WO2016109981A1 - 一种dna合成产物的高通量检测方法 - Google Patents

一种dna合成产物的高通量检测方法 Download PDF

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WO2016109981A1
WO2016109981A1 PCT/CN2015/070459 CN2015070459W WO2016109981A1 WO 2016109981 A1 WO2016109981 A1 WO 2016109981A1 CN 2015070459 W CN2015070459 W CN 2015070459W WO 2016109981 A1 WO2016109981 A1 WO 2016109981A1
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dna
sequencing
mixed
throughput
sequence
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康康
陈世宏
沈玥
王云
徐讯
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深圳华大基因研究院
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Priority to CN201580068286.XA priority Critical patent/CN107002150B/zh
Priority to PCT/CN2015/070459 priority patent/WO2016109981A1/zh
Publication of WO2016109981A1 publication Critical patent/WO2016109981A1/zh
Priority to US15/643,078 priority patent/US10179934B2/en

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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation

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  • the invention relates to the technical field of DNA synthesis, in particular to a high-throughput detection method for DNA synthesis products.
  • the two major challenges facing DNA chip synthesis are the high error rate of oligonucleotide strands and the high complexity of oligonucleotide libraries on assembly. How to further improve the synthesis accuracy of DNA chip synthesis technology, reduce assembly and screening costs, and play a vital role in its commercial application.
  • the correctness detection and positiveness of DNA synthesis fragments Fragment screening is often done by Sanger sequencing, the first generation sequencing technology. Due to the technical bottleneck of DNA chip synthesis itself, the assembled products tend to be complex. Compared with the designed sequence, each assembled DNA molecule may have multiple complexities (such as single base variation and core) in the sequence.
  • the probability of single base variation is about 1 ⁇ 1% due to differences in platform and design. Therefore, when the single base error rate is 5 ⁇ , assembling a 750 bp DNA fragment, the probability of obtaining a completely correct DNA fragment at one time is only 2.33%, and the probability of an intra-error is 11.11%, within two occurrences. The error rate was 27.63%.
  • 98 monoclonals should be selected for Sanger sequencing.
  • the cost of each Sanger reaction is 20 yuan. The cost is about 1,960 yuan, which is 2.61 yuan per bp.
  • the cost is much higher than the traditional synthesis method. If you want to pick at least one clone with at most one error and guarantee a 90% success rate, you need to select 20 monoclonals for Sanger verification.
  • the verification cost is about 400 yuan, which is 0.53 yuan per bp. If you want to pick at least one clone with at most two errors and guarantee a 90% success rate, you need to select 8 monoclonals for Sanger verification.
  • the verification cost is about 160 yuan. 0.21 yuan per bp. With more than three errors, the cost of debugging materials and time is high, DNA chip synthesis technology will lose the cost advantage. It can be seen that the traditional method for detecting the correctness of DNA chip synthesis products based on Sanger sequencing has high cost and is a major cost source for DNA chip synthesis technology, and is also a major bottleneck for further reducing the cost of the technology.
  • the invention provides a high-throughput detection method for DNA synthesis products, which successfully uses high-throughput sequencing technology to detect DNA product synthesis products through a mixed library construction strategy, thereby greatly reducing product detection and correct product screening cost, thereby The cost of DNA chip synthesis is greatly reduced.
  • a high throughput assay for DNA synthesis products comprising the steps of:
  • a plurality of DNA components produced by the synthesis are respectively ligated into the cloning vector and separately transferred into the screening strain, respectively cultured on the screening medium, and the clones to which the DNA components are ligated are selected;
  • the DNA synthesis product is a DNA module synthesized by DNA chip synthesis.
  • the method further comprises: picking out the selected clones for colony PCR verification, and further screening for clones having an insert size consistent with the size of the corresponding DNA component.
  • the cloning liquid mixture is mixed according to the principle that the number of each cloning cell is the same;
  • the respective bacterial cultures cultured from the clones of each DNA component are separately stored for maintenance.
  • the step 4) is specifically: obtaining or amplifying the mixed DNA component from the mixed plasmid sample, and then connecting the sequencing tags to the plurality of mixed DNA components to construct a plurality of mixed sequencing libraries, wherein the different mixed sequencing The sequencing labels of the libraries are different;
  • the method further comprises, after the sequencing tag is attached, ligating the sequencing link to the plurality of mixed DNA components.
  • step 4) obtains or amplifies the mixed DNA component from the mixed plasmid sample by using a digestion or PCR method, respectively.
  • the step 5) is carried out using Illumina HiSeq2000, HiSeq 2500, MiSeq, MiSeqDx, NextSeq500, Hiseq X ten, Life SOLiD, Ion Torrent PGM, Proton, Roche 454, Complete Genomics sequencing device or single molecule sequencing device. Flux sequencing.
  • setting the correct rate means that the base correctness rate is 100%.
  • the method further includes: performing sequence debugging by a single point mutation when the setting correct rate is less than 100%;
  • the set correct rate is less than 100%
  • the clone having the least number of single point mutations, insertions or deletion mutations is selected, and the sequence is debugged by single point mutation
  • the method further comprises: verifying the target DNA component by Sanger sequencing.
  • the steps 2) to 6) are repeated for the DNA components, and the unselected clones are screened and the sequencing and sequencing library is constructed and sequenced, or Repeat steps 4) to 6) to construct and sequence the mixed sequencing library of the mixed plasmid samples that have not been sequenced.
  • the method further comprises: finding a clone and/or cloned bacterial liquid corresponding to the target DNA component having the set correct rate, and then extracting the plasmid and/or performing DNA component amplification.
  • the method of the invention successfully applies high-throughput sequencing technology to the detection of DNA chip synthesis products through the construction of mixed sequencing library and mixed high-throughput sequencing strategy, and utilizes the advantages of low-throughput high-throughput sequencing technology to greatly reduce the synthesis product.
  • the cost of detection and correct product screening It has been calculated that the cost of sequencing detection of DNA component synthesis products is reduced by about two orders of magnitude, thereby greatly reducing the cost of DNA chip synthesis.
  • Figure 1 is a schematic diagram showing the process of detecting a DNA chip synthesis product by a high-throughput sequencing strategy of the present invention.
  • DNAAssembly refers to a double-stranded DNA fragment product produced by DNA synthesis techniques, including but not limited to DNA chip synthesis techniques, generally between 400 bp and 1.5 kbp in length.
  • the target DNA component refers to a double-stranded DNA fragment product produced by DNA synthesis which is detected by the method of the present invention and whose base correctness rate reaches a set correct rate (for example, a base correctness rate of 100%).
  • Mixed Library refers to a mixed DNA sequencing library containing different DNA components, each of which contains only a single kind of DNA molecule, such as a monoclonal A plasmid molecule, a PCR amplification product or a digested product.
  • a high-throughput detection method for a DNA synthesis product generally includes the following processes:
  • a plurality of DNA components are ligated into the vector by restriction enzyme ligation, Gibson assembly or T vector ligation, and transformed into E. coli and other screening strains, and then cultured on the corresponding resistant culture plates to ensure sufficient growth (such as 30 ) Clear and identifiable monoclonal.
  • Vectors with blue-white spot screening or other screening functions can be used as needed to reduce false positive cloning rates.
  • each DNA component is sequentially numbered (for example, for component A, the clones can be sequentially numbered as #1 to #30; for component B, the clones can be sequentially numbered from #1 to #20)
  • the monoclonal antibody is expanded by a 96-well deep-well plate or a test tube to obtain a bacterium, and the bacteria are preserved by a deep-well plate, a centrifuge tube or a plate culture method.
  • the clone number is numbered (eg, DNA#1 to DNA#30).
  • a DNA molecule of a mixed DNA component is obtained or amplified from the mixed plasmid DNA by means of digestion or PCR amplification.
  • This step is an optional step because the sequencing libraries are constructed differently for different high-throughput sequencing platforms.
  • Some sequencing library construction methods can be directly used to link linear plasmid DNA to specific sequencing tags and sequencing junctions.
  • the sequencing library for sequencing on the machine; and the construction method of some sequencing libraries requires the DNA component as the starting molecule, and the specific sequencing tag and sequencing linker are added at both ends to construct the sequencing library.
  • sequencing tags index
  • a sequencing adaptor adaptor
  • the sequencing tag is used to distinguish DNA components from different sample sources, that is, to distinguish DNA components in different mixed sequencing libraries.
  • Each hybrid sequencing library consists of a sequencing tag with a specific sequence, and the sequence of the sequencing tags of different mixed sequencing libraries is different.
  • the general sequencing tag can be a sequence of several or ten bases, which can be a random sequence, such as a random sequence of 8-12 bases; the sequencing linker is a unique linker sequence for each high-throughput sequencing platform, different The sequencing link of the sequencing platform is different.
  • a high-throughput sequencing of a certain number of mixed sequencing sequencing libraries was constructed.
  • the amount of sequencing is relatively low. If each clone of each DNA component is covered by 100 ⁇ , each DNA component is 750 bp in length, and there are 400 mixed DNA components, and 10 mixed sequencing libraries are simultaneously sequenced. The amount is only 300 Mbp; if the sequencing molecule is a vector carrying a DNA component rather than a PCR or a digested product of a DNA component, the required data is 1.1 Gbp.
  • a set correctness rate can be set for the base correctness rate, for example, the base error is not more than 3, 2 or 1, or the base correct rate is 100%. If the sequenced DNA component has a base error, the sequence primer can be designed by single point mutation design.
  • step (3) If the correct rate of some DNA components is still not ideal within several clones selected, for the undesired DNA components, return to step (3) to select clones that have not been sequenced for new hybrid library construction, or return to the step ( 6) Select the mixed plasmid DNA or mixed DNA component molecules that have not been sequenced to construct a new hybrid library (such as L#11 ⁇ L#20), and perform a new round of sequencing verification until the clone with the correct rate is selected.
  • a new hybrid library such as L#11 ⁇ L#20
  • the present invention is based on high-throughput sequencing to detect the correctness of DNA chip synthesis products and to implement correct fragment screening.
  • the advantages of the existing scheme based on Sanger sequencing are as follows:
  • Each chip can synthesize about 400 DNA components.
  • Each DNA component is 750 bases in length, with a total length of 300,000 bases and a single base error rate of 5.
  • the selected number of monoclonal n should satisfy: 1-(1-2.33%) n >0.9, n is at least 98; if at least one clone containing at most one error is guaranteed and a 90% success rate is guaranteed, the selected number of monoclonal n should satisfy: 1-(1-11.11%) n > 0.9, n at least 20; if it is guaranteed to pick at least one clone with at most two errors and guarantee a 90% success rate, the selected number of clones n should satisfy: 1-(1-27.63%) n >0.9, n at least 8 .
  • the sequencing detection cost of the DNA component synthesis product can be reduced by about two orders of magnitude.
  • the successful use of this method can reduce the single base cost of DNA chip synthesis for industrialization by 0.2 to 0.5 yuan.
  • the DNA assembly synthesis product (PCR purified product) for the artificial chromosome I1 to I6 region was synthesized and assembled using the CustomArray B3 synthesizer.
  • the DNA component synthesis product is linked to the pMD18-T vector:
  • the DNA assembly was amplified using Ex Taq enzyme according to the well-known pMD18-T vector ligation instructions. A single base "A" was added to the 3' end to ligate the amplification product to the pMD18-T vector.
  • the reconstituted vector (pMD18-T vector ligation product) was transformed into E. coli DH5 ⁇ according to the well-known E. coli DH5 ⁇ transformation method, and the DNA module was cloned.
  • Each DNA component corresponds to a numbered monoclonal, and 150 ⁇ L of each is mixed into a 15 mL centrifuge tube to form a sample library. Ten clones were picked from each DNA component, that is, there were 10 sets of sample libraries.
  • the plasmids in the sample library were extracted using a plasmid minitonization kit to construct a set of mixed plasmid sample libraries.
  • the subsequent library sequencing was performed directly using this mixed plasmid sample library.
  • the 10 sets of mixed plasmid samples were sent to the Shenzhen Huada Gene Research Institute Sequencing Production Platform (PGM400), and 10 sets of mixed sequencing libraries with sequencing tags and sequencing linkers required for the PGM400 platform were constructed according to well-known operating instructions.
  • the sequencing library has different sequencing tags; then 10 sets of mixed sequencing libraries are mixed and subjected to high-throughput sequencing of PGM400 to obtain sequence information of the DNA components.
  • Variation detection and filtration were performed using GATK (www.broadinstitute.org/gatk). Use the GenomeAnalysisTK.jar-T RealignerTargetCreator command to perform reference sequence corrections near the inserted and missing fragments.
  • the corrected reference sequences were re-aligned using the GenomeAnalysisTK.jar-T IndelRealigner tool.
  • the mutation detection was performed using the GenomeAnalysisTK.jar-T UnifiedGenotyper tool. The parameters are as follows:
  • Table 1 lists the number of errors per DNA component in each clone (including single base variations, nucleotide insertions and deletions, where B (ie, short for Blank) indicates that the selected clone is empty vector or aligned The number of samples with lower coverage, and the number of errors in clones with the fewest errors in the 10 libraries. Of the total 490 clones, 16 were selected for Sanger sequencing verification, and the mutation detection rate was 100%. Of the 49 DNA components, 33 successfully selected exactly the correct ones out of 10 clones, and 12 only had one false segment, and the results were as expected.
  • B ie, short for Blank

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Abstract

本发明公开了一种DNA合成产物的高通量检测方法，包括如下步骤：将多个DNA组件分别连入克隆载体并转入筛选菌株中，筛选出连接有DNA组件的克隆；分别对克隆进行编号，并培养出相应克隆菌液；选取编号相同的克隆菌液混合并提取质粒；连接测序标签，构建多个混合测序文库，其中不同混合测序文库的测序标签不同；进行混样高通量测序，得到DNA组件的序列；与参考序列进行比对，得到DNA合成产物中具有设定正确率的目标DNA组件。本发明的方法通过混合文库构建策略，将高通量测序技术运用于DNA芯片合成产物的检测，降低产物检测和正确产物筛选成本。

Description

一种DNA合成产物的高通量检测方法 技术领域
本发明涉及DNA合成技术领域，尤其涉及一种DNA合成产物的高通量检测方法。
背景技术
随着遗传科学和遗传工程学的发展，DNA合成技术在生命科学领域扮演着日益重要的角色。DNA元件的从头合成，包括调控序列、全基因、人工代谢通路乃至完整的人工基因组合成，将给人类的生命科学研究带来巨大变革。自1961年人类第一次合成寡核苷酸链以来(Nirenberg et al.(1961)Proc.Natl Acad.Sci.USA 54：1588)，DNA的合成和组装技术已经有了长足发展。目前，DNA长片段的合成和组装，由于技术所限，皆通过100bp以下的寡核苷酸链组装而成(Gibson，et al.(2010)Science 329：52；Gibson.(2009)Nucleic.Acids Res.37：6984；Li&Elledge(2007)Nat.Methods4：251；Bang&Church(2008)Nat.Methods5：37；Shao et al.(2009)Nucleic Acids Res.37：e16)，其成本大致稳定在每bp约2.2元人民币。因此，传统的DNA合成和组装方法，碍于成本所限，难以被应用到基因组水平的DNA合成。
2004年，田敬东等人利用寡核苷酸微阵列芯片，成功在DNA芯片上合成了292种不同的寡核苷酸链并将其组装成为14.6kb的DNA片段，使得基于芯片技术的、高效低成本的大规模的DNA长片段合成和组装成为可能(Tian(2004)Nature 432：1050)。2010年，Kosuri等人利用安捷伦的商业DNA微阵列芯片，实现了数百个基因的合成(Kosuri et al.(2010)Nat.Biotech.28：1295)，并提供了完整和完善的技术路线(Eroshenko et al.(2012)Curr.Protoc.in Chem.Biol.4∶1)。基于此相关技术，2012年，GEN9公司在美国成立，成为第一家在全球范围内提供DNA芯片合成服务的商业公司，其DNA合成产品售价约为每bp0.26美元，低于传统DNA合成的市场价。
DNA芯片合成技术面临的两大挑战，分别是寡核苷酸链的高错误率，以及寡核苷酸文库的高复杂度对组装产生的影响。如何进一步提高DNA芯片合成技术的合成正确率、降低组装和筛选成本，对其商业化应用有着至关重要的作用。在现有的DNA芯片合成技术的技术路线中，DNA合成片段的正确性检测和正 确片段筛选，往往通过第一代测序技术——Sanger测序完成。由于DNA芯片合成本身技术瓶颈所限，组装产物往往组成复杂，与设计序列相比，每个组装后的DNA分子可能在序列内存在复杂度较高的多处变异(包括单碱基变异、核苷酸插入和缺失等)，其单碱基变异概率因平台和设计差异，约为1‰～1％。因此，当单碱基错误率在5‰时，组装一条750bp的DNA片段，可一次性获得完全正确的DNA片段的概率仅为2.33％，出现一个以内错误的概率为11.11％，出现两个以内错误的率为27.63％。欲在复杂的DNA组装产物中挑选实际序列与设计序列完全一致的正确分子，并保证有90％的成功率，需挑选98个单克隆进行Sanger测序，每个Sanger反应成本以20元计，验证成本约1,960元，合每bp 2.61元，成本远超过传统合成方法；若保证至少挑到一个含有至多一处错误的克隆，并保证90％的成功率，需挑选20个单克隆进行Sanger验证，验证成本约400元，合每bp 0.53元；若保证至少挑到一个含有至多两处错误的克隆并保证90％的成功率，需挑选8个单克隆进行Sanger验证，验证成本约160元，合每bp 0.21元。错误超过三个时，除错物料和时间成本较高，DNA芯片合成技术将失去成本优势。可见，传统基于Sanger测序的DNA芯片合成产物正确性检测的方法，成本较高，是DNA芯片合成技术的主要成本来源，也是该技术进一步降低成本的重大瓶颈。
发明内容
本发明提供一种DNA合成产物的高通量检测方法，该方法通过混合文库构建策略，成功将高通量测序技术运用于DNA芯片合成产物的检测，大大降低产物检测和正确产物筛选成本，从而大规模降低DNA芯片合成的成本。
本发明通过如下技术方案实现：
一种DNA合成产物的高通量检测方法，包括如下步骤：
1)将合成产生的多个DNA组件分别连入克隆载体并分别转入筛选菌株中，分别在筛选培养基上培养，筛选出连接有DNA组件的克隆；
2)分别对每个DNA组件的各克隆进行顺次编号；
3)分别选取编号相同但DNA组件不同的克隆混合培养出相应混合菌液，再提取质粒得到混合质粒样本；或分别将每个DNA组件的克隆培养出相应菌液，再选取编号相同但DNA组件不同的克隆的菌液混合并提取质粒得到混合质粒样本；
4)对多个混合质粒样本分别连接测序标签，构建多个混合测序文库，其中不同混合测序文库的测序标签不同；
5)将多个混合测序文库进行混样高通量测序，得到所选取的克隆所载的DNA组件的序列；
6)通过将测序得到的DNA组件的序列与参考序列进行比对，得到DNA合成产物中具有设定正确率的目标DNA组件。
作为本发明的优选方案，DNA合成产物为DNA芯片合成组装出的DNA组件。
作为本发明的优选方案，步骤1)和2)之间，还包括：挑取筛选出的克隆进行菌落PCR验证，进一步筛选出插入片段大小与相应DNA组件的大小一致的克隆。
作为本发明的优选方案，步骤3)中，按照各克隆菌体数量相同的原则进行克隆菌液混合；
优选地，分别将每个DNA组件的克隆培养出的相应菌液保菌备用。
作为本发明的优选方案，步骤4)具体为：分别从混合质粒样本中获取或扩增出混合DNA组件，然后对多个混合DNA组件连接测序标签，构建多个混合测序文库，其中不同混合测序文库的测序标签不同；
优选地，还包括，在连接测序标签之后，对多个混合DNA组件连接测序接头。
作为本发明的进一步的优选方案，步骤4)通过使用酶切或PCR方法，分别从混合质粒样本中获取或扩增出混合DNA组件。
作为本发明的优选方案，步骤5)中使用Illumina HiSeq2000、HiSeq2500、MiSeq、MiSeqDx、NextSeq500、Hiseq X ten、Life SOLiD、Ion Torrent PGM、Proton、Roche 454、Complete Genomics测序装置或单分子测序装置进行高通量测序。
作为本发明的优选方案，设定正确率是指碱基正确率为100％。
作为本发明的优选方案，还包括：当设定正确率低于100％时，通过单点突变进行序列除错；
优选地，当设定正确率低于100％时，挑选单点突变、插入或缺失变异数量最少的克隆，通过单点突变进行序列除错；
优选地，还包括，通过Sanger测序验证所述序列除错产物。
作为本发明的优选方案，还包括：通过Sanger测序验证目标DNA组件。
作为本发明的优选方案，还包括：若有DNA组件未达到设定正确率，则针对这些DNA组件重复步骤2)～6)对尚未选择的克隆进行筛选以及混合测序文库的构建和测序，或重复步骤4)～6)对尚未测序的混合质粒样本进行混合测序文库的构建和测序。
作为本发明的优选方案，步骤6)之后还包括：找到具有设定正确率的目标DNA组件所对应的克隆和/或克隆的菌液，然后提取质粒和/或进行DNA组件扩增。
本发明的方法通过混合测序文库的构建和混样高通量测序策略，成功将高通量测序技术运用于DNA芯片合成产物的检测，利用高通量测序技术成本低的优势，大大降低合成产物检测和正确产物筛选的成本。经计算，DNA组件合成产物的测序检测成本降低约两个数量级，从而大规模降低DNA芯片合成的成本。
附图说明
图1为本发明的用高通量测序策略检测DNA芯片合成产物的流程示意图。
具体实施方式
下面通过具体实施例对本发明作进一步详细说明。除非特别说明，下面实施例中所使用的技术均为本领域内的技术人员已知的常规技术；所使用的仪器设备和试剂等，均为本领域内的技术人员可以通过公共途径如商购等获得的。
本发明中使用的术语说明如下：
DNA组件(DNAAssembly)，是指由DNA合成技术(包括但不限于DNA芯片合成技术)产出的双链DNA片段产物，一般长度在400bp～1.5kbp之间。
目标DNA组件，是指通过本发明方法检测出的碱基正确率达到设定正确率(如碱基正确率为100％)的由DNA合成产出的双链DNA片段产物。
混合测序文库(Mixed Sequencing Library)，简称混合文库(Mixed Library)，是指载有来自不同DNA组件的混合DNA测序文库，其中每个DNA组件仅含单一种类的DNA分子，如来源于单克隆的质粒分子、PCR扩增产物或酶切产物。
请参考图1，本发明一个实施方案中，DNA合成产物的高通量检测方法，大致包括如下过程：
(1)将由DNA芯片合成组装产出的多个DNA组件(多个是指两个以上， 如共400个)通过酶切连接、Gibson组装或者T载体连接等方式连入载体，转化入大肠杆菌等筛选菌株，然后在相应抗性的培养平板上培养，保证长出足够多(如30个)清晰可辨的单克隆。根据需要，可采用带有蓝白斑筛选或其它筛选功能的载体，以降低假阳性克隆率。
(2)作为可选步骤，挑取足够多的单克隆，利用载体通用引物或自行设计的引物(如DNA组件自身内部含有的序列)进行菌落PCR验证，筛选插入片段大小与设计组件大小一致的克隆。通过菌落PCR验证能够进一步降低假阳性克隆率，保证用于构建混合测序文库的克隆中均有目标长度的插入片段，减少对不必要的假阳性克隆的测序，从而提高测序的有效性。
(3)对每个DNA组件的克隆进行顺次编号(如针对组件A，其克隆可顺次编号为#1～#30；针对组件B，其克隆可顺次编号为#1～#20)，用96孔深孔板或试管等对单克隆进行扩培得到扩培菌液，并用深孔板、离心管或平板培养方式进行保菌。
(4)将不同DNA组件的相同编号的扩培菌液等量混合(混合前可估计或测量OD值，以保证混入的各种菌体数量大致相近)，并提取混合质粒DNA，根据相应的克隆编号进行编号(如DNA#1～DNA#30)。
(5)作为可选步骤，用酶切或PCR扩增等方式从混合质粒DNA中获取或扩增出混合DNA组件的DNA分子。该步骤为可选步骤，因为对于不同的高通量测序平台，其测序文库的构建方法各不相同，有些测序文库的构建方法可以直接对线性质粒DNA连接特定测序标签和测序接头来构建用于上机测序的测序文库；而有些测序文库的构建方法需要以DNA组件为起始分子，在两端加特定测序标签和测序接头来构建测序文库。
(6)根据不同高通量测序平台(如Hiseq、Miseq、PGM、Proton、CG、PacBio等)的需求，选取一定数量的混合质粒DNA或混合DNA组件，连接不同的测序标签(index)和/或测序接头(adaptor)，构建多个混合测序文库(如L#1～L#10)。其中，测序标签用于区分不同样本来源的DNA组件，即区分不同混合测序文库中的DNA组件，每个混合测序文库由一个具有特定序列的测序标签，不同混合测序文库的测序标签的序列不同，一般测序标签可以是一段几个或十几个碱基的序列，可以是随机序列，比如8-12个碱基的随机序列等；测序接头是每个高通量测序平台特有的接头序列，不同测序平台的测序接头不同。
(7)将构建好的一定数量的混合测序文库混样上机高通量测序。测序量需求相对较低，若保证每个DNA组件每个克隆覆盖100×，每个DNA组件长度为750bp，同时混样的DNA组件有400种，同时测序10个混合测序文库时，所需数据量仅为300Mbp；若测序分子为带有DNA组件的载体而非DNA组件的PCR或酶切产物，则所需数据为1.1Gbp。
(8)通过生物信息学手段分析下机数据(如BWA比对和GATK分析)，对应测序文库编号，获得每个DNA组件每个克隆的组件序列正确性信息。
(9)选取完全正确或正确率最高的克隆，取用步骤(3)中所保藏的菌株进行质粒DNA提取和/或扩增，用于后续实验。根据具体需要，可以为碱基正确率设定一个设定正确率，比如碱基错误不超过3、2或1个，或碱基正确率为100％等。若测序的DNA组件有碱基错误，可通过单点突变设计突变引物进行序列除错。
(10)作为可选步骤，用Sanger测序验证所选的完全正确或正确率最高的克隆进行序列除错后验证序列的正确性。
(11)若所选若干克隆内，某些DNA组件的正确率仍不理想，可针对不理想的DNA组件，返回步骤(3)挑选尚未测序的克隆进行新的混合文库构建，或返回步骤(6)选取尚未测序的混合质粒DNA或混合DNA组件分子进行新的混合文库(如L#11～L#20)构建，进行新一轮测序验证，直至筛选到正确率满意的克隆为止。
本发明基于高通量测序检测DNA芯片合成产物正确性和实施正确片段筛选的方案相比基于Sanger测序的现有方案的优势分析如下：
以CustomArray公司提供的12k芯片为例，每张芯片能够合成的DNA组件约为400种，每个DNA组件长度为750个碱基，合成总长度为300,000个碱基，单碱基错误率为5‰。此时组装一条750bp的DNA片段，可一次性获得完全正确的DNA片段的概率为0.995750＝2.33％，出现一个以内错误的概率为0.995750+750*0.005*0.995749＝11.11％，出现两个以内错误的率为0.995750+0.005*0.995749+0.0052*0.995748＝27.63％。欲在复杂的DNA组装产物中挑选实际序列与设计序列完全一致的正确分子，并保证有90％的成功率，所挑选单克隆数n应满足：1-(1-2.33％)n＞0.9，n至少为98；若保证至少挑到一个含有至多一处错误的克隆，并保证90％的成功率，所挑选单克隆数n应满足：1-(1-11.11％)n＞ 0.9，n至少为20；若保证至少挑到一个含有至多两处错误的克隆，并保证90％的成功率，所挑选单克隆数n应满足：1-(1-27.63％)n＞0.9，n至少为8。
下面比较基于Sanger测序的现有技术和基于高通量测序的本发明的成本：
1.基于Sanger测序的现有技术
1.1当要求挑选到完全正确的DNA组件的概率高于90％时：
Figure PCTCN2015070459-appb-000001
1.2当要求挑选至多有一处错误的DNA组件的概率高于90％时：
Figure PCTCN2015070459-appb-000002
1.3当要求挑选至多有两处错误的DNA组件的概率高于90％时：
Figure PCTCN2015070459-appb-000003
2.基于高通量测序的本发明方案
2.1当要求挑选到完全正确的DNA组件的概率高于90％时：
Figure PCTCN2015070459-appb-000004
2.2当要求挑选至多有一处错误的DNA组件的概率高于90％时：
Figure PCTCN2015070459-appb-000005
2.3当要求挑选至多有两处错误的DNA组件的概率高于90％时：
Figure PCTCN2015070459-appb-000006
通过以上比较可见，使用本发明的基于高通量测序检测DNA芯片合成产物的技术方案，可将DNA组件合成产物的测序检测成本降低约两个数量级。成功使用该方法，可使用于产业化的DNA芯片合成单碱基成本降低0.2～0.5元。
为验证本发明方法的有效性，采用人工酵母基因组中的人工2号染色体中49个成功组装的DNA组件进行验证。所有DNA组件都为人工设计的酵母基因组片段，长度在450～650bp之间。每个组件挑选了10个克隆，共构建10个混合文库并采用PGM测序进行验证。
实验过程如下所示：
1.DNA组件组装PCR产物获取：
按照厂家操作说明，用CustomArray B3合成仪合成和组装用于2号人工染色体I1～I6区域DNA组件合成产物(PCR纯化产物)。
2.DNA组件合成产物与pMD18-T载体连接：
按照公知的pMD18-T载体连接说明书，使用Ex Taq酶将DNA组件扩增并 在3’末端加上一个单碱基“A”，将扩增产物与pMD18-T载体连接。
3.载体转化：
按照公知的大肠杆菌DH5α转化方法，将重构的载体(pMD18-T载体连接产物)转化到大肠杆菌DH5α中，对DNA组件进行克隆。
4.单克隆鉴定：
转化完成后，涂布平板时进行蓝白斑筛选，每个平板加入20％的IPTG 5μL，X-gal 40μL。在37℃培养箱中培养过夜。待菌斑可以观察时，选择白色的菌株。
5.单克隆扩培、保藏：
每个平板挑取适量的单克隆(本实施例挑取10个)，并相应进行编号(1、2、3、4……10)，在37℃下200rpm转速，培养过夜(至平台期)，保存菌株。
6.混合质粒DNA提取：
每个DNA组件对应编号的单克隆，分别取150μL混合到一个15mL离心管中，组成一个样品库。每个DNA组件挑取10个克隆，即有10组样品库。
使用质粒小提中量试剂盒将样品库中的质粒提取出来，构建成一组混合质粒样本库。直接用这个混合质粒样本库进行后续的建库测序。
7.DNA高通量测序文库构建和测序：
将10组混合质粒样本送至深圳华大基因研究院测序生产平台(PGM400)，按照公知的操作说明书，构建带有PGM400平台需要的测序标签和测序接头的10组混合测序文库，其中各组混合测序文库具有不同的测序标签；然后将10组混合测序文库混样，进行PGM400高通量测序，得到DNA组件的序列信息。
8.测序结果分析
8.1参考序列构建：
提取49个DNA组件设计序列，在每个DNA组件两翼添加pMD18-T载体插入位点旁侧翼序列各200bp，保存为含有49条序列信息的fasta格式的参考序列。使用bowtie2(bowtie-bio.sourceforge.net/bowtie2)、picard tools (broadinstitute.github.io/picard)对参考序列进行index构建。
8.2数据过滤
以Filter_ion_bam.pl(深圳华大基因研究院提供)软件，使用以下参数对下机数据进行过滤：
-minlen 30-lowPhred 10-lowRate 0.01-Ns 0-trim-seed 20。
8.3比对
使用bowtie2进行序列比对，所选参数如下：-N 1--mp 10-R 3-D 20-iS，1，0.50。对于比对结果未中，DNA组件序列比对覆盖度未达98％的样本予以舍弃。
8.4bam文件排序、建库
使用samtools(www.htslib.org)的sort功能对生成的bam文件进行排序，用samtools index构建比对序列index。
8.5比对参考序列
使用GATK(www.broadinstitute.org/gatk)进行变异检测和过滤。使用GenomeAnalysisTK.jar-T RealignerTargetCreator命令进行插入、缺失片段附近参考序列修正。
8.6重比对
使用GenomeAnalysisTK.jar-T IndelRealigner工具对修正后的参考序列重新比对。
8.7变异检测
使用GenomeAnalysisTK.jar-T UnifiedGenotyper工具进行变异检测，选用参数如下：
-stand_call_conf 10.0-stand_emit_conf 0-deletions 1.0-glm BOTH-rf BadCigar。
8.8变异过滤
使用GenomeAnalysisTK.jar-T VariantFiltration工具对所得变异信息进行过滤，选用参数如下：
--filterExpression″QD＜10.0||ReadPosRankSum＜-8.0||FS＞10.0||QUAL＜＄MEANQUAL*0.5″--filterName LowQualFilter--missingValueslnExpressionsShouldEvaluateAsFailing--logging_level ERROR。
8.9变异统计：
移除比对于参考序列前200bp和最后200bp的变异信息(T载体侧翼序列)，对每个DNA组件的每个文库测序结果进行统计。
9.Sanger测序验证：
挑选高通量测序反馈完全正确的DNA组件，选用对应的菌液进行扩培，使 用质粒小提试剂盒提取对应的克隆质粒，送华大基因研究院测序生产平台，按照公知的操作说明书，使用pMD18-T载体通用引物进行Sanger测序。
10.Sanger测序的变异检测：
使用blast(www.ncbi.nlm.nih.gov/blast)、DNAman(www.1ynnon.com)和clustal(www.clustal.org)序列比对软件对Sanger测序结果与对应的参考序列进行比对，分析是否有变异存在。
11.比较高通量测序结果和Sanger测序结果，结果均未检测到变异的载体DNA样本用作后续实验。
实验结果分析如下：
下表1列出了每个DNA组件在每个克隆中的错误数量(包括单碱基变异、核苷酸插入和缺失，其中B(即Blank的简写)表示所选克隆为空载体或比对覆盖度较低的样本)，以及10个文库中出现最少错误的克隆的错误数。共计490个克隆中，已挑选16个进行Sanger测序验证，变异检测一致率为100％。49个DNA组件中，有33个成功在10个克隆内挑选到完全正确的片段，12个仅出现一次错误的片段，效果符合预期。
表1.49个成功组装的DNA组件运用高通量测序策略进行准确性检测的结果
Figure PCTCN2015070459-appb-000007
Figure PCTCN2015070459-appb-000008
Figure PCTCN2015070459-appb-000009
以上内容是结合具体的实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换。

Claims (12)

  1. 一种DNA合成产物的高通量检测方法，其特征在于，包括如下步骤：
    1)将合成产生的多个DNA组件分别连入克隆载体并分别转入筛选菌株中，分别在筛选培养基上培养，筛选出连接有所述DNA组件的克隆；
    2)分别对每个DNA组件的各克隆进行顺次编号；
    3)分别选取编号相同但DNA组件不同的克隆混合培养出相应混合菌液，再提取质粒得到混合质粒样本；或分别将每个DNA组件的克隆培养出相应菌液，再选取编号相同但DNA组件不同的克隆的菌液混合并提取质粒得到混合质粒样本；
    4)对多个所述混合质粒样本分别连接测序标签，构建多个混合测序文库，其中不同混合测序文库的测序标签不同；
    5)将所述多个混合测序文库进行混样高通量测序，得到所选取的克隆所载的DNA组件的序列；
    6)通过将所述测序得到的DNA组件的序列与参考序列进行比对，得到所述DNA合成产物中具有设定正确率的目标DNA组件。
  2. 根据权利要求1所述的DNA合成产物的高通量检测方法，其特征在于，所述DNA合成产物为DNA芯片合成组装出的DNA组件。
  3. 根据权利要求1所述的DNA合成产物的高通量检测方法，其特征在于，所述步骤1)和2)之间，还包括：挑取筛选出的克隆进行菌落PCR验证，进一步筛选出插入片段大小与相应DNA组件的大小一致的克隆。
  4. 根据权利要求1所述的DNA合成产物的高通量检测方法，其特征在于，所述步骤3)中，按照各克隆菌体数量相同的原则进行克隆菌液混合；
    优选地，分别将每个DNA组件的克隆培养出的相应菌液保菌备用。
  5. 根据权利要求1所述的DNA合成产物的高通量检测方法，其特征在于，所述步骤4)具体为：分别从所述混合质粒样本中获取或扩增出混合DNA组件，然后对多个所述混合DNA组件连接测序标签，构建多个混合测序文库，其中不同混合测序文库的测序标签不同；
    优选地，还包括，在连接所述测序标签之后，对多个所述混合DNA组件连接测序接头。
  6. 根据权利要求5所述的DNA合成产物的高通量检测方法，其特征在于，通过酶切或PCR方法，分别从所述混合质粒样本中获取或扩增出混合DNA组 件。
  7. 根据权利要求1所述的DNA合成产物的高通量检测方法，其特征在于，所述步骤5)中使用Illumina HiSeq2000、HiSeq2500、MiSeq、MiSeqDx、NextSeq500、Hiseq X ten、Life SOLiD、Ion Torrent PGM、Proton、Roche 454、Complete Genomics测序装置或单分子测序装置进行高通量测序。
  8. 根据权利要求1所述的DNA合成产物的高通量检测方法，其特征在于，所述设定正确率是指碱基正确率为100％。
  9. 根据权利要求1所述的DNA合成产物的高通量检测方法，其特征在于，还包括：当所述设定正确率低于100％时，通过单点突变进行序列除错；
    优选地，当所述设定正确率低于100％时，挑选单点突变、插入或缺失变异数量最少的克隆，通过单点突变进行序列除错；
    优选地，还包括，通过Sanger测序验证所述序列除错产物。
  10. 根据权利要求1所述的DNA合成产物的高通量检测方法，其特征在于，还包括：通过Sanger测序验证所述目标DNA组件。
  11. 根据权利要求1所述的DNA合成产物的高通量检测方法，其特征在于，还包括：若有DNA组件未达到所述设定正确率，则针对这些DNA组件重复步骤2)～6)对尚未选择的克隆进行筛选以及混合测序文库的构建和测序，或重复步骤4)～6)对尚未测序的混合质粒样本进行混合测序文库的构建和测序。
  12. 根据权利要求1所述的DNA合成产物的高通量检测方法，其特征在于，所述步骤6)之后还包括：找到具有所述设定正确率的目标DNA组件所对应的克隆和/或克隆的菌液，然后提取质粒和/或进行DNA组件扩增。
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CN109517882A (zh) * 2018-11-09 2019-03-26 广州燃石医学检验所有限公司 一种用于检测独特双端文库标签组合的质控方法及应用
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CN108866171A (zh) * 2017-05-10 2018-11-23 深圳华大基因研究院 一种基于新一代测序的古生物物种鉴定方法
CN109517882A (zh) * 2018-11-09 2019-03-26 广州燃石医学检验所有限公司 一种用于检测独特双端文库标签组合的质控方法及应用
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