。
把使用后的试剂放回原试剂盒中并在-20℃下保存,将配置好的混合液震荡混匀。首先将Ion XpressTM Barcode X(1.25μM)按照对应编号单加入反应管中,然后再向每个反应管加入55微升酶反应混合液。震荡离心后,置于恒温混匀仪(Thermomixer)中在20℃下温浴30分钟。
温浴完成后,优选地,使用1倍体积(100μL)Ampure XP磁珠纯化,溶20μL的EB。
然后,进行文库混合,根据每个文库所需要的产量,将单库等物质的量混合。
在混合时,例如可以使最终体积为37.2微升。
在混库之后,就可以进行缺口平移。预先从-20℃下保存的试剂盒中取出10×Pfx缓冲液、dNTP混合溶液(10mM)、MgSO4(50mM)和Platinum Pfx DNA聚合酶(2.5U/μL)将其置于冰盒上融解并充分混匀离心(酶不可震荡)。
在1.5毫升离心管中按照以下的使用量配制如下反应体系:
混合后DNA  37.2微升
10×Pfx缓冲液  5微升
dNTP混合溶液(10mM)  5微升
MgSO4(50mM)  2微升
Platinum Pfx DNA聚合酶(2.5U/μL)  0.8微升
配置出的总体积是100微升。
把使用后的试剂放回原试剂盒中并在-20℃下保存,将配置好的混合液震荡混匀,每个反应加入12.8微升酶反应混合液。震荡离心后,置于在PCR仪或恒温混匀仪(Thermomixer)中在72℃下反应20分钟。
反应完成后,优选地,使用1.2倍体积(60μL)Ampure XP磁珠纯化,溶20μL的EB。
最后,在上机测序之前,对经过缺口平移的产物进行质量检测。
效果
在常规文库构建步骤中,对文库进行PCR扩增可以进一步增加有效文库的浓度,通过后续的切胶回收纯化一定大小的片段,可提高上机前制备的质量。但PCR伴随的偏向性扩增有可能严重影响染色体拷贝数变异的真实情况,而且切胶纯化的重复性和稳定性较差,通量低,因此常规建库方案不适用于大量样品检测。
本发明所提供的PF快速建库方法在保证测序产出数据基本一致的前提下,对文库构建步骤进行了优化,去除PCR富集和切胶纯化步骤,并把文库混合后再进行缺口平移,大大地节省了物料试剂的投入并节约耗时,大量样本同时检测时优势更加明显。
下表2 为常规建库(0、2、5、8次循环PCR)测序产出数据的对比。
Figure PCTCN2014087603-appb-000003
其中,“PCR循环数”中,0次循环为不经变性退火延伸的步骤,只进行了缺口平移;“GC含量”为DNA的4种碱基中,鸟嘌呤和胞嘧啶所占的比率。一般情况下,GC含量愈高,DNA的密度也愈高,结构也约复杂;“数据利用率”为唯一序列和过滤后序列数目的商。值越高,表明文库质量越高;“比对率”为序列可比对到参考序列的数目占全部序列数目的比率。值越高,表明WGA产物质量和文库质量越高;“重复率”为两端或完全相同序列占全部序列数目的比率。值越低,表明WGA扩增越均匀,建库过程中人为引入的扩增子越少。
下表3 为常规建库(0、8次循环PCR)切胶回收与不切胶回收测序产出数据的对比。
Figure PCTCN2014087603-appb-000004
Figure PCTCN2014087603-appb-000005
其中,“切胶”中8次循环建库步骤为常规建库步骤,“不切胶”中0次循环建库步骤为本发明建库步骤。
根据本发明的另一方面,提供了一种胚胎染色体的CNV检测方法,如图4所示,该方法包括以下步骤:根据本发明第一方面的PF快速建库方法构建文库;对所构建的文库进行上机测序,得到测序结果;以及对所述测序结果进行信息分析。
本领域的技术人员可以理解,可以通过本领域已知的任何高通量测序技术进行上机测序,根据本发明的具体示例,优选地使用Ion Proton测序仪进行测序。本发明的发明人们发现,利用Ion Proton测序仪能够有效地获得测序结果,且测序用时少,效率高,测序结果准确,可重复性好。
根据本发明的再另一方面,提供了一种胚胎染色体的CNV检测装置。如图5所示,该装置包括建库单元100、测序单元200、以及分析单元300。
根据本发明的实施方式,建库单元100采用上述的PF快速建库技术构建文库且输出。
测序单元200连接于建库单元100且对建库单元100输出的文库进行上机测序以输出测序结果
分析单元300连接于测序单元200且对测序单元200输出的测序结果进行信息分析。
本领域技术人员能够理解的是,可以采用本领域中已知的任何适于进行上 述操作的装置作为上述各个单元的组成部件。在本文中所使用的术语“连接”应作广义解释,可以是直接相连,也可以通过中间媒介简介相连,对于本领域的普通技术人员而言,可以根据具体情况理解上述的具体含义。
此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种PF快速建库方法,其特征在于,包括以下步骤:
    对基因组DNA进行打断以获得DNA片段的第1步骤;
    对所述DNA片段进行末端修复的第2步骤;
    在经过末端修复的所述DNA片段的末端添加接头的第3步骤;
    根据每个文库所需要的产量,进行文库混合的第4步骤;
    对混合后的文库进行缺口平移的第5步骤;以及
    对经过缺口平移的产物进行质量检测的第6步骤。
  2. 如权利要求1所述的PF快速建库方法,其特征在于,
    在第1步骤中以Covaris LE220打断仪对基因组DNA进行打断。
  3. 如权利要求1所述的PF快速建库方法,其特征在于,
    在第2步骤中利用多聚核苷酸激酶缓冲液、dNTP混合溶液、T4DNA聚合酶、Klenow大片段所进行末端修复,
    在第3步骤中所述接头是P1和BarcodeX接头。
  4. 如权利要求1所述的PF快速建库方法,其特征在于,
    在第2步骤之后第3步骤之前,第3步骤之后第4步骤之前,第5步骤之后第6步骤之前,分别进行磁珠纯化。
  5. 一种胚胎染色体的CNV检测方法,其特征在于,包括以下步骤:
    根据权利要求1至4中任意一项所述的PF快速建库方法构建文库;
    对所构建的文库进行上机测序,得到测序结果;以及
    对所述测序结果进行信息分析。
  6. 如权利要求5所述的CNV检测方法,其特征在于,
    所述上机测序是用高通量测序技术进行的。
  7. 如权利要求5所述的CNV检测方法,其特征在于,
    所述上机测序是利用Ion Proton测序仪进行的。
  8. 一种胚胎染色体的CNV检测装置,其特征在于,具备:
    建库单元,该建库单元根据权利要求1至4中任意一项所述的PF快速建库方法构建文库且输出;
    测序单元,该测序单元连接于建库单元且对建库单元输出的文库进行上机测序以输出测序结果;以及
    分析单元,该分析单元连接于测序单元且对测序单元输出的测序结果进行信息分析。
  9. 如权利要求8所述的CNV检测装置,其特征在于,
    所述上机测序是用高通量测序技术进行的。
  10. 如权利要求8所述的CNV检测装置,其特征在于,
    所述上机测序是利用Ion Proton测序仪进行的。
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