WO2023030259A1 - 一种基于二代测序技术检测微单倍型基因座的引物组合物、试剂盒和方法及其应用 - Google Patents

一种基于二代测序技术检测微单倍型基因座的引物组合物、试剂盒和方法及其应用 Download PDF

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WO2023030259A1
WO2023030259A1 PCT/CN2022/115540 CN2022115540W WO2023030259A1 WO 2023030259 A1 WO2023030259 A1 WO 2023030259A1 CN 2022115540 W CN2022115540 W CN 2022115540W WO 2023030259 A1 WO2023030259 A1 WO 2023030259A1
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pcr
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primer composition
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张素华
李成涛
陈安琪
陶瑞旸
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司法鉴定科学研究院
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Definitions

  • the present invention relates to the technical field of forensic medicine, in particular to a primer composition, kit, method and application thereof for detecting microhaplotypes (microhaplotype, MH) based on next-generation sequencing technology.
  • the primer composition is used to amplify and cover 22 pairs of 163 MHs of autosomes.
  • Forensic physical evidence mainly relies on the detection and analysis of DNA genetic markers to solve problems related to individual identification and paternity identification in forensic identification.
  • STR has good polymorphism and simple typing method, and is the most commonly used type of genetic marker in the field of forensic medicine for individual identification and paternity testing.
  • the SNP and InDel of the two alleles have the advantages of low mutation rate and short amplified fragments, which can make up for the shortcomings of high STR mutation rate, large amplified fragments, and stutter peaks in typing. It is more advantageous in the direction of source inference.
  • MH a polymorphic site containing two or more SNPs within a DNA segment of 200-300 bp.
  • Micro-haplotypes composed of SNPs not only have high polymorphisms comparable to STR loci, but do not produce stutter peaks, while retaining the characteristics of low mutation rate and short fragments of SNPs.
  • the 74 micro-haplotype marker complex systems constructed by Oldoni et al. and the 118 micro-haplotype marker complex systems constructed by La Puente et al. have individual recognition ability far superior to the existing STR amplification system, and have good individual Identification and paternity competency.
  • MH markers can also be used in paternity cases involving STR mutations as well as involving close relatives involved.
  • STR typing detection For the analysis and detection of mixture samples, traditional STR typing detection often shows multiple equal allele peaks, and it is difficult to distinguish the stutter peak from the allele peak with a small contribution ratio.
  • the splitting of the results and the interpretation of the evidence value are both Difficulties in the industry.
  • MH has no stutter peak interference, high allelic polymorphism, has the advantages of both STR and SNP markers, and has high sensitivity. It is an ideal genetic marker for mixture detection and analysis.
  • MH composed of ancestry informative SNP (AI-SNP) can provide an important basis for population structure research and forensic ancestry inference.
  • AI-SNP ancestry informative SNP
  • the system consisting of 31 MH markers originally established by Kidd can better distinguish the five major geographical regions of Africa, Europe, Southeast Asia, East Asia, America and Pacific Islands, showing the superiority of MH as an ancestry information marker.
  • Next generation sequencing also known as massively parallel sequencing
  • NGS Next generation sequencing
  • Detection and application provide the platform.
  • MH is composed of multiple SNPs, which are sequence polymorphisms in nature, and next-generation sequencing can obtain all MH types in the multiple amplification system at one time, and realize parallel analysis and detection of a large number of genetic markers.
  • the present invention screens the MH loci with forensic application value, and based on the next-generation sequencing technology, develops and establishes a primer composition and kit that can simultaneously detect 163 MH loci in a single pass .
  • the present invention adopts the following technical solutions:
  • the first aspect of the present invention is to provide a primer composition for detecting MH loci based on next-generation sequencing technology, which primer composition includes one or more pairs of amplification primers for 163 MH loci;
  • the above-mentioned amplification primer composition includes one or more pairs of primers whose nucleotide sequences are SEQ ID No.1-326.
  • the above-mentioned primer composition includes a primer whose nucleotide sequence is SEQ ID No.1-326.
  • the second aspect of the present invention is to provide a kit for detecting MH based on next-generation sequencing technology including the above primer composition, which also includes a PCR mixture and a PCR reaction solution.
  • the third aspect of the present invention is to provide a method for detecting MH based on next-generation sequencing technology using the above kit, including the following steps:
  • Step 1 taking the sample to be tested, extracting the sample DNA and quantifying it;
  • Step 2 Prepare a complex amplification system and perform the first round of multiplex PCR; after the reaction is completed, add the purified reaction solution to purify the product, and then perform magnetic bead sorting;
  • Step 3 perform filling and repairing, add A (adenine) and linker connection, and purify the product again by using purified magnetic beads;
  • Step 4 performing PCR reaction on the purified eluted product to construct a library, the reaction system used includes eluted product, PCR mixture, QU reagent, mixed capture post-P5 primer and mixed capture pre-p7 primer;
  • Step 5 library purification and quantification: use purified magnetic beads to purify the product, and use Qubit for library quantification and quality control;
  • Step 6 on-machine sequencing and data analysis: Place the constructed library on the MiSeq FGx TM platform for sequencing analysis; for the obtained sequencing data, use the Trimmomatic software to sequence the adapters, and then use the BWA software to compare the sequencing sequences with the human reference genome hg19 For comparison, use the Python tool to obtain the MH typing.
  • the concentration of sample DNA is 5 ng/ ⁇ L.
  • the above compound amplification system is 20 ⁇ L, including 8 ⁇ L PCR mixture, 2 ⁇ L PCR reaction solution, 8 ⁇ L primer mixture and 2 ⁇ L sample DNA.
  • the concentration of the primer mixture is 0.5 ⁇ M.
  • reaction conditions of the multiplex PCR in step 2 are: 95°C, pre-denaturation for 15 minutes; 95°C, denaturation for 30 seconds, 60°C, annealing for 90 seconds, 72°C, extension for 30s, cycle 24 times; 72°C, heat preservation 10 minutes.
  • reaction system of filling and repairing plus A in step 3 is 50 ⁇ L, including 42 ⁇ L of the purified product in step 2, 6.8 ⁇ L of end repairing and tailing buffer, and 1.2 ⁇ L of end repair and tailing enzyme.
  • reaction conditions for filling and repairing plus A in step three are: 30°C, 30 minutes; 65°C, 30 minutes; 4°C, keep warm.
  • the adapter ligation reaction system in step 3 is 80 ⁇ L, including 50 ⁇ L of the purified product in step 3, 2.5 ⁇ L of adapter mixture, 16 ⁇ L of ligation buffer, 10 ⁇ L of ligase, and 1.5 ⁇ L of Nuclease-free water.
  • reaction conditions for linker connection in step three are: 25°C, 15 minutes; 4°C, keep warm.
  • the PCR reaction system of step four is 50 ⁇ L, including 14 ⁇ L of the eluted product of step three, 25 ⁇ L of PCR mixture, 3 ⁇ L of QU reagent, 5 ⁇ L of mixed capture post-P5 primer, and 5 ⁇ L of mixed capture pre-p7 primer.
  • the PCR reaction conditions in step 4 are: 37°C, 15 minutes; 98°C, pre-denaturation for 45 seconds; 98°C, denaturation for 15 seconds, 60°C, annealing for 30 seconds, 72°C, extension for 30 seconds, cycle 10 times ; 72°C, 5 minutes; 4°C, keep warm.
  • the fourth aspect of the present invention is to provide the application of the above primer composition or kit in individual identification, kinship identification, mixture analysis and family origin inference.
  • MH01CP008 MH01CP012, MH01CP016, MH01KK117, MH01KK205, MH01KK211, MH02KK134, MH02KK136 ⁇ MH04CP002 ⁇ MH04CP003 ⁇ MH04CP007 ⁇ MH04KK030 ⁇ MH05CP004 ⁇ MH05CP006 ⁇ MH05KK020 ⁇ MH05KK170 ⁇ MH06CP003 ⁇ MH06CP007 ⁇ MH09KK153 ⁇ MH10CP003 ⁇ MH10KK163 ⁇ MH11CP003 ⁇ MH11CP005 ⁇ MH11KK180 ⁇ MH12KK046 ⁇ MH12KK202 ⁇ MH13CP008 ⁇ MH13KK213 ⁇ MH13KK217 ⁇ MH13KK218 ⁇ MH13KK225 ⁇ MH14CP003 ⁇ MH14CP004 , MH15CP001, MH15KK066, MH16KK255, MH16KK30
  • the primer composition is used to construct, purify and quantify the genomic DNA samples extracted from biological samples or mixed biological samples, and place them on the MiSeq FGx TM platform for sequencing analysis, and finally analyze the obtained sequencing data, Obtain MH typing.
  • the present invention adopts the above technical scheme, and compared with the prior art, it has the following technical effects:
  • the primer composition for detecting MH loci based on the next-generation sequencing technology provided by the present invention involves 163 MH loci covering 22 pairs of autosomes, and can provide more new genetic information compared with systems constructed in the past. At the same time, compared with the next-generation sequencing kit of the STR loci, the present invention has better detection ability of the mixture.
  • the MH loci involved in the present invention all have high ancestry information content and can distinguish populations in Africa, Europe, South Asia and East Asia. Therefore, in addition to individual identification and kinship identification, they can also be applied to family origin inference.
  • Fig. 1 is the statistics of the sequencing results of detecting DNA with different concentration gradients by the method provided in Example 1 of the present invention
  • Figure 2 is the result of detecting the sequencing uniformity of DNA with different concentration gradients by the method provided in Example 1 of the present invention
  • Fig. 3 is the principal component analysis results of the method provided in Example 1 of the present invention in 27 global populations.
  • the present invention relates to a primer composition for detecting MH markers based on next-generation sequencing technology, which includes one or more pairs of amplification primers for 163 MHs;
  • the 163 MHs are all derived from the MH loci recorded in the ALFRED website and the MHs published in the literature, distributed in the intron region, polymorphic in the Asian population, and the distribution length is ⁇ 300bp. Its name, chromosome information and locus information are shown in Table 1:
  • Table 1 The names, chromosome information and SNP information of 163 MH loci
  • the above-mentioned primer composition includes one or more pairs of primers whose nucleotide sequence is SEQ ID No.1-326, and the specific primer sequence information is as follows:
  • the above-mentioned primer composition includes primers whose nucleotide sequences are SEQ ID No.1-326.
  • the present invention also relates to a kit for detecting MH based on next-generation sequencing technology, including the above-mentioned primer composition, which also includes a PCR mixture and a PCR reaction solution.
  • the methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared according to conventional methods unless otherwise specified.
  • This embodiment provides a method for detecting MH markers using the above primer composition or kit based on next-generation sequencing technology, including the following steps:
  • the PCR reaction conditions are: 95°C, pre-denaturation for 15 minutes; 95°C, denaturation for 30 seconds, 60°C, annealing for 90 seconds, 72°C, extension for 30 seconds, cycle 24 times; 72°C, incubation for 10 minutes.
  • After the reaction is completed add 1 ⁇ L of purified reaction solution to purify the product, and complete the following reactions: 37°C for 10 minutes, 50°C for 10 minutes, 65°C for 10 minutes, 4°C, and keep warm. Then perform magnetic bead sorting.
  • the PCR reaction conditions are: 30°C for 30 minutes, 65°C for 30 minutes, 4°C, and keep warm.
  • the PCR reaction conditions are: 25°C, 15 minutes, 4°C, heat preservation.
  • the reaction product is then purified using purification magnetic beads.
  • the PCR reaction conditions are: 37°C, 15 minutes; 98°C, pre-denaturation for 45 seconds; 98°C, denaturation for 15 seconds, 60°C, annealing for 30 seconds, 72°C, extension for 30 seconds, cycle 10 times; 72°C, 5 minutes; 4°C, keep warm.
  • On-machine sequencing and data analysis Place the constructed library on the MiSeq FGx TM platform for sequencing analysis; for the obtained sequencing data, use Trimmomatic software to sequence the joints, and then use BWA software for sequence comparison to compare the sequences with human The reference genome (hg19) was compared, and the MH was obtained using Python tools.
  • This method can be used for family origin inference, specifically based on the analysis of the MH typing results of all 163 loci.
  • the average heterozygosity of the 48 loci reached 0.7227
  • the polymorphic information content was greater than 0.60
  • the average individual identification probability reached 0.8692
  • the cumulative individual identification probability was 1-8.26 ⁇ 10 -44
  • the cumulative non-parent exclusion rate of doublets and triplets were 1-1.26 ⁇ 10 -8 and 1-8.27 ⁇ 10 -16 , respectively.
  • This example is a comparison of the analysis performance of the method provided in Example 1 and the STR kit of the next-generation sequencing platform for mixture samples.
  • Table 7 The unique allele detection rate of the minor contributors of the two genetic markers in DNA mixture samples with different mixing ratios
  • primer composition, kit and method provided by the present invention provide new detection means for individual identification, kinship identification, mixture analysis and family origin inference in the field of forensic science.

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Abstract

本发明属于法医学技术领域，提供了一种基于二代测序技术检测微单倍型基因座的引物组合物、试剂盒和方法及其应用，用于扩增人类基因组上163个微单倍型基因座；该引物组合物包括序列如SEQ ID NO.1～326所示的引物中的一对或多对。本发明可用于个人识别、亲缘鉴定和族源推断。

Description

一种基于二代测序技术检测微单倍型基因座的引物组合物、试剂盒和方法及其应用 技术领域
本发明涉及法医学技术领域，具体涉及一种基于二代测序技术检测微单倍型(microhaplotype,MH)的引物组合物、试剂盒和方法及其应用，该引物组合物用于扩增覆盖22对常染色体的163个MH。
背景技术
法医物证学主要依靠检测和分析DNA遗传标记，解决司法鉴定中有关个体识别和亲权鉴定方面的问题。在多类遗传标记中，STR多态性好，分型方法简单，是法医学领域进行个体识别与亲权鉴定时最常用的一类遗传标记。二等位基因的SNP与InDel具有突变率低、扩增片段短等优势，可弥补STR突变率高、扩增片段大、分型存在stutter峰等缺点，在降解检材的分析以及生物地理族源推断方向上更具优势。然而，二等位基因遗传标记由于单个位点多态性较低，常常需要通过增加检测位点数目方可达到与STR体系相近的检测效能。因此，一些学者提出了复合遗传标记的概念，包括连锁遗传标记SNP-STR、InDel-STR、multi-InDel等。
2013年，美国耶鲁大学Kidd教授提出了MH的概念，即在200-300bp的DNA区段内，包含2个或以上SNP的多态性位点。由SNP构成的微单倍型，不仅拥有足以与STR基因座比拟的高多态性，不会产生stutter峰，同时保留了SNP突变率低、片段短的特点，在亲权鉴定和个体识别中具有优势。例如，Oldoni等构建的74个微单倍型标记复合体系以及由La Puente等构建的118个微单倍型标记复合体系，其个体识别能力远超现有的STR扩增体系，具有良好的个体识别和亲权鉴定能力。此外，MH标记还可用于涉及STR突变以及涉及近亲属参与的亲权鉴定案件。
对于混合物检材的分析检测，传统STR分型检测常表现为多个等个等位基因峰，难以区分stutter峰与贡献比例较小的等位基因峰，结果的拆分及证据价值诠 释均是行业难点。MH不存在stutter峰干扰，等位基因多态性高，兼具STR和SNP标记的优势，灵敏度高，是混合物检测分析的理想遗传标记。
一些SNP由于长期迁徙进化，在不同种群间频率分布差异极大，含有丰富的族源信息。筛选由族源信息SNP(Ancestry informative SNP,AI-SNP)组成的MH可为群体结构研究以及法医学族源推断(ancestry inference)提供重要依据。Kidd最初建立的包含31个MH标记的体系便可较好的区分非洲、欧洲、东南亚、东亚以及美洲和太平洋岛屿这五个主要地理区域，显示了MH作为祖先信息标记的优越性。
二代测序(next generation sequencing,NGS)，又称大规模并行测序，有着高通量和高准确性等优点，不仅可以验证并深入研究传统遗传标记分型技术的结果，也为新型遗传标记的检测和应用提供了平台。MH由多个SNP组成，其本质上是序列多态性，而二代测序能一次性获得复合扩增体系中所有的MH分型，实现大量遗传标记的并行分析检测。
发明内容
为了克服现有技术中的缺陷，本发明筛选具有法医学应用价值的MH基因座，并基于二代测序技术，开发并建立了一个单次可同时检测163个MH基因座的引物组合物和试剂盒。
为实现上述目的，本发明采用如下技术方案：
本发明的第一方面是提供一种基于二代测序技术检测MH基因座的引物组合物，该引物组合物包括163个MH基因座的扩增引物中的一对或多对；
上述163个MH基因座包括MH01CP007、MH01CP008、MH01CP012、MH01CP016、MH01KK001、MH01KK070、MH01KK072、MH01KK106、MH01KK117、MH01KK172、MH01KK205、MH01KK210、MH01KK211、MH02CP004、MH02KK003、MH02KK004、MH02KK073、MH02KK102、MH02KK105、MH02KK131、MH02KK134、MH02KK136、MH02KK138、MH02KK139、MH02KK201、MH02KK202、MH02KK213、MH02KK215、MH03KK006、MH03KK007、MH03KK008、MH03KK009、MH03KK216、 MH04CP002、MH04CP003、MH04CP007、MH04KK010、MH04KK011、MH04KK013、MH04KK015、MH04KK016、MH04KK017、MH04KK019、MH04KK028、MH04KK029、MH04KK030、MH04KK074、MH05CP004、MH05CP006、MH05CP010、MH05KK020、MH05KK022、MH05KK062、MH05KK078、MH05KK079、MH05KK122、MH05KK123、MH05KK124、MH05KK170、MH06CP003、MH06CP007、MH06KK026、MH06KK030、MH06KK031、MH06KK080、MH06KK101、MH07KK030、MH07KK031、MH07KK081、MH07KK082、MH08KK032、MH09KK020、MH09KK033、MH09KK034、MH09KK152、MH09KK153、MH09KK157、MH09KK161、MH10CP003、MH10KK083、MH10KK084、MH10KK085、MH10KK086、MH10KK087、MH10KK088、MH10KK101、MH10KK163、MH10KK170、MH11CP003、MH11CP004、MH11CP005、MH11KK036、MH11KK037、MH11KK038、MH11KK039、MH11KK040、MH11KK041、MH11KK089、MH11KK090、MH11KK091、MH11KK180、MH11KK187、MH11KK191、MH12KK042、MH12KK043、MH12KK045、MH12KK046、MH12KK092、MH12KK093、MH12KK202、MH13CP008、MH13KK047、MH13KK213、MH13KK217、MH13KK218、MH13KK225、MH13KK226、MH14CP003、MH14CP004、MH14KK048、MH14KK101、MH15CP001、MH15CP003、MH15CP004、MH15KK066、MH15KK067、MH15KK069、MH15KK095、MH16KK053、MH16KK062、MH16KK096、MH16KK255、MH16KK302、MH17CP001、MH17CP006、MH17KK014、MH17KK052、MH17KK053、MH17KK054、MH17KK055、MH17KK077、MH17KK105、MH17KK110、MH17KK272、MH18CP003、MH18CP005、MH18KK285、MH18KK293、MH19CP007、MH19KK056、MH19KK057、MH19KK299、MH19KK301、MH20KK058、MH20KK059、MH20KK307、MH21KK313、MH21KK315、MH21KK316、MH21KK324、MH22KK060、MH22KK064和MH22KK303。
进一步地，上述扩增引物组合物包括核苷酸序列为SEQ ID No.1～326的引物的一对或多对。
进一步优选地，上述引物组合物包括核苷酸序列为SEQ ID No.1～326的引 物。
本发明的第二方面是提供一种包括上述引物组合物的基于二代测序技术检测MH的试剂盒，其还包括PCR混合液和PCR反应液。
本发明的第三方面是提供采用上述试剂盒的基于二代测序技术检测MH的方法，包括如下步骤：
步骤一，取待测样品，提取样本DNA并进行定量；
步骤二，配制复合扩增体系，并进行第一轮多重PCR；反应完成后，加入纯化反应液纯化产物，再进行磁珠分选；
步骤三，进行补平修复加A(腺嘌呤)和接头连接，再次利用纯化磁珠纯化产物；
步骤四，对纯化后的洗脱产物进行PCR反应构建文库，采用的反应体系包括洗脱产物、PCR混合液、QU试剂、混捕post-P5引物和混捕pre-p7引物；
步骤五，文库的纯化和定量：利用纯化磁珠纯化产物，采用Qubit进行文库定量与质控；
步骤六，上机测序及数据分析：将构建好的文库放置于MiSeq FGx TM平台进行测序分析；对于获得的测序数据，采用Trimmomatic软件去测序接头，然后采用BWA软件将测序序列与人类参考基因组hg19进行比对，利用Python工具获取MH分型。
进一步地，样本DNA的浓度为5ng/μL。
进一步地，上述复合扩增体系为20μL，包括8μL PCR混合液、2μL PCR反应液、8μL引物混合液和2μL样本DNA。
进一步地，引物混合液的浓度为0.5μM。
进一步地，步骤二中的多重PCR的反应条件为：95℃，预变性15分钟；95℃，变性30秒，60℃，退火90秒，72℃，延伸30s，循环24次；72℃，保温10分钟。
进一步地，步骤三中的补平修复加A的反应体系为50μL，包括步骤二的纯化产物42μL，末端修复加尾缓冲液6.8μL，末端修复加尾酶1.2μL。
进一步地，步骤三中的补平修复加A的反应条件为：30℃，30分钟；65℃，30分钟；4℃，保温。
进一步地，步骤三中的接头连接的反应体系为80μL，包括步骤三中的纯化产物50μL，接头混合液2.5μL，连接缓冲液16μL，连接酶10μL，Nuclease-free water 1.5μL。
进一步地，步骤三中的接头连接的反应条件为：25℃，15分钟；4℃，保温。
进一步地，步骤四的PCR反应体系为50μL，包括步骤三的洗脱产物14μL，PCR混合液25μL，QU试剂3μL，混捕post-P5引物5μL，混捕pre-p7引物5μL。
进一步地，步骤四中的PCR反应条件为：37℃，15分钟；98℃，预变性45秒；98℃，变性15秒，60℃，退火30秒，72℃，延伸30秒，循环10次；72℃，5分钟；4℃，保温。
本发明的第四方面是提供上述引物组合物或者试剂盒在个体识别、亲缘关系鉴定、混合物分析及族源推断中的应用。
进一步地，上述个体识别和亲缘关系鉴定主要根据48个多态性较好的MH基因座的分型结果，48个MH基因座包括：MH01CP008、MH01CP012、MH01CP016、MH01KK117、MH01KK205、MH01KK211、MH02KK134、MH02KK136、MH04CP002、MH04CP003、MH04CP007、MH04KK030、MH05CP004、MH05CP006、MH05KK020、MH05KK170、MH06CP003、MH06CP007、MH09KK153、MH10CP003、MH10KK163、MH11CP003、MH11CP005、MH11KK180、MH12KK046、MH12KK202、MH13CP008、MH13KK213、MH13KK217、MH13KK218、MH13KK225、MH14CP003、MH14CP004、MH15CP001、MH15KK066、MH16KK255、MH16KK302、MH17CP001、MH17CP006、MH17KK272、MH18CP003、MH18CP005、MH19CP007、MH19KK299、MH20KK058、MH20KK307、MH21KK315、MH21KK324。
进一步地，采用引物组合物对生物检材或混合生物检材提取得到的基因组DNA样本进行文库构建、纯化及定量，并放置于MiSeq FGx TM平台进行测序分析，最后对获得的测序数据进行分析，获取MH分型。
本发明采用以上技术方案，与现有技术相比，具有如下技术效果：
本发明提供的基于二代测序技术检测MH基因座的引物组合物涉及覆盖22对常染色体的163个MH基因座，与过去构建的体系相比能够提供更多新的遗 传信息。同时，本发明相较于STR基因座的二代测序试剂盒表现出更优秀的混合物检测能力。此外，本发明涉及的MH基因座均具有较高的祖先信息含量，能够区分非洲、欧洲、南亚和东亚地区的群体，因此除个体识别和亲缘鉴定外，还可应用于族源推断。
附图说明
图1为本发明实施例1提供的方法检测不同浓度梯度DNA的测序结果统计；
图2为本发明实施例1提供的方法检测不同浓度梯度DNA的测序均一度的结果；
图3为本发明实施例1提供的方法在全球27个种群中的主成分分析结果。
具体实施方式
本发明涉及一个基于二代测序技术检测MH标记的引物组合物，其包括163个MH的扩增引物一对或多对；
其中，所述的163个MH均源自ALFRED网站中已收录的MH基因座和文献中已发表的MH，分布于内含子区域、在亚洲人群中多态性较好、分布长度≤300bp，其名称、染色体信息及其位点信息如表1所示：
表1 163个MH基因座的名称、染色体信息及包含的SNP信息
Figure PCTCN2022115540-appb-000001
Figure PCTCN2022115540-appb-000002
Figure PCTCN2022115540-appb-000003
Figure PCTCN2022115540-appb-000004
Figure PCTCN2022115540-appb-000005
Figure PCTCN2022115540-appb-000006
Figure PCTCN2022115540-appb-000007
根据筛选MH标记的物理位置，设计多重PCR引物。设计原则包括：(1)最优的熔链温度；(2)避免引物二聚体和发夹结构；(3)GC含量在20％～80％之间；(4)进行脱靶分析以减少引物脱靶杂交；(5)进行重叠分析以减少引物的数量。在本发明一优选的实施方式中，上述引物组合物包括核苷酸序列为SEQ ID No.1～326的引物一对或多对，具体的引物序列信息如下：
表2 163个MH标记的扩增引物序列号以及引物序列
Figure PCTCN2022115540-appb-000008
Figure PCTCN2022115540-appb-000009
Figure PCTCN2022115540-appb-000010
Figure PCTCN2022115540-appb-000011
Figure PCTCN2022115540-appb-000012
Figure PCTCN2022115540-appb-000013
Figure PCTCN2022115540-appb-000014
Figure PCTCN2022115540-appb-000015
Figure PCTCN2022115540-appb-000016
Figure PCTCN2022115540-appb-000017
Figure PCTCN2022115540-appb-000018
Figure PCTCN2022115540-appb-000019
Figure PCTCN2022115540-appb-000020
在本发明一优选的实施方式中，上述引物组合物包括核苷酸序列为SEQ ID No.1～326的引物。
本发明还涉及一种基于二代测序技术检测MH的试剂盒，包括上述引物组合物，其还包括PCR混合液和PCR反应液。
下面通过具体实施例和附图对本发明进行详细和具体的介绍，以使更好的理解本发明，但是下述实施例并不限制本发明范围。
实施例中方法如无特殊说明的采用常规方法，使用的试剂如无特殊说明的使用常规市售试剂或按常规方法配制的试剂。
实施例1
本实施例提供一种利用上述引物组合物或者试剂盒的基于二代测序技术检测MH标记的方法，包括如下步骤：
(1)取待测样品，提取样本DNA，定量样本浓度为5ng/μL；
(2)进行第一轮多重PCR，PCR扩增体系及扩增条件如表3所示。
表3文库构建第一轮PCR复合扩增反应体系
Figure PCTCN2022115540-appb-000021
Figure PCTCN2022115540-appb-000022
PCR反应条件为：95℃，预变性15分钟；95℃，变性30秒，60℃，退火90秒，72℃，延伸30s，循环24次；72℃，保温10分钟。反应完成后，向其中加入1μL纯化反应液纯化产物，并完成如下反应：37℃，10分钟，50℃，10分钟，65℃，10分钟，4℃，保温。然后进行磁珠分选。
(3)补平修复加A：反应体系如表4所示：
表4文库构建补平修复加A反应体系
Figure PCTCN2022115540-appb-000023
PCR反应条件为：30℃，30分钟，65℃，30分钟，4℃，保温。
(4)接头连接：反应体系如表5所示：
表5文库构建接头连接反应体系
Figure PCTCN2022115540-appb-000024
PCR反应条件为：25℃，15分钟，4℃，保温。然后利用纯化磁珠纯化反应产物。
(5)再次进行PCR扩增，PCR反应体系如表6所示：
表6文库构建第二轮PCR反应体系
Figure PCTCN2022115540-appb-000025
Figure PCTCN2022115540-appb-000026
PCR反应条件为：37℃，15分钟；98℃，预变性45秒；98℃，变性15秒，60℃，退火30秒，72℃，延伸30秒，循环10次；72℃，5分钟；4℃，保温。
(6)文库的纯化和定量：再次利用纯化磁珠纯化产物，采用Qubit进行文库定量及质控。
(7)上机测序及数据分析：将构建好的文库放置于MiSeq FGx TM平台进行测序分析；对于获得的测序数据，采用Trimmomatic软件去测序接头，然后采用BWA软件进行序列比对将序列与人类参考基因组(hg19)进行比对，利用Python工具获取MH。
该方法可用于个体识别和亲缘鉴定，具体是挑选多态性较好的48个MH基因座：MH01CP008、MH01CP012、MH01CP016、MH01KK117、MH01KK205、MH01KK211、MH02KK134、MH02KK136、MH04CP002、MH04CP003、MH04CP007、MH04KK030、MH05CP004、MH05CP006、MH05KK020、MH05KK170、MH06CP003、MH06CP007、MH09KK153、MH10CP003、MH10KK163、MH11CP003、MH11CP005、MH11KK180、MH12KK046、MH12KK202、MH13CP008、MH13KK213、MH13KK217、MH13KK218、MH13KK225、MH14CP003、MH14CP004、MH15CP001、MH15KK066、MH16KK255、MH16KK302、MH17CP001、MH17CP006、MH17KK272、MH18CP003、MH18CP005、MH19CP007、MH19KK299、MH20KK058、MH20KK307、MH21KK315、MH21KK324，采用对应表2中的引物序列按照上述步骤进行检测分析。
该方法可用于族源推断，具体是基于全部163个基因座的MH分型结果进行分析。
实施例2
本实施例为实施例1提供的方法的法医学验证工作，具体的实验和结果如下：
按照DNA分析方法科学工作组(Scientific Working Group for DNA Analysis  Methods,SWGDAM)要求对实施例1构建的复合扩增体系进行灵敏度、准确性、重复性以及法医学参数计算。
结果显示实施例1构建的方法(针对163个MH)具有高灵敏度，所有浓度均可以获得MH基因座的完整基因分型，不同浓度梯度DNA投入量下二代测序数据统计见图1和图2；准确性高，采用Sanger测序法进行验证，结果显示MH体系中所有SNP位点分型均与二代测序结果一致；重复性好。
针对48个多态性好的MH基因座，48个基因座平均杂合度达到0.7227，多态性信息含量均大于0.60，平均个体识别概率达到0.8692，累计个体识别概率为1-8.26×10 -44，二联体累积非父排除率和三联体累积非父排除率分别为1-1.26×10 -8和1-8.27×10 -16。
实施例3
本实施例为实施例1提供的方法与二代测序平台的STR试剂盒对混合物样本分析效能的比较。
基于二代测序平台的ForenSeq TM DNA Signature Prep Kit中常染色体STR基因座，由于其高敏感性以及stutter峰扩增的影响，在20:1的混合比以下开始出现次要贡献者等位基因的大量丢失。制备不同混合比例的DNA混合物样本，分别使用实施例1提供的方法(针对163个MH)与ForenSeq TM DNA Signature Prep Kit进行检测，比较两者的混合物检测效能。表7是两者在不同混合比例的DNA混合物样本中的次要贡献者独特等位基因检出率。结果显示，实施例1提供的方法对混合物的检测效果明显优于二代测序平台的STR试剂盒。
表7两种遗传标记在不同混合比例的DNA混合物样本中的次要贡献者独特等位基因检出率
Figure PCTCN2022115540-appb-000027
Figure PCTCN2022115540-appb-000028
实施例4
本实施例为实施例1提供的方法在族源推断方面的应用，具体的操作步骤和结果如下：
利用千人基因组计划中26个群体联合华东汉族群体共27个群体的MH分型数据，比较27个群体间基因型频率分布差异，计算MH标记在27个群体中的I n值，评估基因座的族源信息含量，并进行主成分分析。
结果显示，163个MH标记的I n值均大于0.185，具备较高的祖先信息含量，可用于族源推断。由图3可知，本发明所包含的163个MH标记可将世界范围主要地区的人群较为清晰地区分，非洲、东亚、南亚与欧洲群体之间有明显的分离。
由上述实施例可知，本发明提供的引物组合物、试剂盒和方法为法医学领域中个体识别、亲缘鉴定、混合物分析以及族源推断等提供了新的检测手段。
以上对本发明的具体实施例进行了详细描述，但其只作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。

Claims (10)

  1. 一种基于二代测序技术检测微单倍型(microhaplotype,MH)的引物组合物，其特征在于，包括163个MH的扩增引物中的一对或多对；
    所述163个MH包括MH01CP007、MH01CP008、MH01CP012、MH01CP016、MH01KK001、MH01KK070、MH01KK072、MH01KK106、MH01KK117、MH01KK172、MH01KK205、MH01KK210、MH01KK211、MH02CP004、MH02KK003、MH02KK004、MH02KK073、MH02KK102、MH02KK105、MH02KK131、MH02KK134、MH02KK136、MH02KK138、MH02KK139、MH02KK201、MH02KK202、MH02KK213、MH02KK215、MH03KK006、MH03KK007、MH03KK008、MH03KK009、MH03KK216、MH04CP002、MH04CP003、MH04CP007、MH04KK010、MH04KK011、MH04KK013、MH04KK015、MH04KK016、MH04KK017、MH04KK019、MH04KK028、MH04KK029、MH04KK030、MH04KK074、MH05CP004、MH05CP006、MH05CP010、MH05KK020、MH05KK022、MH05KK062、MH05KK078、MH05KK079、MH05KK122、MH05KK123、MH05KK124、MH05KK170、MH06CP003、MH06CP007、MH06KK026、MH06KK030、MH06KK031、MH06KK080、MH06KK101、MH07KK030、MH07KK031、MH07KK081、MH07KK082、MH08KK032、MH09KK020、MH09KK033、MH09KK034、MH09KK152、MH09KK153、MH09KK157、MH09KK161、MH10CP003、MH10KK083、MH10KK084、MH10KK085、MH10KK086、MH10KK087、MH10KK088、MH10KK101、MH10KK163、MH10KK170、MH11CP003、MH11CP004、MH11CP005、MH11KK036、MH11KK037、MH11KK038、MH11KK039、MH11KK040、MH11KK041、MH11KK089、MH11KK090、MH11KK091、MH11KK180、MH11KK187、MH11KK191、MH12KK042、MH12KK043、MH12KK045、MH12KK046、MH12KK092、MH12KK093、MH12KK202、MH13CP008、MH13KK047、MH13KK213、MH13KK217、MH13KK218、MH13KK225、MH13KK226、MH14CP003、MH14CP004、MH14KK048、MH14KK101、MH15CP001、MH15CP003、MH15CP004、MH15KK066、MH15KK067、MH15KK069、MH15KK095、MH16KK053、MH16KK062、MH16KK096、MH16KK255、MH16KK302、MH17CP001、 MH17CP006、MH17KK014、MH17KK052、MH17KK053、MH17KK054、MH17KK055、MH17KK077、MH17KK105、MH17KK110、MH17KK272、MH18CP003、MH18CP005、MH18KK285、MH18KK293、MH19CP007、MH19KK056、MH19KK057、MH19KK299、MH19KK301、MH20KK058、MH20KK059、MH20KK307、MH21KK313、MH21KK315、MH21KK316、MH21KK324、MH22KK060、MH22KK064和MH22KK303。
  2. 根据权利要求1所述的引物组合物，其特征在于，包括核苷酸序列为SEQ ID No.1～326的引物的一对或多对；较佳地，包括核苷酸序列为SEQ ID No.1～326的引物。
  3. 一种包括权利要求1或2所述引物组合物的基于二代测序技术检测MH的试剂盒，其特征在于，还包括PCR混合液和PCR反应液。
  4. 一种采用如权利要求3所述试剂盒的基于二代测序技术检测MH的方法，其特征在于，包括如下步骤：
    步骤一，取待测样品，提取样本DNA并进行定量；
    步骤二，配制复合扩增体系，并进行第一轮多重PCR；反应完成后，加入纯化反应液纯化产物，再进行磁珠分选；
    步骤三，进行补平修复加A和接头连接，再次利用纯化磁珠纯化产物；
    步骤四，对纯化后的洗脱产物进行PCR反应构建文库，采用的反应体系包括洗脱产物、PCR混合液、QU试剂、混捕post-P5引物和混捕pre-p7引物；
    步骤五，文库的纯化和定量：利用纯化磁珠纯化产物，采用Qubit进行文库定量与质控；
    步骤六，上机测序及数据分析：将构建好的文库放置于MiSeq FGx TM平台进行测序分析；对于获得的测序数据，采用Trimmomatic软件去测序接头，然后采用BWA软件将测序序列与人类参考基因组hg19进行比对，利用Python工具获取微单倍型分型。
  5. 根据权利要求4所述的方法，其特征在于，样本DNA的浓度为5ng/μL。
  6. 根据权利要求4所述的方法，其特征在于，所述复合扩增体系为20μL，包括8μL PCR混合液、2μL PCR反应液、8μL引物混合液和2μL样本DNA；较佳地，步骤二中的多重PCR的反应条件为：95℃，预变性15分钟；95℃，变性30秒，60℃，退火90秒，72℃，延伸30s，循环24次；72℃，保温10分钟。
  7. 根据权利要求4所述的方法，其特征在于，步骤三中的补平修复加A的反应体系为50μL，包括步骤二的纯化产物42μL，末端修复加尾缓冲液6.8μL，末端修复加尾酶1.2μL；较佳地，步骤三中的补平修复加A的反应条件为：30℃，30分钟；65℃，30分钟；4℃，保温。
  8. 根据权利要求4所述的方法，其特征在于，步骤三中的接头连接的反应体系为80μL，包括步骤三中的纯化产物50μL，接头混合液2.5μL，连接缓冲液16μL，连接酶10μL，Nuclease-free water 1.5μL；较佳地，步骤三中的接头连接的反应条件为：25℃，15分钟；4℃，保温。
  9. 根据权利要求4所述的方法，其特征在于，步骤四的PCR反应体系为50μL，包括步骤三的洗脱产物14μL，PCR混合液25μL，QU试剂3μL，混捕post-P5引物5μL，混捕pre-p7引物5μL；较佳地，步骤四中的PCR反应条件为：37℃，15分钟；98℃，预变性45秒；98℃，变性15秒，60℃，退火30秒，72℃，延伸30秒，循环10次；72℃，5分钟；4℃，保温。
  10. 如权利要求1或2所述的引物组合物或者如权利要求3所述的试剂盒在个体识别、亲缘关系鉴定、混合物分析及族源推断中的应用；所述个体识别和亲缘关系鉴定优选根据48个多态性较好的MH的分型结果，所述48个MH包括：MH01CP008、MH01CP012、MH01CP016、MH01KK117、MH01KK205、MH01KK211、MH02KK134、MH02KK136、MH04CP002、MH04CP003、MH04CP007、MH04KK030、MH05CP004、MH05CP006、MH05KK020、 MH05KK170、MH06CP003、MH06CP007、MH09KK153、MH10CP003、MH10KK163、MH11CP003、MH11CP005、MH11KK180、MH12KK046、MH12KK202、MH13CP008、MH13KK213、MH13KK217、MH13KK218、MH13KK225、MH14CP003、MH14CP004、MH15CP001、MH15KK066、MH16KK255、MH16KK302、MH17CP001、MH17CP006、MH17KK272、MH18CP003、MH18CP005、MH19CP007、MH19KK299、MH20KK058、MH20KK307、MH21KK315和MH21KK324。
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