WO2023284768A1 - 融合引物直扩法人类线粒体全基因组高通量测序试剂盒 - Google Patents

融合引物直扩法人类线粒体全基因组高通量测序试剂盒 Download PDF

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WO2023284768A1
WO2023284768A1 PCT/CN2022/105384 CN2022105384W WO2023284768A1 WO 2023284768 A1 WO2023284768 A1 WO 2023284768A1 CN 2022105384 W CN2022105384 W CN 2022105384W WO 2023284768 A1 WO2023284768 A1 WO 2023284768A1
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primer
sequencing
fusion
kit
seq
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潘雅姣
曲保旺
陈朝辉
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北京爱普益生物科技有限公司
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    • 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
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    • C12Q1/6869—Methods for sequencing

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  • the invention relates to the technical fields of forensic medicine, criminal investigation and physical evidence identification, in particular to a high-throughput sequencing kit for human mitochondrial whole genome by fusion primer direct expansion method.
  • Mitochondrial DNA plays an important role in the study of human origin evolution and forensic identification.
  • mitochondrial DNA has the characteristics of maternal inheritance and no recombination, which can be used to study human origin evolution.
  • mitochondrial DNA Compared with nuclear DNA, mitochondrial DNA has a high copy number and stable structure, and can still be preserved intact under harsh conditions. It is suitable for the identification of old and highly degraded samples in forensic medicine.
  • the human mitochondrial genome sequence is a circular genetic material with a length of 16569bp. According to the function, it can be divided into a control region with a length of 1122bp and a coding region with a length of 15447bp.
  • the control region contains 3 hypervariable regions with good polymorphism .
  • HTS high-throughput sequencing
  • MPS massive parallel sequencing technology
  • the purpose of the present invention is to provide a high-throughput sequencing kit for human mitochondrial whole genome by fusion primer direct expansion method, which has low detection cost and is easy to operate.
  • the present invention provides a high-throughput sequencing kit for human mitochondrial whole genome by fusion primer direct expansion method, including a library preparation kit, a sequencing template preparation kit and a sequencing kit;
  • the library preparation kit contains multiple PCR primer pools labeled with different sample labels, DNA extraction-free PCR amplification enzyme, PCR reaction buffer, 2800 control DNA and DNA purification magnetic beads.
  • the multiplex PCR primer pool includes the sample tag sequences shown in SEQ ID NO: 91-129 and tcacgaata.
  • the sequencing template preparation kit is from Thermo Fisher Corporation of the United States.
  • the sequencing kit is from Thermo Fisher Scientific, USA.
  • the PCR system consists of a special amplification enzyme against PCR inhibitory components in blood and a corresponding buffer buffer;
  • Emulsion DNA polymerase chain reaction obtains the sequencing template, and forms an independent PCR micro-reaction pool by covering the particles carrying a single DNA fragment with emulsion, so as to realize the independent parallel amplification of the entire fragment library;
  • the present invention adopts the above-mentioned human mitochondrial whole genome high-throughput sequencing kit by the direct expansion method of fusion primers to form a complete set of kits suitable for high-throughput DNA sequencing platforms to realize parallel and stable testing of multiple samples of mitochondrial whole genomes.
  • the kit is free of DNA extraction and connection-free library construction. A single measurement can be completed within one working day. One measurement can realize the whole genome detection of mitochondria from dozens to hundreds of people. The cost and operation time of the test allow large-scale library construction. .
  • the human mitochondrial whole genome detection kit based on high-throughput DNA sequencing of the present invention includes all reagents for library preparation, water-in-oil PCR sequencing template preparation and high-throughput sequencing process, and has the following technical effects:
  • Fig. 1 is a technical flow chart of the preparation of a human mitochondrial whole genome high-throughput sequencing kit by fusion primer direct expansion method of the present invention
  • Fig. 2 is under the free DNA extraction PCR system (10mL), the electrophoresis schematic diagram of multiplex PCR amplification efficiency of different template types;
  • Figure 3 is a schematic diagram of the sequencing results of the full mitochondrial genome sequence using the Ion S5XLTM sequencing platform.
  • Proliferator library refers to DNA fragments with different adapters connected at both ends, of which one side is a sequencing adapter: it can contain sample tags to distinguish the sequencing results of different samples; the other side is a fixed adapter: used to connect to capture particles.
  • the structure of the proliferator library is as follows: P linker-target amplicon-sample tag-A linker general part.
  • Fusion primers composed of target fragment-specific primers, adapters and sample tags, PCR amplification enzymes and buffers capable of amplifying blood sources, blood samples can be directly obtained by multiplex PCR amplification, composed of multiple STR target fragments, And the two ends are connected with different adapter sequences, and the DNA library with sample tags saves multiple steps of DNA extraction, single-plex PCR, PCR product mixing and adapter connection in the construction of existing high-throughput DNA sequencing libraries.
  • Table 1 Composition of PCR system for constructing sequencing library by direct amplification method
  • Fusion primers are long primer sequences that include sequencing adapters, fixed adapters, and sample tags in addition to target fragment-specific primers.
  • the structure of the fusion primer is as follows, where the A linker is the sequencing primer region, the P linker is the capture particle binding region, and the sample label is used to distinguish different samples.
  • target fragment-specific primers are used to amplify target fragments of the whole human mitochondrial genome;
  • adapter sequences include fixed adapters and sequencing adapters, which are used to bind capture magnetic beads and sequencing primers respectively to complete subsequent water-in-oil PCR and sequencing reactions.
  • the sample label sequence is used to distinguish different samples.
  • NC_012920.1 Cambridge revised version of the human mitochondrial genome sequence Primer5.0 online tool was used to design primers for the target fragment.
  • the full length of the NC_012920.1 reference genome is 16569bp.
  • Table 2 lists the design distribution of primers. Sequence numbers 1 to 44 are upstream primers, and Sequence numbers 45 to 88 are corresponding downstream primers.
  • the primers include the end and beginning of the mitochondrial reference genome (mitochondrion is circular ).
  • the specificity and content of the amplified product were detected by agarose electrophoresis, and the accuracy of the sequence of the amplified product was detected by sequencing to prove its usability.
  • the present invention selects the Thermo Fisher Scientific sequencing system as the detection platform, and its corresponding linker sequence is shown in Table 3.
  • the P adapter is a fixed adapter, which is used to bind DNA capture beads
  • the A adapter is a sequencing adapter, which is used for sequencing with universal primers.
  • serial number connector sequence 89
  • a connector CCATCTCATCCCTGCGTGTCTCCGACTCAG 90
  • P connector CCTTCCTATGGGCAGTCGGTGAT
  • the samples tested in parallel are all distinguished by unique sample labels. On the premise of sufficient throughput, the number of samples tested in parallel is determined by the number of available sample labels. Table 4 lists 40 sample labels verified by the present invention.
  • the Taq enzyme protein product of the engineering bacteria has the function of resisting blood PCR inhibitory components.
  • a PCR enhancer such as (NH 4 ) 2 SO 4 and having a higher pH.
  • the performance verification results show that the PCR reaction system can still maintain the ideal multiplex PCR amplification efficiency in the case of containing anti-blood-derived PCR inhibitor components.
  • lanes 1 and 2 are 10 ng genomic DNA templates
  • lanes 3 and 4 are blood sheet templates with a diameter of 1 mm
  • M is a 100 bp marker.
  • DNA polymerase chain reaction emPCR
  • emPCR DNA polymerase chain reaction
  • the PCR reagent in the water phase is emulsified with the reagent in the oil phase to form an emulsion, and the magnetic beads carrying the template are mixed with the emulsion and enter into the droplets, wherein each droplet is a water-in-oil micro-reaction pool.
  • dNTP deoxyribonucleoside triphosphate
  • Steps 2-4 are repeated until the sequencing of the entire DNA fragment is completed.
  • Data quality control filter the raw data according to the length and quality of the sequencing
  • mtDNA Human mitochondrial whole genome
  • Library preparation kit including 40 sets of multiple PCR primer pools labeled with different sample labels, DNA extraction-free PCR amplification enzyme, PCR reaction buffer, 2800 control DNA, DNA purification magnetic beads;
  • Sample Take whole blood spotted on the filter paper matrix as the sample.
  • 10mL multiplex PCR system includes the following components:
  • step c Equilibrate the mixed solution in step a for 5 minutes at room temperature to achieve the highest recovery effect.
  • high-throughput DNA sequencing templates were prepared by water-in-oil PCR and positive product enrichment.
  • the reagents used are 3 small product numbers in 510/520/530TM Kit- chefs (A34019): Ion S5 chefsupplies (A27755), Ion chef solutions (A27754), and Ion 510/520/530chef regents (A34018).
  • the experimental steps are as follows, and you can also refer to the operating instructions of the 510/520/530 TM Kit- chefs (A34019) kit.
  • the 2 library holes in the upper right corner of the Ion PGM TM Hi-Q TM View Chef Reagents reagent chuck are used to add sequencing libraries, namely the purified products of step 2
  • Chip Type 520chip
  • the reagents used are 2 small product numbers in 510/520/530 TM Kit- chefs (A34019): Ion S5 sequencing solutions (A27767), IonS5 sequencing regents (A27768).
  • the experimental steps are as follows, for details, please refer to the 510/520/530 TM Kit- chefs (A34019) kit instructions.
  • the operation steps of the S5 device are as follows:
  • the amount of data obtained by different chips is different. Taking one of the 520 chips as an example, the result is shown in Figure 3.
  • the effective area in the chip reaches 94%, and 99% of which is connected with the library.
  • the final effective library was 59.7%, with a total of 6.9 ⁇ 106 read fragments, and the average read length of the fragments was 310bp, generating the total amount of raw sequencing data 2.13Gb.
  • the sequencing data is classified into different sample folders, the results are as follows:
  • sample number Number of point mutations sample number Number of point mutations sample number Number of point mutations 1 33 14 15 27 10 2 34 15 9 28 34 3 10 16 26 29 34 4 9 17 26 30 33 5 9 18 14 31 35 6 15 19 twenty two 32 40 7 15 20 6 33 29 8 33 twenty one 34 34 40 9 26 twenty two 33 35 39 10 27 twenty three 10 36 9 11 15 twenty four twenty three 37 10 12 10 25 twenty two 38 10 13 9 26 twenty one 39 39
  • sample 1 Compared with the reference sequence (NC_012920.1 Cambridge Revised Version Human Mitochondrial Whole Genome Sequence), the sample’s mitochondrial 4883, 5153, 5178, 5301, 7028, 8701, 8857, 8860, 9180, 9540, 9667, 10397, 10398, 10400, 11176, 11719, 12705, 15043, 15301, 15326, 15724, 16223, 165192 A total of 33 point mutations occurred (Table 8). Six of them occurred in mitochondrial hypervariable regions (hypervariable region 1:16024-16569, hypervariable region 2:1-576), and the remaining 27 occurred in coding regions.
  • the reference sequence NC_012920.1 Cambridge Revised Version Human Mitochondrial Whole Genome Sequence
  • the human mitochondrial whole genome (mtDNA) high-throughput sequencing kit with fusion primer direct expansion method increases the polymorphic information content, reduces the probability of random matching of personnel, and improves the ability of mitochondrial maternity screening.
  • the present invention adopts the above-mentioned human mitochondrial whole genome high-throughput sequencing kit by the direct amplification method of fusion primers, which has low detection cost and is easy to operate.

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Abstract

本发明公开了一种融合引物直扩法人类线粒体全基因组高通量测序试剂盒，包括文库制备试剂盒、测序模板制备试剂盒和测序试剂盒；其中所述文库制备试剂盒包含不同样本标签标记的多重PCR引物池、免DNA提取PCR扩增酶、PCR反应缓冲液、2800对照DNA和DNA纯化磁珠。本发明的融合引物直扩法人类线粒体全基因组高通量测序试剂盒，检测成本低，操作方便。

Description

融合引物直扩法人类线粒体全基因组高通量测序试剂盒
本申请要求于2021年07月13日提交中国专利局、申请号为“202110788220.5”、发明名称为“融合引物直扩法人类线粒体全基因组高通量测序试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及法医学、刑侦及物证鉴定技术领域，尤其是涉及一种融合引物直扩法人类线粒体全基因组高通量测序试剂盒。
背景技术
线粒体DNA在人类起源进化研究和法医身份识别领域具有重要作用，其中，线粒体DNA具有母系遗传不重组的特点，可利用该遗传特征进行人类起源进化研究。与核DNA相比，线粒体DNA拷贝数高、结构稳定，在恶劣的条件下仍能完整保存，适用于法医陈旧检材和高度降解检材鉴定。
人类线粒体基因组序列是一个长度为16569bp的环状遗传物质，按照功能可分长度为1122bp的控制区和长度为15447bp的编码区，其中在控制区内包含3个高变区，具有良好多态性。
目前，对线粒体的研究常常局限于控制区的高变区、部分特征性编码区单核苷酸多态性(single nucleotide polymorphism，SNP)。但是，仅对线粒体部分区域进行检测会降低多态信息含量，导致随机匹配人数增加并降低***排除能力，增加冤假错案增加的可能。
毫无疑问，线粒体全基因组数据将会比现在提供更准确的信息。目前多数的线粒体基因组数据是由传统的Sanger测序法获得，但这个工作费时费力。高通量测序技术(high-throughput sequencing，HTS)，又称大量并行测序技术(Massive parallelsequencing，MPS)，一次可对多个样本的几十万到几百万条DNA分子进行序列测定，为DNA分析提供了一种全新的技术手段。但是，MPS技术需要进行DNA提取、连接等操作，过程复杂，工作效率低。
发明内容
本发明的目的是提供一种融合引物直扩法人类线粒体全基因组高通量测序试剂盒，检测成本低，操作方便。
为实现上述目的，本发明提供了一种融合引物直扩法人类线粒体全基因组高 通量测序试剂盒，包括文库制备试剂盒、测序模板制备试剂盒和测序试剂盒；
文库制备试剂盒包含不同样本标签标记的多重PCR引物池、免DNA提取PCR扩增酶、PCR反应缓冲液、2800对照DNA和DNA纯化磁珠。
优选的，多重PCR引物池包括SEQ ID NO：91～129所示的样本标签序列以及tcacgaata。
优选的，测序模板制备试剂盒来自美国赛默飞公司。
优选的，测序试剂盒来自美国赛默飞公司。
融合引物直扩法人类线粒体全基因组高通量测序试剂盒，制备步骤如下：
(1))设计由特异引物、样本标签和接头序列组成的融合引物；
(2)建立免DNA提取的PCR体系：PCR体系由抗血液中PCR抑制成分的特殊扩增酶和对应缓冲液buffer组成；
(3)建立免DNA提取、融合引物直扩的基因座文库；
(4)乳液DNA聚合酶链式反应(emulsion PCR，ePCR)获得测序模板，通过携带单一DNA片段的颗粒经乳液覆盖形成独立的PCR微反应池，实现整个片段文库的独立平行扩增；
(5)高通量DNA测序；
(6)数据分析及展示报告结果。
因此，本发明采用上述一种融合引物直扩法人类线粒体全基因组高通量测序试剂盒，形成全套适用于高通量DNA测序平台，实现多样本线粒体全基因组平行、稳定测试的试剂盒。
试剂盒免DNA提取、免连接文库构建，单次测定可在一个工作日内完成，一次测定实现几十到几百人份的线粒体全基因组检测，测定成本和操作时间容许大批量的建库使用。
本发明的基于高通量DNA测序的人类线粒体全基因组检测试剂盒，包含文库制备、油包水PCR测序模板制备和高通量测序流程的全部试剂，具有如下技术效果：
(1)线粒体全基因组序列检测，提高多态信息含量；
(2)实现多样本的平行测试，提高检测效率；
(3)免DNA提取、免连接的直扩法建库，文库构建时间压缩至2个小时，单次测定时间压缩至一个工作日。
下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。
附图说明
图1是本发明一种融合引物直扩法人类线粒体全基因组高通量测序试剂盒的制备技术流程图；
图2是免DNA提取PCR体系(10mL)下，不同模板类型对多重PCR扩增效率电泳示意图；
图3是应用Ion S5XLTM测序平台对线粒体基因组全序列测序结果示意图。
具体实施方式
以下通过附图和实施例对本发明的技术方案作进一步说明。
实施例一
直扩法测序文库的构建
增殖子文库指两端连有不同接头的DNA片段，其中，一侧为测序接头：可以含样本标签，以区分不同样本的测序结果；另外一侧为固定接头：用于连接捕获颗粒。
增殖子文库结构如下：P接头-目的扩增子-样本标签-A接头通用部分。
由目的片段特异性引物、接头和样本标签组成的融合引物，具有血源扩增能力的PCR扩增酶和缓冲液，血液样本经多重PCR扩增即可直接获得由多个STR目的片段组成，并且两端连有不同接头序列，带有样本标签的DNA文库，节约了现有高通量DNA测序文库构建的DNA提取、单重PCR、PCR产物混合和接头连接多个步骤。
表1直扩法构建测序文库的PCR体系组成
Figure PCTCN2022105384-appb-000001
一、融合引物设计
融合引物是除了目的片段特异性引物外，还含有其他包括测序接头、固定接 头、样本标签的长引物序列。融合引物结构如下，其中A接头为测序引物区，P接头为捕获颗粒结合区，样本标签用以区分不同样本。
上游融合引物：
5'-A接头(30个碱基)样本标签(10个碱基)目的片段上游引物(19-23碱基)-3'
下游融合引物：
5'-P接头(23个碱基)目的片段下游引物(19-23碱基)-3'
其中，目的片段特异引物用于扩增人类线粒体全基因组目的片段；接头序列包括固定接头和测序接头，分别用于绑定捕获磁珠及测序引物，以完成后续的油包水PCR和测序反应。样本标签序列用于区分不同样本。
(1)人线粒体全基因组目的片段引物设计及验证
以NC_012920.1剑桥修正版人类线粒体全基因组序列为基础，采用Primer5.0在线工具进行目的片段引物设计。NC_012920.1参考基因组全长16569bp，表2列出引物设计分布，序列编号1～44是上游引物，序列编号45～88是对应的下游引物，引物包含线粒体参考基因组末尾及开头(线粒体为环状)。采用琼脂糖电泳对扩增产物的特异性和含量进行检测，采用测序法对扩增产物序列的准确性进行检测，证明其可用性。
表2特异引物的序列对应线粒体基因组位置
Figure PCTCN2022105384-appb-000002
(2)接头序列设计(选择高通量测序平台)
不同的高通量测序平台均具有特定的接头序列，具有以下特点：①基于离子 半导体测序原理，测序成本低；②快速，上机测序只需2～3小时③测序读长200～400碱基，可以满足测序读长需要；④灵活性强，具有满足不同通量需求的多种芯片。
本发明选择赛默飞测序***为检测平台，其对应的接头序列见表3。P接头即固定接头，用于绑定DNA捕获磁珠，A接头即测序接头用于以通用引物测序。
表3接头序列
序列编号 接头 序列
89 A接头 CCATCTCATCCCTGCGTGTCTCCGACTCAG
90 P接头 CCTCTCTATGGGCAGTCGGTGAT
(3)样本标签序列设计及验证
平行检测的样本均由唯一的样本标签加以区分，在通量足够的前提下，平行检测的样本数由可用的样本标签数决定，表4列出经过本发明验证的40个样本标签。
表4样本标签序列
Figure PCTCN2022105384-appb-000003
二、建立血源直扩的PCR扩增体系(包括PCR酶和缓冲液)
筛选基因突变的Taq工程菌，由于N端含约10个氨基酸的缺失突变，该工程菌的Taq酶蛋白产物具有抗血液PCR抑制成分的功能。配合含有(NH 4) 2SO 4等PCR增强剂、且具有较高的pH值的PCR缓冲液。性能验证结果显示，在含有抗血源PCR抑制成分的情况下，该PCR反应体系仍能保持理想的多重PCR扩增效 率。在图2中，1、2泳道为10ng基因组DNA模板，3、4泳道为直径1mm血片模板，M为100bp marker。
三、建立平衡的多重PCR体系
通过调整多重PCR扩增体系中各引物对的比例，实现各STR目的扩增产物量的平衡，多重PCR体系的各引物池内44对融合引物比例见表5。
表5各引物池内44对融合引物比例表
Figure PCTCN2022105384-appb-000004
实施例二
油包水微反应器中的DNA聚合酶链式反应(emPCR)以获得测序模板
(1)上述多重PCR直接扩增生成的文库内容经P接头被固定在捕获磁珠上，使每个磁珠携带一个单一的DNA片段。
(2)将水相的PCR试剂与油相的试剂乳化，形成了乳液，携带模板的磁珠与乳液混合后进入液滴中，其中每个液滴为一个油包水的微反应池。
(3)整个片段文库的扩增在每个油包水微反应池中平行进行，形成测序模板。
实施例三
平行批量式DNA测序
采用高通量DNA测序仪进行，例如赛默飞的PGM或S5/S5XL，测序反应采用边合成边测序的方式进行。
(1)上述文库内容的emPCR产物经A接头与测序通用引物结合；
(2)4种脱氧核糖核苷三磷酸(dNTP，N为A、G、C、T)依次参入PCR合成体系；
(3)当加入的dNTP与测序模板配对时，发生DNA聚合反应；
(4)DNA聚合反应释放的H离子引发pH变化被识别，完成1个碱基的测序；
(5)重复步骤2～4，直至整条DNA片段的测序完成。
实施例四
数据分析及报告结果
(1)数据质控：根据测序长度和质量，对原始数据进行过滤；
(2)测序信息归类：根据测序结果中的样本标签序列进行，测序结果能够被有效归类至不同样本文件夹中。
(3)根据与标准参考序列比对，发现样本线粒体基因组序列的变异。
实施例五
40份样品的线粒体全基因组序列平行检测
(一)实验材料
试剂：融合引物直扩法人类线粒体全基因组(mtDNA)高通量测序试剂盒，包括：
1)文库制备试剂盒，含40套由不同样本标签标记的多重PCR引物池，免DNA提取PCR扩增酶，PCR反应缓冲液，2800对照DNA、DNA纯化磁珠；
2)测序模板制备试剂盒(购自美国赛默飞公司)；
3)测序试剂盒(购自美国赛默飞公司)。
样本：以点制于滤纸基质的全血为样本。
(二)实验步骤
1.文库制备
1)样本制备
以直径1mm打孔器，按一定顺序将39份血片打入96孔PCR板，每个样本取血片1份，每个96孔板的第1孔加入1ng 2800 control DNA。
2)多重PCR
将保存于96孔板的40个多重PCR体系，以每孔10mL加入到对应的装有血片的PCR板中。10mL多重PCR体系包括如下组分：
Figure PCTCN2022105384-appb-000005
按照如下程序进行PCR：
Figure PCTCN2022105384-appb-000006
3)PCR产物纯化
a、每孔取5μL PCR产物混合，放入1.5mL的EP管中，振荡混匀后取50μL用于纯化。
b、吸取50μL PCR混合产物到一个1.5mL EP管中，再加入60mL的纯化磁珠(磁珠需提前平衡至室温)，将移液器调至150μL吸打10次混匀。
c、将步骤a中的混合液在室温下平衡5分钟以达到最高回收效果。
d、将混合物放在磁力架上，静置10分钟。
e、移除上清液后，将离心管从磁力架取下来。
f、吸取200μL 70％乙醇到离心管中，吸打10次以充分清洗磁珠，然后将离心管放置磁力架上静置2分钟，并移除上清。
g、重复步骤e一遍。
h、磁力架静置10分钟以充分干燥磁珠。
i、将EP管从磁力架拿出，加入50μL无核酸酶水洗脱，吸打混匀，室温静 置30分钟，期间吸打混匀2-3次。
j、EP管放回磁力架，静置2分钟。取48μL上清液转移到一个新的1.5mL EP管，-20℃保存备用。
4)测序模板制备
以纯化后PCR产物0.75ng为模板，通过油包水PCR和阳性产物富集，制备高通量DNA测序模板。所用试剂为510/520/530TM Kit-Chef(A34019)中的3个小货号组分：Ion S5 chefsupplies(A27755)、Ion chef solutions(A27754)、Ion 510/520/530chef regents(A34018)。实验步骤如下，也可参照510/520/530 TM Kit-Chef(A34019)试剂盒操作说明。
Chef油包水PCR及芯片loading
a、试剂及耗材入位
将Ion PGM TM Hi-Q TM View Chef Reagents、Ion PGM TM Hi-Q TM Chef Solutions分别放入Ion 520/530Kit chef里的27756和27754试剂卡盘；
Ion PGM TM Hi-Q TM View Chef Reagents试剂卡盘的右上角2个文库孔，用来加测序文库，即步骤2的纯化产物
放入耗材、芯片。
b、创建运行程序
登录Server开始Plan创建
Plan→Templates→Whole Genome→Plan New Run→IonReporter→Application→Next
参数设置
Sample Preparation Kit：空白；
Library Kit Type：空白；
Template Kit：Ion 520&530Kit Chef
Templating Size：400；
Sequencing Kit：Ion S5 Sequencing Kit
Flow：520或840；
Chip Type：520chip
Plugins：FileExporter
输入项目名称，开始运行
Projects→添加新的“Project”→Plan→Run Plan Name填写实验名称。
5)高通量DNA测序
将上述loading好的芯片放入S5或S5XL设备开始测序，所用试剂为510/520/530 TM Kit-Chef(A34019)中的2个小货号组分：Ion S5 sequencing solutions(A27767)、IonS5 sequencing regents(A27768)。实验步骤如下，具体也可参考参照510/520/530 TM Kit-Chef(A34019)试剂盒说明书。
S5设备的操作步骤如下：
a、打开电源开关；
b、触屏点initialize；
c、放Ion 520/530Kit chef里小货号27767的Ion S5 Wash solution bottle；
d、放Ion 520/530Kit chef里小货号27767的Ion S5 cleaning solutionbottle；
e、废液缸清空；
f、放Ion 520/530Kit chef里小货号27768的Ion S5 sequencing regentcartridge；
g、放步骤3loading好的芯片；
h、一切就绪后点击Next开始。
6)数据分析
a、数据质控结果
不同芯片得出的数据量有所差别，以其中一块520芯片为例，结果如图3。芯片中的有效区域达94％，其中连接有文库的为99％。除去28.4％的多克隆，11.9％的低质量文库和0％的测试片段，最终有效文库为59.7％，共6.9×10 6个读取片段，片段平均读长为310bp，产生原始测序数据总量为2.13Gb。
b、测序信息归类
根据样本标签序列信息，测序数据被归类至不同样本文件夹，结果如下：
表6样本标签归类结果
Figure PCTCN2022105384-appb-000007
Figure PCTCN2022105384-appb-000008
c、序列微变异识别
有937Mb与参考序列匹配，占总序列数的44％，平均测序深度为1500×，其中达到与参考序列99％匹配的数据量为851Mb。以突变比例≥80％，测序条数≥50作为筛选标准，对线粒体基因组内序列微变异进行筛查。除去barcode001为质控品2800，其他39个血卡样本的突变位点分布如表7。
表739个血卡样本的点突变个数分布
样本编号 点突变个数 样本编号 点突变个数 样本编号 点突变个数
1 33 14 15 27 10
2 34 15 9 28 34
3 10 16 26 29 34
4 9 17 26 30 33
5 9 18 14 31 35
6 15 19 22 32 40
7 15 20 6 33 29
8 33 21 34 34 40
9 26 22 33 35 39
10 27 23 10 36 9
11 15 24 23 37 10
12 10 25 22 38 10
13 9 26 21 39 39
以样本1为例，与参考序列(NC_012920.1剑桥修正版人类线粒体全基因组序列)相比，该样本在线粒体基因组的第73、150、489、1048、1107、1438、2706、4048、4769、4883、5153、5178、5301、7028、8701、8857、8860、9180、9540、9667、10397、10398、10400、11176、11719、12705、15043、15301、15326、15724、16223、16362、16519号碱基共发生33个点突变(表8)。其中6个发生在线粒体高变区(高变区1：16024-16569，高变区2：1-576)，其余27个均发生在编码区。相比局限于高变区的线粒体测序方法，融合引物直扩法人类线粒体全基因组(mtDNA)高通量测序试剂盒提高了多态信息含量，减少了人员随机匹配几率， 提高线粒体母系排查能力。
表81号样本线粒体全基因组测序的单碱基突变
Figure PCTCN2022105384-appb-000009
Figure PCTCN2022105384-appb-000010
因此，本发明采用上述一种融合引物直扩法人类线粒体全基因组高通量测序试剂盒，检测成本低，操作方便。
最后应说明的是：以上实施例仅用以说明本发明的技术方案而非对其进行限制，尽管参照较佳实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对本发明的技术方案进行修改或者等同替换，而这些修改或者等同替换亦不能使修改后的技术方案脱离本发明技术方案的精神和范围。

Claims (12)

  1. 一种融合引物，包括上游融合引物和下游融合引物；所述上游融合引物从5'至3'端依次串联有A接头、样本标签和目的片段上游引物；
    所述下游融合引物从5'至3'端依次串联有P接头和目的片段下游引物；
    所述A接头为测序接头，用于以通用引物测序；所述P接头为捕获颗粒结合区，用于绑定捕获磁珠；
    所述目的片段上游引物和目的片段下游引物用于扩增人类线粒体全基因组目的片段；
  2. 根据权利要求1所述的融合引物，其特征在于，所述A接头的长度为30bp；所述样本标签的长度为10bp；所述目的片段上游引物的长度为19～23bp；所述P接头的长度为23bp；所述目的片段下游引物的长度为19～23bp。
  3. 根据权利要求1所述的融合引物，其特征在于，所述A接头的核苷酸序列如SEQ ID NO：89所示；所述P接头的核苷酸序列如SEQ ID NO：90所示。
  4. 多重PCR引物池，包括不同样本标签标记的权利要求1～3任意一项所述融合引物。
  5. 根据权利要求4所述的多重PCR引物池，其特征在于，所述融合引物包括SEQ ID NO：1～88所示的目的片段特异性引物；SEQ ID NO：1～44所示的目的片段特异性引物为上游引物，SEQ ID NO：45～88是对应的下游引物，其中SEQ ID NO：1所示的上游引物和SEQ ID NO：45所示的下游引物对应，SEQ ID NO：2所示的上游引物和SEQ ID NO：46所示的下游引物对应，依次类推。
  6. 根据权利要求5所述的多重PCR引物池，其特征在于，各引物池内44对融合引物比例表如下所示：
    Figure PCTCN2022105384-appb-100001
    Figure PCTCN2022105384-appb-100002
  7. 根据权利要求4所述的多重PCR引物池，其特征在于，所述样本标签的核苷酸序列为tcacgaata和SEQ ID NO：91～129所示核苷酸序列。
  8. 一种文库制备试剂盒，包括权利要求4～7任意一项所述的多重PCR引物池。
  9. 根据权利要求8所述的文库制备试剂盒，其特征在于，还包括：免DNA提取PCR扩增酶、PCR反应缓冲液、2800对照DNA和DNA纯化磁珠。
  10. 一种融合引物直扩法人类线粒体全基因组高通量测序试剂盒，包括权利要求8或9所述的文库制备试剂盒、测序模板制备试剂盒和测序试剂盒。
  11. 根据权利要求10所述的融合引物直扩法人类线粒体全基因组高通量测序试剂盒，其特征在于，所述测序模板制备试剂盒来自美国赛默飞公司。
  12. 根据权利要求10所述的融合引物直扩法人类线粒体全基因组高通量测序试剂盒，其特征在于，所述测序试剂盒来自美国赛默飞公司。
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