WO2014079002A1 - Dna二代测序技术中待测样品的追踪方法及检测试剂盒 - Google Patents

Dna二代测序技术中待测样品的追踪方法及检测试剂盒 Download PDF

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WO2014079002A1
WO2014079002A1 PCT/CN2012/084988 CN2012084988W WO2014079002A1 WO 2014079002 A1 WO2014079002 A1 WO 2014079002A1 CN 2012084988 W CN2012084988 W CN 2012084988W WO 2014079002 A1 WO2014079002 A1 WO 2014079002A1
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dna
sample
sequencing
tag
tested
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PCT/CN2012/084988
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English (en)
French (fr)
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陈迪
宋卓
张建光
周代星
高扬
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北京贝瑞和康生物技术有限公司
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Priority to US14/346,413 priority Critical patent/US20150252359A1/en
Application filed by 北京贝瑞和康生物技术有限公司 filed Critical 北京贝瑞和康生物技术有限公司
Priority to CN201280003514.1A priority patent/CN104024426B/zh
Priority to PCT/CN2012/084988 priority patent/WO2014079002A1/zh
Publication of WO2014079002A1 publication Critical patent/WO2014079002A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to the field of clinical detection, and in particular to a tracking method and a detection kit for a sample to be tested in a DNA second generation sequencing technology.
  • BACKGROUND OF THE INVENTION With the advancement of sequencing technology, the traditional Sanger sequencing has not fully met the needs of research, and the second generation sequencing technology with lower cost, higher throughput, faster speed and complete genome sequencing has emerged.
  • the core idea of the second-generation sequencing technology is high-throughput side-synthesis sequencing, which is to determine the sequence of DNA by capturing the newly synthesized end-labels.
  • the existing technology platforms mainly include Roche/454 FLX, Illumina/Genome Analyzer/ Hiseq/Miseq Applied Biosystems SOLID and Life Technologies/Ion Torrent et al.
  • the HiSeq 2000 is currently running at a sequencing throughput of up to 6 people in a genome of 30X, running about 600 G of data at a time, and reducing the operating time of the machine to 30 minutes.
  • the second-generation sequencing technology its application to clinical research has developed rapidly. Studies have shown that the genetic health of the fetus can be judged by sequencing the plasma DNA of pregnant women, and the plasma DNA of the sequencing tester can be used for early screening of cancer, and has a strong application prospect.
  • the present invention is to provide a tracking method and a detection kit for a sample to be tested in a DNA second generation sequencing technology, to solve the technical problem that the sample to be tested in the prior art is confusing and cannot be found in time during manual operation. .
  • a method for tracking a sample to be tested in a DNA second generation sequencing technique is provided.
  • the method comprises the following steps: 1) incorporating a DNA molecule tag of a known sequence into a sample to be tested to obtain a sequenced sample; 2) performing DNA sequencing on the sequenced sample; 3) extracting step 2) molecular tag sequence and molecule in the sequencing result
  • the known sequences of the tags are aligned.
  • the DNA molecular tag is a DNA sequence having a similarity to the DNA to be tested of less than 20% within the length of the sequencing.
  • the sample to be tested is a human blood and/or plasma sample, and the DNA molecular tag is a foreign species DNA or synthetic DNA having a similarity to human genomic DNA of less than 20% within the range of sequencing; the length of the DNA molecular tag is 120 ⁇ 200bp.
  • step 1) further comprising: phosphorylating the 5' end of the DNA molecule tag; and/or pre-phosphorylating the 5' end of the amplification primer of the DNA molecule tag.
  • the ratio of the DNA molecule tag to the blood is lpg ⁇ 1000pg: 1ml of blood; the ratio of the DNA molecule tag to the plasma is 0.1pg ⁇ 1000pg: lml plasma.
  • a detection kit for a sample to be tested in a DNA second generation sequencing technique comprises: a DNA molecule tag of a known sequence, a DNA sequencing primer to be tested, and a sequencing primer for the DNA molecule tag.
  • the DNA molecular tag is a DNA sequence having a similarity to the DNA to be tested of less than 20% within the length of the sequencing.
  • the sample to be tested is a human blood and/or plasma sample
  • the DNA molecular tag is a foreign species DNA or synthetic DNA having a similarity to human genomic DNA of less than 20% within the range of sequencing.
  • the length of the DNA molecular tag is 120 to 200 bp. Since the plasma DNA fragment is about 166 bp long, the length of the DNA molecule tag needs to be adapted.
  • the DNA molecule tag of the known sequence is incorporated into the sample to be tested in the DNA second generation sequencing technology, and then the molecular tag sequence in the sequencing result is compared with the known sequence of the molecular tag to determine the test to be tested. Whether the sample is confusing.
  • FIG. 1 shows a flow chart of the tracking of a plasma/blood sample according to an embodiment of the invention
  • Figure 2 shows a flow chart of the preparation of a DNA molecule tag according to an embodiment of the invention
  • Figure 3A shows A DNA molecule tag gel electrophoresis pattern according to an embodiment of the present invention
  • FIG. 3B shows a gel electrophoresis pattern of a PCR amplification product according to an embodiment of the present invention.
  • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It should be noted that the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict. The invention will be described in detail below with reference to the drawings in conjunction with the embodiments. According to an exemplary embodiment of the present invention, a method for tracking a sample to be tested in a DNA second generation sequencing technique is provided.
  • the tracking method comprises the following steps: 1) incorporating a DNA molecule tag of a known sequence into a sample to be tested to obtain a sequenced sample; 2) performing DNA sequencing on the sequenced sample; 3) extracting step 2) molecular tag sequence in the sequencing result and The known sequences of the molecular tags are aligned. If the information of the molecular tag sequence in the sequencing result coincides with the information of the corresponding molecular tag incorporated in the sample to be tested, it indicates that the sample has no tag sample mark error and cross-contamination between samples. Because the sequencing process of this tag is performed simultaneously in the sequencing process of the DNA molecule to be tested, this method is convenient to operate, and can immediately discover the technical problem that the sample may be confused due to manual operation, which is not only important for scientific research.
  • the DNA molecule tag is a DNA sequence with a similarity to the DNA to be tested of less than 20% within the length of the sequence. In this way, it is convenient to quickly distinguish the DNA molecular tag detection sequence from the sequence of the DNA molecule to be tested from the sequencing result, thereby improving the detection and analysis speed and facilitating the analysis of the batch sample.
  • the sample to be tested is a human blood and/or plasma sample
  • the DNA molecular tag is exogenous DNA or synthetic DNA having a similarity to human genomic DNA of less than 20% within the range of sequencing. If the technical solution of the present invention is applied to clinical testing, the obtained effect is more prominent. Because, in view of the rigor of clinical testing, it is necessary to fundamentally find and eliminate false negatives and false positives caused by sample confusion, and use the technical solution of the present invention to add a certain proportion of exogenous DNA (DNA molecular tag) to plasma and/or blood.
  • the foreign DNA sequence is compared with the incorporation record without affecting the detection, and the sample is determined to be a mixed sample when the sample record is inconsistent with the actual infiltration.
  • the plasma DNA fragment is about 166 bp long
  • the length of the foreign DNA incorporated in the plasma and/or blood sample detection is 120 to 200 bp, preferably 166 ⁇ 10 bp, in order to facilitate the sequencing after constructing the library.
  • the foreign DNA may be derived from other species having poor homology with the human genome, or may be artificially synthesized.
  • step 1) further comprising: phosphorylating the 5' end of the DNA molecule tag; and/or pre-phosphorylating the 5' end of the amplification primer of the DNA molecule tag Processing, is conducive to improve the efficiency of TA clone connection.
  • the ratio of the DNA molecular tag to the blood is lpg ⁇ 1000pg: 1ml blood; the ratio of the DNA molecular tag to the plasma is 0.1pg ⁇ 1000pg: 1ml plasma, and the ratio of the addition amount can effectively and accurately detect the molecule.
  • the label does not affect the rapid and efficient sequence detection of the DNA molecule to be tested.
  • the sequencing in step 2) uses the second-generation sequencing technology, because the second-generation sequencing technology not only has high sequencing throughput, but also accurate results, and is very suitable for application in the technical solution of the present invention.
  • a detection kit for a sample to be tested in a DNA second generation sequencing technique comprises: a DNA molecule tag of a known sequence, a DNA sequencing primer to be tested, and a sequencing primer for the DNA molecule tag.
  • the DNA molecular tag is a DNA sequence having a similarity to the DNA to be tested of less than 20% within the length of the sequencing, thereby facilitating rapid separation of the DNA molecular tag detection sequence from the sequence of the DNA molecule to be detected from the sequencing result, Improve the speed of detection and analysis, and facilitate the analysis of batch samples.
  • the sample to be tested in the DNA second generation sequencing technology is a human blood and/or plasma sample
  • the DNA molecular tag is a foreign species DNA or synthetic DNA having a similarity to human genomic DNA of less than 20% within the range of sequencing.
  • the foreign DNA sequence is compared with the incorporation record without affecting the detection, and the sample is determined to be a mixed sample when the sample record is inconsistent with the actual infiltration. Since the plasma DNA fragment is about 166 bp long, the length of the foreign DNA incorporated in the plasma and/or blood sample detection is 120 to 200 bp, preferably 166 ⁇ 10 bp.
  • the foreign DNA may be derived from other species having poor homology with the human genome, or may be artificially synthesized.
  • the method further comprises: a phosphorylation reagent that phosphorylates the 5' end of the DNA molecule tag; and/or a prephosphorylation reagent that prephosphorizes the 5' end of the amplification primer of the DNA molecule tag.
  • a phosphorylation reagent that phosphorylates the 5' end of the DNA molecule tag
  • prephosphorylation reagent that prephosphorizes the 5' end of the amplification primer of the DNA molecule tag.
  • the exogenous genomic phix fragment with poor homology to human is fragmented, a fragment of a specific length is selected, a single phix fragment sequence is obtained by TA cloning, sequence composition is determined by Sanger sequencing, and the PCR plasmid is amplified to a corresponding length.
  • the 167 bp phix fragment acts as a molecular tag. See Figure 2 for the flow of DNA molecular tag preparation.
  • the exogenous genomic DNA used in this embodiment is the phix genome, but not limited to Phix. Any genome with poor homology to the human genome can be used as a molecular tag. For example, a manually designed synthetic sequence can be used as a molecule. label.
  • the reagents and operation steps used in this embodiment are as follows:
  • the TA cloning vector is TAKARA pMD19-T Vector
  • the plasmid amplification primers are Barcode-F and Barcode-R
  • the 5' end of the primer needs to be phosphorylated:
  • Barcode-F 5' pCCGGGGATCCTCTAGAGAT3 '
  • Barcode-R 5' p ATGCCTGC AGGTCGACGAT3 'DNA tag sequence structure:
  • Sample Plasma samples ImL normal human plasma was added to the corresponding amount of label DNA to extract plasma free DNA.
  • Whole blood sample 5 ng of label DNA was added to the peripheral blood of 1 mL normal human, plasma was separated, and plasma free DNA was extracted. The relationship between sample and label penetration is shown in Table 1: Table 1
  • Klenow enzyme 0.5 ⁇ 1 sterile H20 0 ⁇ total volume 50 ⁇ 1 a.
  • b. Purify the DNA sample on a purification column and elute in 42 ⁇ l of sterile dH 2 0 or elution buffer to obtain blunt-ended DNA.
  • Phusion DNA polymerase (Phusion DNA polymerization 25 ⁇ enzyme mixture)
  • Amplification was carried out using the following PCR protocol: a. 98 ° C for 30 seconds; b. 18 cycles of: 98 ° C for 10 seconds, 65 ° C for 30 seconds, 72 ° C for 30 seconds; c 72 ° C for 5 minutes d. Maintain at 4 ° C.
  • PCR product from step 6 was electrophoresed on a 2% agarose gel. The results are shown in Figure 3b. Then, a 300 bp target band (DNA library) was recovered using Qiagnen kit and eluted at 30 ⁇ . In the elution buffer.
  • Fig. 3a is a gel electrophoresis diagram of a DNA molecular tag used in an embodiment of the present invention.
  • the library of the sample to be tested is sequenced. Although one sample is incorporated with a DNA label, the DNA label incorporated into the other sample can be simultaneously detected at the time of detection, and if only the DNA label incorporated in the sample is detected, The addition of other DNA notes is 0, which can better explain the accuracy of the sample. It can be seen from Table 2 that there is a linear relationship between the amount of label incorporation and the label. From the actual application, the ratio of molecular label infiltration into whole blood is estimated to be lpg ⁇ 100pg: 1ml whole blood; the ratio of molecular label infiltration into whole blood is 0.1pg ⁇ 10pg: Lml plasma is the most efficient.

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Abstract

本发明提供了一种DNA二代测序技术中待测样品的追踪方法及检测试剂盒。其中，该方法包括以下步骤：1）将已知序列的DNA分子标签掺入待测样品，得到测序样品；2）对测序样品进行DNA测序；3）提取步骤2）测序结果中的分子标签序列与分子标签的已知序列进行比对。

Description

DNA二代测序技术中待测样品的追踪方法及检测试剂盒 技术领域 本发明涉及临床检测领域， 具体而言， 涉及一种 DNA二代测序技术中待测样品 的追踪方法及检测试剂盒。 背景技术 随着测序技术的进步， 传统的 Sanger测序已经不能完全满足研究的需要， 费用更 低、 通量更高、 速度更快、 可完成全基因组测序的第二代测序技术应运而生。 第二代 测序技术的核心思想是高通量的边合成边测序， 即通过捕捉新合成的末端的标记来确 定 DNA 的序列， 现有的技术平台主要包括 Roche/454 FLX、 Illumina/Genome Analyzer/Hiseq/Miseq Applied Biosystems SOLID禾口 life Technologies/Ion Torrent等。 以 Illumina产品为例， HiSeq 2000目前运行一次可以达到 6个人基因组 30X覆盖的测 序通量， 运行一次约 600 G数据， 上机操作时间也减少到了 30分钟。 而且随着第二代 测序技术的成熟， 将其应用于临床的研究迅猛发展。研究表明通过测序孕妇血浆 DNA 可以判断胎儿遗传健康状况， 而测序检测者血浆 DNA可以进行癌症早期筛查， 并具 有很强的应用前景。 但是， 随着血浆 DNA检测的普及， 大样品量集中检测时， 样品检测步骤较多， 涉及不少人工操作， 样本混淆的概率会逐渐增加， 样本的追踪和及时发现样品混淆变 得越来越重要。 目前尚没有一种有效方法能够发现血浆 /血液检测过程中的样品混淆问 题。 发明内容 本发明旨在提供一种 DNA二代测序技术中待测样品的追踪方法及检测试剂盒， 以解决现有技术中待测样品在人工操作时易产生混淆且不能被及时发现的技术问题。 为了实现上述目的， 根据本发明的一个方面， 提供了一种 DNA二代测序技术中 待测样品的追踪方法。 该方法包括以下步骤： 1 ) 将已知序列的 DNA分子标签掺入待 测样品， 得到测序样品； 2)对测序样品进行 DNA测序； 3 )提取步骤 2)测序结果中 的分子标签序列与分子标签的已知序列进行比对。 进一步地， DNA分子标签是在测序长度内与待测 DNA相似性低于 20%的 DNA 序列。 进一步地， 待测样品为人的血液和 /或血浆样品， DNA分子标签是在测序范围内 与人基因组 DNA相似性低于 20%的外源物种 DNA或人工合成 DNA; DNA分子标签 的长度为 120~200bp。 进一步地，在步骤 1 )之前进一步包括：对 DNA分子标签的 5'端进行磷酸化处理； 和 /或对 DNA分子标签的扩增引物的 5'端进行预磷酸化处理。 进一步地， DNA分子标签掺入血液的比例为 lpg~1000pg： 1ml血液； DNA分子 标签掺入血浆的比例为 0.1pg~1000pg： lml血浆。 根据本发明的另一个方面， 提供一种 DNA二代测序技术中待测样品的检测试剂 盒。 该试剂盒包括： 已知序列的 DNA分子标签、 待测 DNA测序引物及 DNA分子标 签的测序引物。 进一步地， DNA分子标签是在测序长度内与待测 DNA相似性低于 20%的 DNA 序列。 进一步地， 待测样品为人的血液和 /或血浆样品， DNA分子标签是在测序范围内 与人基因组 DNA相似性低于 20%的外源物种 DNA或人工合成 DNA。 进一步地， DNA分子标签的长度为 120~200bp。 由于血浆 DNA片断长约 166bp， 所以 DNA分子标签的长度需与之相适配。 进一步包括:对 DNA分子标签的 5'端进行磷酸化处理的磷酸化试剂;和 /或对 DNA 分子标签的扩增引物的 5'端进行预磷酸化处理的预磷酸化试剂。 应用本发明的技术方案，将已知序列的 DNA分子标签掺入 DNA二代测序技术中 待测样品， 然后通过测序结果中的分子标签序列与分子标签的已知序列进行比对， 判 断待测样品是否混淆。 因为此标签的测序过程是在待测 DNA分子的测序过程中同时 进行的， 所以此方法操作方便， 且能即时发现可能因人工操作造成的将样品混淆的技 术问题， 这不但对科学研究有重要意义， 如果应用于临床检测， 将极大的提高临床检 测的严谨性。 附图说明 说明书附图用来提供对本发明的进一步理解， 构成本发明的一部分， 本发明的示 意性实施例及其说明用于解释本发明， 并不构成对本发明的不当限定。 在附图中： 图 1示出了根据本发明实施例的血浆 /血液样品的追踪流程图； 图 2示出了根据本发明实施例的 DNA分子标签制备的流程图； 图 3 A示出了根据本发明实施例中的 DNA分子标签凝胶电泳图； 以及 图 3B示出了根据本发明实施例中的 PCR扩增产物的凝胶电泳图。 具体实施方式 需要说明的是， 在不冲突的情况下， 本发明中的实施例及实施例中的特征可以相 互组合。 下面将参考附图并结合实施例来详细说明本发明。 根据本发明一种典型的实施方式， 提供一种 DNA二代测序技术中待测样品的追 踪方法。 该追踪方法包括以下步骤： 1 ) 将已知序列的 DNA分子标签掺入待测样品， 得到测序样品； 2)对测序样品进行 DNA测序； 3 )提取步骤 2)测序结果中的分子标 签序列与分子标签的已知序列进行比对。 如果测序结果中的分子标签序列的信息与待 测样品掺入的相应分子标签的信息相吻合， 说明该样品不存在标签样品标记错误和样 品间交叉污染。 因为此标签的测序过程是在待测 DNA分子的测序过程中同时进行的， 所以此方法操作方便， 且能即时发现可能因人工操作造成的将样品混淆的技术问题， 这不但对科学研究有重要意义， 如果应用于临床检测， 将极大的提高临床检测的严谨 性。 DNA分子标签在测序长度内与待测 DNA序列的相似性越差越便于检测分析准确 率及速度， 优选地， DNA分子标签是在测序长度内与待测 DNA相似性低于 20%的 DNA序列， 这样便于从测序结果中迅速地将 DNA分子标签检测序列与待测 DNA分 子的序列区分开来， 提高检测分析速度， 方便批量化样品的分析。 根据本发明一种典型的实施方式， 待测样品为人的血液和 /或血浆样品， DNA分 子标签是在测序范围内与人基因组 DNA相似性低于 20%的外源物种 DNA或人工合成 DNA。若将将本发明的技术方案应用于临床检测，其所得到的有效果更为突出。因为， 鉴于临床检测的严谨性， 需要从根本上发现并消除样本混淆带来的假阴性和假阳性， 采用本发明的技术方案向血浆和 /或血液中加入一定比例外源 DNA (DNA分子标签）， 通过第二代测序技术， 在不影响检测的情况下， 将外源 DNA序列与掺入记录比较， 当样品记录与实际渗入不一致时确定为混样。 由于血浆 DNA片断长约 166bp， 为了构 建文库后测序的方便， 在进行血浆和 /或血液样品检测时掺入的外源 DNA 长度为 120~200bp, 优选地， 166±10bp。 其中， 外源 DNA可以来自于与人基因组同源性差的 其他物种， 也可以人工合成。 为了方便测序的高效进行， 优选地， 在步骤 1 )之前进一步包括： 对 DNA分子标 签的 5'端进行磷酸化处理； 和 /或对 DNA分子标签的扩增引物的 5'端进行预磷酸化处 理， 有利于提高 TA克隆连接效率。 优选地， DNA分子标签掺入血液的比例为 lpg~1000pg： 1ml血液； DNA分子标 签掺入血浆的比例为 0.1pg~1000pg ： 1ml血浆， 这种比例的添加量既能有效准确的检 测到分子标签， 又不会影响待测 DNA分子快速有效的序列检测。 步骤 2) 中的测序采 用的是二代测序技术， 因为二代测序技术不仅测序通量高， 而且结果准确， 非常适合 在本发明的技术方案中应用。 根据本发明一种典型的实施方式， 提供一种 DNA二代测序技术中待测样品的检 测试剂盒。 该检测试剂盒包括： 已知序列的 DNA分子标签、 待测 DNA测序引物及 DNA分子标签的测序引物。 采用此时试剂盒， 应用本发明的技术方案对 DNA二代测 序技术中待测样品进行检测， 可以极大的提高检测的严谨性。 优选地， DNA分子标签是在测序长度内与待测 DNA相似性低于 20%的 DNA序 列，这样便于从测序结果中迅速地将 DNA分子标签检测序列与待测 DNA分子的序列 区分开来， 提高检测分析速度， 方便批量化样品的分析。 优选地， DNA二代测序技术中待测样品为人的血液和 /或血浆样品， DNA分子标 签是在测序范围内与人基因组 DNA相似性低于 20%的外源物种 DNA或人工合成 DNA。 因为， 鉴于临床检测的严谨性， 需要从根本上发现并消除样本混淆带来的假阴 性和假阳性，采用本发明的技术方案向血浆和 /或血液中加入一定比例外源 DNA(DNA 分子标签）， 通过第二代测序技术， 在不影响检测的情况下， 将外源 DNA序列与掺入 记录比较， 当样品记录与实际渗入不一致时确定为混样。 由于血浆 DNA 片断长约 166bp，在进行血浆和 /或血液样品检测时掺入的外源 DNA长度为 120~200bp，优选地， 166±10bp。 其中， 外源 DNA可以来自于与人基因组同源性差的其他物种， 也可以人 工合成。 优选地， 进一步包括： 对 DNA分子标签的 5'端进行磷酸化处理的磷酸化试剂； 和 /或对 DNA分子标签的扩增引物的 5'端进行预磷酸化处理的预磷酸化试剂。 下面将结合实施例进一步说明本发明的有益效果。 实施例 本实施例的血浆 /血液样品的追踪流程参见图 1。 本实施例采用的是将与人同源性差的外源基因组 phix片断破碎，选取特定长度的 片断， 通过 TA克隆得到单一的 phix片断序列， Sanger测序确定序列组成， PCR质粒 扩增出相应长约 167bp 的 phix片断作为一个分子标签。 DNA分子标签制备的流程参 见图 2。 当然， 本实施例中采用的外源基因组 DNA为 phix基因组， 但不局限于 Phix, 任何与人的基因组同源性差的基因组均可以作为分子标签， 例如， 可以将人工设计合 成的序列做为分子标签。 本实施例中所采用的试剂及操作步骤如下：
1. TA 克隆载体为 TAKARA pMD19-T Vector, 质粒扩增引物为 Barcode-F 和 Barcode-R, 引物 5'端需做磷酸化处理：
Barcode-F: 5' pCCGGGGATCCTCTAGAGAT3 '
Barcode-R: 5' p ATGCCTGC AGGTCGACGAT3 ' DNA标签序列结构：
5' ATGCCTGCAGGTCGACGATT NN... NNNAATCTCTAGAGGATCCCCGG3 '
3' TACGGACGTCCAGCTGCTAANNN... NNTTAGAGATCTCCTAGGGGCC5
DNA标签 1序列:
Figure imgf000007_0001
2. 样品 血浆样品： 取 ImL正常人血浆加入相应量标签 DNA， 抽提血浆游离 DNA。 全血样品： 取 ImL正常人外周血加入 5ng标签 DNA，进行血浆分离，抽提血浆游 离 DNA。 样品与标签渗入量的关系见表 1 : 表 1
Figure imgf000008_0001
3. 末端补平 制备如下的反应混合液 待测样品 DNA溶液 38.5 μΐ
T4 DNA磷酸化缓冲液 （10X) 5 μ1 lO mM dNTP混合液 2 μΐ
T4 DNA聚合酶 2 μΐ
T4 DNA磷酸化酶 2 μΐ
Klenow酶 0.5μ1 无菌 H20 0 μΐ 总体积 50μ1 a. 在 20。C温浴 30分钟； b. 在纯化柱上纯化 DNA样品， 并在 42μ1的无菌 dH20或洗脱缓冲液中洗脱， 得 到平末端 DNA。
4. 在 DNA片段的 3'末端加多聚腺嘌吟尾 制备如下的反应混合液 平末端 DNA 32μ1
Klenow反应缓冲液 （10X) 5μ1 dATP溶液 ΙΟμΙ klenow ex- (3'-5'外切活性缺失） 3μ1 无菌 H20 Ομΐ 总体积 50μ1 a. 在 37°C温浴 30分钟； b. 在柱上纯化 DNA样品， 并在 25μ1的无菌 dH20或洗脱缓冲液中洗脱， 得到末 端补平的、 dA-尾 DNA。
5. 为 DNA片段连接接头 制备如下的反应混合液 末端补平的、 dA-尾 DNA 33μ1 快速连接反应缓冲液 （5X) ΙΟμΙ μΜ DNA接头 2μ1 快速 T4 DNA连接酶 （ EB) 5μ1 总体积 50μ1 a. 在 20°C温浴 15分钟; b. 在 Qiagen柱上纯化回收 DNA样品，并在 25μ1的无菌 dH20或洗脱缓冲液中洗
6. 通过 PCR预扩增富集接头修饰的 DNA片段 制备如下的 PCR反应混合液
DNA (步骤 5中得到的） 12.5 μΐ
Phusion DNA聚合酶（Phusion DNA聚合 25 μΐ 酶混合物）
PCR引物混合物 2 μΐ 超纯水 10.5μ1 总体积 50 μΐ
用如下的 PCR实验方案进行扩增: a. 98 °C 30 秒； b. 18个如下的循环： 98 °C 10 秒， 65 °C 30秒， 72°C 30 秒； c 72 °C 5 分钟； d. 保持在 4°C。
7. 将步骤 6中的 PCR产物置于 2%的琼脂糖凝胶上进行电泳， 结果见图 3b所示， 然后采用 Qiagnen kit切胶回收 300bp目标条带 （DNA文库）， 洗脱于 30 μΐ的洗脱缓 冲液中。 图 3a为本发明实施例采用的 DNA分子标签凝胶电泳图。
8. 将制作好的文库质控后， Illumina Hiseq 2000进行 36bp单端测序。 测序分析结果见表 2。 表 2
Figure imgf000011_0001
对待测样品的文库进行测序， 尽管一个样品掺入的是一种 DNA标签， 但在检测 时可以同时检测加入其他样品所掺入的 DNA标签， 如果只检测出本样品掺入的 DNA 标签， 而其他 DNA便签的掺入量为 0， 则可以更好的说明样品的准确性。 由上表 2可以看出标签掺入量与标签存在线性关系， 从实际应用推算分子标签渗 入全血的比例在 lpg~100pg： 1ml全血； 分子标签渗入全血的比例在 0.1pg~10pg： lml 血浆检测效率最高。 从表 2的数据也可以看出， 在本实施例的样品检测过程中没有样 品混淆的情况出现。 以上所述仅为本发明的优选实施例而已， 并不用于限制本发明， 对于本领域的技 术人员来说， 本发明可以有各种更改和变化。 凡在本发明的精神和原则之内， 所作的 任何修改、 等同替换、 改进等， 均应包含在本发明的保护范围之内。

Claims

权 利 要 求 书
1. 一种 DNA二代测序技术中待测样品的追踪方法， 其特征在于， 包括以下步骤：
1 ) 将已知序列的 DNA分子标签掺入所述待测样品， 得到测序样品；
2) 对所述测序样品进行 DNA测序；
3 )提取步骤 2)测序结果中的分子标签序列与所述分子标签的已知序列进 行比对。
2. 根据权利要求 1所述的追踪方法， 其特征在于， 所述 DNA分子标签是在测序 长度内与待测 DNA相似性低于 20%的 DNA序列。
3. 根据权利要求 1 所述的追踪方法， 其特征在于， 所述待测样品为人的血液和 / 或血浆样品，所述 DNA分子标签是在测序范围内与人基因组 DNA相似性低于 20%的外源物种 DNA 或人工合成 DNA; 所述 DNA 分子标签的长度为 120~200bp。
4. 根据权利要求 3所述的追踪方法， 其特征在于， 在所述步骤 1 ) 之前进一步包 括：
对所述 DNA分子标签的 5'端进行磷酸化处理； 和 /或
对所述 DNA分子标签的扩增引物的 5'端进行预磷酸化处理。
5. 根据权利要求 3所述的追踪方法， 其特征在于， 所述 DNA分子标签掺入血液 的比例为 lpg~1000pg ： lml 血液； 所述 DNA 分子标签掺入血浆的比例为 0.1pg~1000pg： lml血浆。
6. 一种 DNA二代测序技术中待测样品的检测试剂盒， 其特征在于， 包括： 已知 序列的 DNA分子标签、待测 DNA测序引物及所述 DNA分子标签的测序引物。
7. 根据权利要求 6所述的检测试剂盒， 其特征在于， 所述 DNA分子标签是在测 序长度内与待测 DNA相似性低于 20%的 DNA序列。
8. 根据权利要求 6所述的检测试剂盒， 其特征在于， 所述待测样品为人的血液和 / 或血浆样品，所述 DNA分子标签是在测序范围内与人基因组 DNA相似性低于 20%的外源物种 DNA或人工合成 DNA。
9. 根据权利要求 8所述的检测试剂盒， 其特征在于， 所述 DNA分子标签的长度 为 120~200bp。
10. 根据权利要求 8所述的检测试剂盒， 其特征在于， 进一步包括： 对所述 DNA 分子标签的 5'端进行磷酸化处理的磷酸化试剂； 和 /或对所述 DNA分子标签的 扩增引物的 5 '端进行预磷酸化处理的预磷酸化试剂。
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