WO2016049878A1 - Snp profiling-based parentage testing method and application - Google Patents

Snp profiling-based parentage testing method and application Download PDF

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WO2016049878A1
WO2016049878A1 PCT/CN2014/087988 CN2014087988W WO2016049878A1 WO 2016049878 A1 WO2016049878 A1 WO 2016049878A1 CN 2014087988 W CN2014087988 W CN 2014087988W WO 2016049878 A1 WO2016049878 A1 WO 2016049878A1
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base
snp
parent
ratio
sequencing
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芦静
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深圳华大基因科技有限公司
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    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Abstract

Provided in the present invention is a parentage testing method based on an SNP profiling result of a second-generation high-throughput sequencing. The present invention selects a third-generation nucleic acid molecular genetic marker SNP to serve as a genetic marker for parentage testing and, by means of multiple steps such as SNP loci selection, primer designing, target nucleic acid extraction, high-throughput PCR amplification, second-generation sequencing, SNP profiling, and parentage testing, produces profiling results and parentage testing of increased accuracy.

Description

一种基于SNP分型的亲子鉴定方法及应用A paternity identification method based on SNP typing and its application 技术领域Technical field
本本发明涉及生物医学领域。更具体而言,本发明涉及亲子鉴定的新方法。The present invention relates to the field of biomedicine. More specifically, the present invention relates to a novel method of paternity testing.
背景技术Background technique
亲子鉴定是采用医学、遗传学等学科的理论和现代化DNA检测技术来判断有争议的父母与子女之间是否存在亲生血缘关系。Parent-child identification uses the theory of medicine, genetics and other disciplines and modern DNA detection technology to determine whether there is a blood relationship between controversial parents and children.
目前国内外进行亲子鉴定的手段主要有:At present, the means of paternity testing at home and abroad mainly include:
(一)血型检验,即血液中各种成分的遗传多态性标记检验。此种检验方法操作和判读结果依靠人工,操作相对复杂。(1) Blood type test, that is, genetic polymorphism labeling test for various components in the blood. The operation of this test method and the interpretation result depend on labor, and the operation is relatively complicated.
(二)DNA多态性检验:是目前国际公认的最有效的用于亲子鉴定和个体识别的方法。而且采用的检材可以是血液、血痕、唾液、毛发、骨骼等几乎人体任何组织或器官。(2) DNA polymorphism test: It is currently the most internationally recognized method for paternity testing and individual identification. Moreover, the sample used may be any tissue or organ of the human body such as blood, blood marks, saliva, hair, bones and the like.
1900年,ABO血型被发现,运用ABO血型系统,可以起到排除亲子关系的作用。人类DNA分子结构的发现以及分子生物学理论的技术飞速发展为亲子鉴定带来了前所未有的契机。In 1900, the ABO blood type was discovered, and the use of the ABO blood group system can eliminate the parent-child relationship. The discovery of the molecular structure of human DNA and the rapid development of the technology of molecular biology have brought unprecedented opportunities for paternity testing.
DNA具有个体的唯一性和群体的多样性,没有任何两个个体的DNA完全一致(同卵双生的双胞胎除外)。也因此,通过DNA进行亲子鉴定,能够达到准确的程度。自1985年英国遗传学家Alec Jeffreys建立了DNA指纹技术以来,新技术、新方法不断出现,可应用的各种遗传标记与日俱增,今天的法医DNA分析技术已成为个人识别和血缘鉴定最有效的方法。DNA has individual uniqueness and group diversity, and no two individuals have identical DNA (except twins of identical twins). Therefore, the paternity test by DNA can achieve an accurate degree. Since the establishment of DNA fingerprinting technology by British geneticist Alec Jeffreys in 1985, new technologies and methods have emerged, and various genetic markers have been applied. Today's forensic DNA analysis technology has become the most effective method for personal identification and blood identification. .
在法医学上,STR位点和单核苷酸(SNP)位点检测分别是第二代、第三代DNA分析技术的核心,是继RFLP(限制性片段长度多态性)和VNTR(可变数量串联重复序列多态性)研究而发展起来的检测技术。作为最前沿的刑事生物技术, DNA分析为法医物证检验提供了科学、可靠和快捷的手段,使物证鉴定从个体排除过渡到了可以作同一认定的水平,DNA亲子鉴定在司法刑事中能直接认定犯罪、强(暴)奸致孕案等重大疑难案件的侦破提供准确可靠的依据。随着DNA技术的发展和应用,DNA标志系统的检测将成为破案的重要手段和途径。此方法作为亲子鉴定已经是非常成熟的,也是国际上公认的最好的一种方法。In forensic science, STR locus and single nucleotide (SNP) locus detection are the core of second- and third-generation DNA analysis techniques, respectively, followed by RFLP (restriction fragment length polymorphism) and VNTR (variable Detection techniques developed by the study of quantitative tandem repeat polymorphisms. As the most advanced criminal biotechnology, DNA analysis provides a scientific, reliable and fast means for forensic evidence testing, so that the identification of physical evidence can be transferred from individual exclusion to the level that can be used for the same identification. DNA paternity testing can directly identify criminals and strong (violent) gestation in judicial criminals. The detection of major difficult cases such as the case provides an accurate and reliable basis. With the development and application of DNA technology, the detection of DNA marker system will become an important means and means to solve the case. This method is very mature as a paternity test and is the best recognized method in the world.
当前DNA亲子鉴定利用人类基因组中的重复碱基序列(STR作为第二代分子标记)和PCR技术进行个体识别,但STR具有很大的局限性,SNP是第三代分子标记技术是将来的发展方向,美国911尸体辨认即利用了此技术。STR作为第二代分子标记,势必会被90年代后期诞生的第三代分子标记技术:一种被称为单核苷的多态(SNP)的技术所取代。目前科学家正在开发用于亲子鉴定的SNP检测技术,志在跨越把SNP技术和基因芯片结合的技术难点,完成亲子鉴定基因芯片的设计和制造,并将其率先运用到中国的亲子鉴定事业中去。Current DNA paternity testing utilizes repeated base sequences in the human genome (STR as a second-generation molecular marker) and PCR technology for individual recognition, but STR has great limitations, and SNP is a third-generation molecular marker technology that is future development. Direction, the United States 911 body recognition is the use of this technology. As a second-generation molecular marker, STR is bound to be replaced by the third-generation molecular marker technology that was born in the late 1990s: a polymorphism (SNP) technique called mononucleoside. At present, scientists are developing SNP detection technology for paternity testing, aiming at the technical difficulties of combining SNP technology and gene chip, completing the design and manufacture of paternity test gene chip, and applying it to China's paternity testing business. .
如今,STR的方法主要是用毛细管测序仪进行检测,SNP也只是用基因芯片或者试剂盒分型的方法来检测,这些检测方法虽然准确度高,但是通量小,成本高,不利于大规模样本的鉴定。Nowadays, the STR method is mainly detected by capillary sequencer. SNP is only detected by gene chip or kit typing. Although these methods are highly accurate, the flux is small and the cost is high, which is not conducive to large scale. Identification of the sample.
发明内容Summary of the invention
本发明针对当今亲子鉴定通量小,成本高的难点,提供了一种基于二代高通量测序的SNP分型结果进行亲子鉴定的方法,所述方法包括如下步骤:The present invention provides a method for performing paternity testing based on SNP typing results of second-generation high-throughput sequencing, and provides a method for performing paternity testing based on the second-generation high-throughput sequencing SNP typing method. The method includes the following steps:
1)选择SNP位点作为分子标记,1) Select the SNP locus as a molecular marker,
优选地,所述SNP满足以下标准:Preferably, the SNP meets the following criteria:
MAF>0.4的位点,a site with a MAF > 0.4,
能通过设计引物进行扩增,扩增产物长度在150bp-250bp,优选180-220,特别优选200bp;Can be amplified by designing primers, the length of the amplified product is from 150 bp to 250 bp, preferably from 180 to 220, particularly preferably 200 bp;
无连锁不平衡;No linkage imbalance;
SNP位点之间符合Hardy-Weinberg平衡; The SNP sites are in harmony with the Hardy-Weinberg equilibrium;
2)对来自待测子代和父母的样品核酸构建文库:2) Construct a library of sample nucleic acids from the progeny and parents to be tested:
优选地,对来自待测子代和父母的样品DNA,利用内外双引物扩增的方法,内引物扩增目的产物,外引物扩增加入建库接头使得扩增产物可以直接上机测序达到快速建库的目的(例如SEQ ID NO.1和SEQ ID NO.2-[Barcode]-SEQ ID NO.143);Preferably, the sample DNA from the progeny and the parent to be tested is amplified by the internal and external double primers, the internal primer is used to amplify the target product, and the external primer is amplified and added to the ligated adaptor so that the amplified product can be directly sequenced on the machine to achieve rapid Purpose of library construction (eg SEQ ID NO. 1 and SEQ ID NO. 2- [Barcode] - SEQ ID NO. 143);
3)对步骤2)建的库进行测序;3) Sequencing the library built in step 2);
4)将去除外接头的数据在含有扩增目的序列的目的库中进行序列的比对,获得比对后的序列数据集;4) comparing the data of the outer linker in the target library containing the amplification target sequence, and obtaining the aligned sequence data set;
5)对4)中比对后的序列,进行SNP基因型的分型;5) performing the typing of the SNP genotype on the sequence after the comparison in 4);
6)对于5)中得到的分型,统计在父母子三者中统计所述子代与父母在排除因为错误率之后不一致的位点数n,n等于0认定为亲子关系,否则有1-n*10-8的可行度确认无亲子关系。6) For the classification obtained in 5), the statistics in the parents and the children are statistically calculated. The child and the parent are inconsistent with the number of sites n after the error rate, n is equal to 0, and is determined to be a parent-child relationship, otherwise there is 1-n *10 -8 viability confirms no parent-child relationship.
在一个实施方案中,所选择的SNP位点是表2中显示的70个SNP位点。在这些位点中随机选择35个位点,用11个样本做准确性评估In one embodiment, the selected SNP sites are the 70 SNP sites shown in Table 2. Randomly select 35 loci in these loci and use 11 samples for accuracy assessment
表2中显示的70个SNP位点是:The 70 SNP loci shown in Table 2 are:
rs11239930、rs10801520、rs3899750、rs11714239、rs1397228、rs472728、rs7429010、rs4478233、rs2172651、rs325238、rs7715674、rs1337823、rs574202、rs7741536、rs4719491、rs13438255、rs7834428、rs6994603、rs10124916、rs4606122、rs7035090、rs2038597、rs1484443、rs518357、rs895648、rs1939904、rs991718、rs7306163、rs10860402、rs11146962、rs1147437、rs4789817、rs8083190、rs2829066、rs2076039、rs4076086、rs1106201、rs3756050、rs11123823、rs2274212、rs3829868、rs2276967、rs9821880、rs1049500、rs3811474、rs2292564、rs2013162、rs1997660、rs14134、rs26821、rs7690296、rs5745448、rs1343469、rs1699798、rs1698647、rs2293195、rs3805392、rs227368、rs6909306、rs62431284、rs562381、rs10734685、rs929310、rs1355634、rs2356027、 rs1657741、rs8076154、rs2235907、rs228104、rs5749426。Rs11239930, rs10801520, rs3899750, rs11714239, rs1397228, rs472728, rs7429010, rs4478233, rs2172651, rs325238, rs7715674, rs1337823, rs574202, rs7741536, rs4719491, rs13438255, rs7834428, rs6994603, rs10124916, rs4606122, rs7035090, rs2038597, rs1484443, rs518357, rs895648, Rs1939904, rs991718, rs7306163, rs10860402, rs11146962, rs1147437, rs4789817, rs8083190, rs2829066, rs2076039, rs4076086, rs1106201, rs3756050, rs11123823, rs2274212, rs3829868, rs2276967, rs9821880, rs1049500, rs3811474, rs2292564, rs2013162, rs1997660, rs14134, rs26821, Rs7690296, rs5745448, rs1343469, rs1699798, rs1698647, rs2293195, rs3805392, rs227368, rs6909306, rs62431284, rs562381, rs10734685, rs929310, rs1355634, rs2356027, Rs1657741, rs8076154, rs2235907, rs228104, rs5749426.
在一个实施方案中,扩增所述SNP位点的引物是SEQ ID N0.3-142,每两个引物依次扩增所述SNP位点,如表2所示。In one embodiment, the primer that amplifies the SNP site is SEQ ID N 0.3-142, and each of the two primers sequentially amplifies the SNP site, as shown in Table 2.
表2中显示的前35个SNP位点:The first 35 SNP loci shown in Table 2:
rs11239930、rs10801520、rs3899750、rs11714239、rs1397228、rs472728、rs7429010、rs4478233、rs2172651、rs325238、rs7715674、rs1337823、rs574202、rs7741536、rs4719491、rs13438255、rs7834428、rs6994603、rs10124916、rs4606122、rs7035090、rs2038597、rs1484443、rs518357、rs895648、rs1939904、rs991718、rs7306163、rs10860402、rs11146962、rs1147437、rs4789817、rs8083190、rs2829066、rs2076039。Rs11239930, rs10801520, rs3899750, rs11714239, rs1397228, rs472728, rs7429010, rs4478233, rs2172651, rs325238, rs7715674, rs1337823, rs574202, rs7741536, rs4719491, rs13438255, rs7834428, rs6994603, rs10124916, rs4606122, rs7035090, rs2038597, rs1484443, rs518357, rs895648, Rs1939904, rs991718, rs7306163, rs10860402, rs11146962, rs1147437, rs4789817, rs8083190, rs2829066, rs2076039.
在一个实施方案中,扩增所述SNP位点的引物是SEQ ID NO.3-72,每两个引物依次扩增所述SNP位点,如表2所示。In one embodiment, the primer that amplifies the SNP site is SEQ ID NO. 3-72, and each of the two primers sequentially amplifies the SNP site, as shown in Table 2.
在一个实施方案中,建库通过WaferGen平台进行。In one embodiment, the library is built using the WaferGen platform.
本发明的方法可以适用于三联体家系亲子鉴定或者有创亲子鉴定。The method of the invention can be applied to paternity identification or invasive paternity testing of triplet families.
理论上可以提取出核酸,并优先二代测序平台(包括Hiseq,Miseq,Proton,PGM等相关二代测序机型)进行样本测序做三联体的亲子鉴定。所述样本包括、但不限于毛发、口腔细胞、血液、尿液、羊水、指甲、烟头等。In theory, nucleic acids can be extracted, and the second-generation sequencing platform (including the second-generation sequencing models such as Hiseq, Miseq, Proton, and PGM) is preferentially used for sample sequencing to perform paternity paternity testing. The sample includes, but is not limited to, hair, oral cells, blood, urine, amniotic fluid, nails, cigarette butts, and the like.
在一个实施方案中,对于SNP基因型的分型,提取出所测SNP位点的包括碱基类型及数目的碱基信息,如果该点的主要碱基支持数目低于50X则判定为低覆盖度不足以准确分型,当主要碱基支持数目高于50X则按照如下判定:In one embodiment, for the typing of the SNP genotype, base information including the base type and number of the tested SNP site is extracted, and if the number of primary base supports at the point is less than 50X, it is determined to be low coverage. The degree is not sufficient for accurate typing. When the number of major base supports is higher than 50X, it is determined as follows:
如果主要碱基与第二碱基所占比大于所有碱基的4/5,则进行如下杂合型的判定:如果第二碱基数目与第三碱基数目的比值超过表9中的阈值,则对杂合型判定给予一个高的宽容度(主要碱基与第二碱基比值在15倍之内),反之给予一个中等宽容度(主要碱基与第二碱基比值在10倍之内),If the ratio of the primary base to the second base is greater than 4/5 of all bases, the following heterozygous type is determined: if the ratio of the number of second bases to the number of third bases exceeds the threshold in Table 9. , giving a high tolerance to the heterozygous determination (the primary base to the second base ratio is within 15 times), and vice versa giving a medium latitude (the ratio of the primary base to the second base is 10 times Inside),
如果主要碱基与第二碱基所占比不大于4/5,则对杂合型的判定给予一 个低的宽容度(主要碱基与第二碱基比值在2倍之内)。相比传统的STR亲子鉴定,本发明的方法通量高,一次扩增多达5184个位点,每个样本只需70个SNP位点,单次扩增可以做74个样本,适合大规模的样本鉴定分析,成本低,每个家系的鉴定成本不足200元,用时短,单次鉴定两天内可以出报告结果,同时所用的遗传标记SNP的突变率比STR更低,鉴定结果的可信度更高。If the ratio of the main base to the second base is not more than 4/5, the judgment of the heterozygous type is given A low latitude (the ratio of the primary base to the second base is within 2 times). Compared with the traditional STR paternity test, the method of the invention has high throughput, and up to 5184 loci in one amplification, only 70 SNP loci per sample, and 74 samples in a single amplification, suitable for large scale. The sample identification analysis has low cost. The identification cost of each family is less than 200 yuan. The time is short. The results can be reported within two days of single identification. At the same time, the genetic marker SNP has a lower mutation rate than STR, and the identification result is credible. Higher degrees.
本发明还提供一种SNP基因型分型判定亲子关系方法:The invention also provides a method for determining the parent-child relationship of SNP genotyping:
1)选取测序数据不低于50X的目标SNP位点进行统计1) Select the target SNP locus with no less than 50X sequencing data for statistical analysis.
2)根据SNP定点位置的主要碱基,第二碱基,第三碱基数量之间的关系确定该位点的基因型2) Determine the genotype of the site based on the relationship between the major base, the second base, and the number of third bases at the SNP site.
3)根据步骤2)确定的基因型以及选取的错误率的阈值,在三联体家系中统计不一致的位点数进行亲子关系的判定3) According to the genotype determined in step 2) and the threshold of the selected error rate, the number of statistically inconsistent sites in the triplet family is used to determine the parent-child relationship.
在一个实施方案中,所述的主要碱基,第二碱基,第三碱基数量之间的关系确定基因型的方法为:如果主要碱基与第二碱基所占比大于所有碱基的4/5,则进行如下杂合型的判定:In one embodiment, the relationship between the major base, the second base, and the third base number determines the genotype: if the ratio of the primary base to the second base is greater than all bases For 4/5, the following heterozygous type is determined:
如果第二碱基数目与第三碱基数目的比值超过表9中的阈值,则对杂合型判定给予一个高的宽容度(主要碱基与第二碱基比值在15倍之内),反之给予一个中等宽容度(主要碱基与第二碱基比值在10倍之内),If the ratio of the number of second bases to the number of third bases exceeds the threshold in Table 9, a high tolerance is given to the heterozygous determination (the primary base to the second base ratio is within 15 times), Conversely giving a moderate tolerance (the ratio of the primary base to the second base is within 10 times),
如果主要碱基与第二碱基所占比不大于4/5,则对杂合型的判定给予一个低的宽容度(主要碱基与第二碱基比值在2倍之内)。If the ratio of the primary base to the second base is not more than 4/5, the determination of the heterozygous type is given a low tolerance (the ratio of the primary base to the second base is within 2 times).
优选地,所述错误率阈值选择范围为:3-6%,优选6%Preferably, the error rate threshold selection range is: 3-6%, preferably 6%
进一步优选地,所述亲子关系判定方法还包括:统计在父母子三者中所述子代与父母排除由于实验和测序错误后不一致的位点数n,若n等于0认定为亲子关系,否则有1-n*10-8的可行度确认无亲子关系。Further preferably, the parent-child relationship determining method further comprises: counting, in the parent and the child, that the child and the parent exclude the number of sites n that are inconsistent after the experiment and the sequencing error, and if n is equal to 0, the parent-child relationship is determined, otherwise The feasibility of 1-n*10 -8 confirms that there is no parent-child relationship.
本发明的方法利用了二代测序平台,采用高通量位点扩增和快速建库的方法,通过大规模测序的方法和对测序数据的SNP位点的分型结果进行亲子的鉴 定。本方法灵活简便,可根据要求随意增加位点,同时通量高,成本低,能有效的进行亲子的鉴定,可信度相比STR亲子鉴定更高。The method of the invention utilizes a second-generation sequencing platform, adopts a method of high-throughput site amplification and rapid database construction, and performs parent-child identification by large-scale sequencing method and typing results of SNP sites of sequencing data. set. The method is flexible and simple, and can increase the site at random according to requirements. At the same time, the flux is high and the cost is low, and the identification of the parent and child can be effectively performed, and the credibility is higher than the STR paternity test.
具体实施方式detailed description
本方法针对当今亲子鉴定通量小,成本高的难点,提供了一种基于二代高通量测序的SNP分型结果进行亲子鉴定的方法。This method provides a method for paternity testing based on SNP typing results of second-generation high-throughput sequencing for the difficulty of low-throughput and high cost of paternity testing.
为实现本发明的方法,本发明人选取了第三代的DNA分子遗传标记SNP作为亲子鉴定的遗传标记,具有扩增片段小,突变率低等优势,通过SNP位点的选取、引物的设计、目的DNA的提取、高通量PCR扩增、二代测序、SNP分型、亲子鉴定等多个步骤,可获得较为准确的分型结果和亲子鉴定。In order to realize the method of the present invention, the present inventors selected the third generation DNA molecular genetic marker SNP as a genetic marker for paternity identification, which has the advantages of small amplified fragments and low mutation rate, and selection of SNP sites and design of primers. , DNA extraction of the target, high-throughput PCR amplification, second-generation sequencing, SNP typing, paternity testing and other steps, can obtain more accurate typing results and paternity testing.
定义definition
本文所用的“条形码(barcode)”是指由一组按某种规则排列的碱基序列构成的标记,用以表示一定的信息。As used herein, "barcode" refers to a mark consisting of a set of base sequences arranged in a certain order to indicate certain information.
本发明所称的“内引物”是指加入特定连接序列的针对某一特定位点的引物,即在原有的普通引物F和R的5’加入特定的公共接头。The term "internal primer" as used in the present invention refers to a primer for a specific site to which a specific linker sequence is added, i.e., a specific common linker is added to 5' of the original common primers F and R.
本发明所称的“外引物”(包括外侧正向引物和反向引物)是指含有特定的公共接头和适应于二代测序序列的引物。在一个具体实施方案中,其处理方法包括如下步骤:As used herein, "external primers" (including lateral forward primers and reverse primers) refer to primers containing specific public linkers and sequences adapted to the second generation sequencing. In a specific embodiment, the processing method comprises the following steps:
1)根据90个炎黄个体的数据(数据来源为炎黄项目的数据)和1500个银屑病正常对照组数据(数据来源为银屑病研究项目的数据)分型出的SNP位点,选取MAF>0.4的位点,经过设计引物,扩增效能测试,平台兼容性测试以及连锁不平衡的进一步筛查测试,在其中筛选出可用的70个无连锁不平衡的SNP位点作为分子标记;1) According to the data of 90 Yanhuang individuals (data from Yanhuang project) and 1500 psoriasis normal control data (data from the data of psoriasis research project), choose MAF. >0.4 sites, through design primers, amplification potency tests, platform compatibility tests, and further screening tests for linkage disequilibrium, in which 70 available SNPs without linkage disequilibrium were screened as molecular markers;
2)通过WaferGen公司的高通量PCR扩增平台,采用基于高通量快速建库的方法,利用内外双引物扩增的方法,内引物扩增目的产物,外引物扩增加入建 库接头使得扩增产物可以直接上机测序达到快速建库的目的,同时,5184个反应同时进行,增加了扩增反应的通量;2) Through the high-throughput PCR amplification platform of WaferGen, using the method based on high-throughput rapid database construction, using the method of internal and external double primer amplification, the internal primers amplify the target product, and the external primers are amplified and added. The library linker allows the amplification products to be directly sequenced on the machine for rapid database construction. At the same time, 5184 reactions are simultaneously performed, increasing the flux of the amplification reaction;
3)将下机数据进行处理,统计数据的有效性,包括每条读段(read)对应的位点、每个位点对应的读段的长度和数量,有效读段数及百分比,碱基深度等,对测序数据进行合理的评估;3) Processing the offline data, the validity of the statistical data, including the corresponding points of each read (read), the length and number of reads corresponding to each position, the number of valid reads and the percentage, the base depth Etc., a reasonable evaluation of the sequencing data;
4)将去除外接头的数据在含有扩增目的序列的目的库中进行序列的比对,获得比对后的序列数据集;4) comparing the data of the outer linker in the target library containing the amplification target sequence, and obtaining the aligned sequence data set;
5)对4)中比对后的序列,采用自己研发的个体定点SNP分型程序,依据在定点位置上的出现的四种碱基的个数比例进行基因型的分型,并对分型后的结果进行准确率的验证;5) For the sequence after comparison in 4), use the self-developed individual fixed-point SNP typing program to perform genotyping according to the proportion of the four bases appearing at the fixed point position, and classify the genotypes. The subsequent results are verified for accuracy;
6)将5)中得到的分型经过,统计在父母子三联体中统计不一致的位点数,根据不一致的位点数进行亲子的判定,判定的标准是依据排除实验和测序错误后的位点数在子代和亲代中不符合孟德尔遗传规律的位点数,如果为0则判定为亲子关系,如果位点数为n(n>0),则有1-n*10-8的概率说明不是亲子关系。6) After the classification obtained in 5), the number of statistically inconsistent sites in the parental triplet is counted, and the parental judgment is performed according to the number of inconsistent sites. The criterion for the determination is based on the number of sites after the exclusion experiment and the sequencing error. The number of loci that do not conform to the Mendelian inheritance law in the offspring and the parental family. If it is 0, it is judged as a parent-child relationship. If the number of loci is n (n>0), the probability of 1-n*10 -8 indicates that it is not a parent-child relationship. .
在上述步骤1)中常用的位点是我们经过精心筛选的,有很强的鉴别力度,但位点的选取和数量不局限于此,可以灵活的增加和减少。选取高效的SNP位点尽量满足MAF>0.4,位点之间无连锁不平衡,同时位点尽量从已知的数据库中选取,SNP位点之间符合Hardy-Weinberg平衡。The sites commonly used in the above step 1) are carefully selected and have strong discrimination strength, but the selection and number of sites are not limited thereto, and can be flexibly increased and decreased. Efficient SNP loci were selected to satisfy MAF>0.4 as much as possible, and there was no linkage disequilibrium between the loci. At the same time, the loci were selected from known databases, and the SNP loci were consistent with Hardy-Weinberg equilibrium.
在本发明中,MAF是指Mionr Allele Frequency,即最小等位基因频率,是指在指定人群中的不常见的等位基因发生频率。例如TT,TC,CC三个基因型,在人群中C的频率为0.36,T的频率为0.64,则等位基因C就为最小等位基因频率,MAF=0.36。MAF可通过如下方式进行计算:MAF=Minor Allele/(Minor+Major Allele)In the present invention, MAF refers to the Mionr Allele Frequency, the minimum allele frequency, which refers to the frequency of occurrence of unusual alleles in a given population. For example, TT, TC, CC three genotypes, the frequency of C in the population is 0.36, the frequency of T is 0.64, then the allele C is the minimum allele frequency, MAF=0.36. The MAF can be calculated as follows: MAF=Minor Allele/(Minor+Major Allele)
在本发明中,Hardy-Weinberg平衡是指在一个群体无限大,且又具备以下条件:随机交配、没有突变没有选择、没有遗传漂变的情况下,群体内一个位 点上的基因型频率和基因频率将代代保持不变,处于遗传平衡状态,这一平衡状态就称之为Hardy-Weinberg平衡。In the present invention, Hardy-Weinberg balance refers to an infinite population in a group, and has the following conditions: random mating, no mutation without selection, no genetic drift, one position within the group The genotype frequency and gene frequency at the point will remain unchanged from generation to generation, in a genetic equilibrium state, which is called the Hardy-Weinberg equilibrium.
在上述步骤2)中对位点的引物设计应使扩增的产物较短,扩增条件尽量一致,如此才能提高在高通量扩增平台扩增的成功率,对设计好的引物先预实验成功后才可上高通量扩增平台,扩增产物长度在150bp-1000bp,优选150-500bp,特别优选180-220bp。In the above step 2), the primer design of the site should be such that the amplified product is shorter and the amplification conditions are as uniform as possible, so as to improve the success rate of amplification in the high-throughput amplification platform, and the designed primer is pre-prepared. After the experiment is successful, a high-throughput amplification platform can be obtained, and the amplification product has a length of 150 bp to 1000 bp, preferably 150 to 500 bp, and particularly preferably 180 to 220 bp.
在上述步骤5)中,可以用所选取SNP位点前6-10bp的序列作为定位依据,提取出相应位点的特定位置碱基,并统计每种碱基个数,依照相应的阈值(阈值即为下面SNP分型方法的参数)进行SNP分型。In the above step 5), the sequence of 6-10 bp in front of the selected SNP site can be used as a positioning basis, and the specific position base of the corresponding site can be extracted, and the number of each base can be counted according to the corresponding threshold (threshold value) That is, the parameters of the following SNP typing method are performed for SNP typing.
分型阈值的确定要针对性的根据测序数据的有效深度进行评估并调整参数,通过与金标准(sanger测序结果)的比较确定不同阈值的准确率,并根据不同深度的数据对主要等位基因,次要等位基因,第三等位基因设定不同的权重和阈值,以最准确的分型结果作为后续亲子鉴定的依据。针对我们的分型程序,我们总结了在不同深度数据基础上的最优参数组合,参照我们的参数能够在很大程度上提高位点分型的准确性。The determination of the typing threshold should be based on the effective depth of the sequencing data and the parameters should be adjusted. The accuracy of the different thresholds is determined by comparison with the gold standard (sanger sequencing results), and the major alleles are based on the data of different depths. The minor allele, the third allele sets different weights and thresholds, and uses the most accurate typing results as the basis for subsequent paternity testing. For our classification program, we summarize the optimal combination of parameters based on different depth data. The accuracy of the site classification can be greatly improved by referring to our parameters.
上述步骤6)是对步骤5)所得分型结果的统计。根据孟德尔定律,子代的基因型的两个等位基因分别来自父母,将父母双方基因型的所有组合与孩子的基因型进行比较,统计父母双方基因型的所有组合中不包含孩子基因型的位点个数,在排除因为错误率(错误率为选取测试中的几个样本和35个位点进行准确性评估后推测出的一个保守的错误率)产生的不一致位点,如果仍存在不一致的位点,理论上可以认为是由于基因突变产生的,而SNP的突变率为10-8[1]以往亲子鉴定用的分子标记STR要低很多,STR的突变率为10-3-10-5[2,3]。在排除了错误率之后不一致的位点个数n与亲子鉴定的可信度关系为:可信度=1-n*10-8,远高于传统的亲子鉴定标准。The above step 6) is a statistic of the scored result of step 5). According to Mendel's law, the two alleles of the genotype of the offspring are from the parents, and all combinations of the genotypes of the parents are compared with the genotype of the child. The parental genotype is not included in all combinations of the genotypes of the parents. The number of sites, in the exclusion of the inconsistency due to the error rate (the error rate is a conservative error rate after several samples and 35 sites in the selection test), if it still exists Inconsistent sites can theoretically be thought to be due to gene mutations, while the SNP mutation rate is 10 -8 [1] In the past, the molecular marker STR used for paternity testing was much lower, and the mutation rate of STR was 10 -3 -10. -5 [2,3]. The relationship between the number of inconsistent sites and the paternity of the paternity test after eliminating the error rate is: credibility = 1 - n * 10 -8 , which is much higher than the traditional paternity test.
虽然不希望拘囿于理论,但发明人考虑:在本发明中,子代与父母不一致的位点数n理论上完全符合孟德尔遗传定律,如果出现的不一致的位点便是可以 认定是因为基因突变产生的,而SNP的基因突变率是非常低的,大约为10-8,如果子代中有n个位点与所选的父母不符合,则表明有1-n*10-8的概率说明不是亲子关系,正常的亲子关系在排除错误率后的位点数与父母的位点数不符合孟德尔遗传规律的个数为0,此时可有认定为亲子关系。Although it is not desirable to be bound by theory, the inventors considered that in the present invention, the number of sites in which the progeny is inconsistent with the parent is theoretically in full accordance with Mendel's law of inheritance, and if the inconsistent site appears, it can be determined that the gene is Mutations are produced, and the mutation rate of SNPs is very low, about 10 -8 . If there are n loci in the offspring that do not match the selected parents, there is a probability of 1-n*10 -8 . It is not a parent-child relationship. The number of normal parent-child relationships after the error rate is excluded from the number of parents is not in accordance with the Mendelian inheritance law. At this time, it can be considered as a parent-child relationship.
本方法通过选取的70个SNP位点通过子代与父母不一致的位点个数统计来进行高可信度的亲子鉴定,所提及的位点支持证据不局限于本方法中选取的位点。凡有易于扩增并利于进行鉴定的位点同样适用于本方法,均可作为支持证据加入到亲子鉴定的过程。The method performs high-confidence paternity testing by selecting the 70 SNP loci through the statistics of the number of loci in which the progeny is inconsistent with the parent. The supported locus support evidence is not limited to the locus selected in the method. . Sites that are easy to amplify and facilitate identification are equally applicable to this method and can be used as supporting evidence for the process of paternity testing.
本方法中所采用的利用第三代分子遗传标记联合多位点证据进行亲子鉴定的方法,有效提高了亲子鉴定的可信度,排除率至少提升了4个数量级。而通过使用高通量扩增、高通量测序,个体定点的SNP分型更是保证了大样本量的工作和效率的提高,能够极大降低亲子鉴定的成本。同时,所用平台的兼容性和扩展性能够保证位点灵活随意的增加,可以进一步提升亲子鉴定的力度,能够最大程度的保证结果的准确性,同时能够最大程度的获取家系的遗传信息,以及以后可以用来进行家系溯祖的检测和家系图谱的建立。因此本发明有益效果在于,在综合了第三代分子遗传标记优势基础之上得到了更加丰富、成本更低的基因信息,能够很好的应用于亲子鉴定的工作中,减少因为大样本量而额外投入的劳动量。The method of using the third-generation molecular genetic marker combined with multi-site evidence for paternity testing in the method effectively improves the credibility of the paternity test, and the elimination rate is improved by at least four orders of magnitude. By using high-throughput amplification and high-throughput sequencing, individual SNP typing ensures that the work and efficiency of large sample sizes is improved, which can greatly reduce the cost of paternity testing. At the same time, the compatibility and scalability of the platform used can ensure the flexible and random increase of the site, which can further enhance the strength of paternity testing, ensure the accuracy of the results to the greatest extent, and at the same time maximize the genetic information of the family, and in the future. It can be used for the detection of family ancestors and the establishment of family maps. Therefore, the beneficial effects of the present invention are that a richer and lower cost genetic information is obtained on the basis of combining the advantages of the third generation molecular genetic marker, which can be well applied to the work of paternity testing, and the reduction of the large sample size. The amount of labor invested.
本方法是一种联合多位点的综合性基因分型方法。为了使本方法的目的、技术方案及优点更加清楚明白,以下结合实施例,对本方法进行进一步详细说明。The method is a comprehensive genotyping method combining multiple sites. In order to make the purpose, technical solution and advantages of the method more clear, the method will be further described in detail below with reference to the embodiments.
本实施例以两组三联体家系为例,其中一个家系为唇腭裂遗传病家系(父亲、母亲、孩子),另一个家系为正常家系(父亲、母亲、孩子)。In this embodiment, two families of triplets are taken as an example. One family is a family of cleft lip and palate genetic diseases (father, mother, child), and the other family is a normal family (father, mother, child).
本发明的实施例的具体方案如下:The specific scheme of the embodiment of the present invention is as follows:
1.实验方法 Experimental method
1)引物1) Primers
本方法实验所用的高通量快速建库实验所用的外引物序列(接头序列)分别为:The external primer sequences (linker sequences) used in the high-throughput rapid database experiments used in the experiments of this method are:
外引物正向(SEQ ID NO.1)External primer forward (SEQ ID NO. 1)
AATGATACGGCGACCACCGAGATCTACACTGACGACATGGTTCTACA;AATGATACGGCGACCACCGAGATCTACACTGACGACATGGTTCTACA;
外引物反向(SEQ ID NO.2-[Barcode]-SEQ ID NO.143)External primer reverse (SEQ ID NO. 2-[Barcode]-SEQ ID NO. 143)
TCTGGTTCAGAGACGATGGCAT-[Barcode]-TAGAGCATACGGCAGAAGACGAACTCTGGTTCAGAGACGATGGCAT-[Barcode]-TAGAGCATACGGCAGAAGACGAAC
本方法实验所用的70个SNP位点的扩增引物(内引物)序列如下表2所示:The amplification primers (inner primers) sequences of the 70 SNP sites used in the experiments of this method are shown in Table 2 below:
表2:Table 2:
Figure PCTCN2014087988-appb-000001
Figure PCTCN2014087988-appb-000001
Figure PCTCN2014087988-appb-000002
Figure PCTCN2014087988-appb-000002
Figure PCTCN2014087988-appb-000003
Figure PCTCN2014087988-appb-000003
Figure PCTCN2014087988-appb-000004
Figure PCTCN2014087988-appb-000004
Figure PCTCN2014087988-appb-000005
Figure PCTCN2014087988-appb-000005
Figure PCTCN2014087988-appb-000006
Figure PCTCN2014087988-appb-000006
Figure PCTCN2014087988-appb-000007
Figure PCTCN2014087988-appb-000007
Figure PCTCN2014087988-appb-000008
Figure PCTCN2014087988-appb-000008
Figure PCTCN2014087988-appb-000009
Figure PCTCN2014087988-appb-000009
2)建库的实验步骤:2) Experimental steps for building a library:
(1)高通量PCR扩增目的片段(1) High-throughput PCR amplification of the target fragment
本实施例以72个样品为例,其中47个样本作为随机样本,6个为2个家系的样本。每个样品检测70个SNP位点总共只需要30ng的基因组DNA。应用含有 barcode的外引物和本发明设计的内引物进行一轮PCR反应,扩增出目的片段,同时将每个样品加上barcode和测序接头,使PCR产物不必再进行测序文库的构建过程,直接进行二代高通量测序。本发明应用的高通量PCR扩增芯片大大节省了测序文库构建的时间和成本。应用高通量PCR芯片配套的试剂及外引物进行目的片段扩增及建库,具体的操作步骤如下:In this example, 72 samples are taken as an example, of which 47 samples are taken as random samples and 6 are samples of 2 families. A total of only 30 ng of genomic DNA was required to detect 70 SNP sites per sample. Application contains The outer primer of the barcode and the inner primer designed by the invention perform a round of PCR reaction, and the target fragment is amplified, and the barcode and the sequencing linker are added to each sample, so that the PCR product does not need to be subjected to the sequencing library construction process directly. High-throughput sequencing. The high throughput PCR amplification chip used in the present invention greatly saves the time and cost of sequencing library construction. The reagents and external primers of the high-throughput PCR chip are used to amplify and build the target fragment. The specific steps are as follows:
a.配制PCR Mix:a. Prepare PCR Mix:
根据高通量芯片固定模式,按如下表体系配制PCR Mix,震荡混匀后,放在冰盒上备用:According to the high-throughput chip fixation mode, the PCR Mix is prepared according to the following table system, shaken and mixed, placed on the ice box for use:
Figure PCTCN2014087988-appb-000010
Figure PCTCN2014087988-appb-000010
b.准备样品板:b. Prepare the sample plate:
将72个待测样品浓度全部稀释至10ng/μL,每孔20μL分装至96孔PCR板中,再根据芯片固定模式,分别将PCR Mix,模板DNA、外引物反向加入到384孔板相应的70个孔位,Outer Reverse Primer连有barcode,注意按事先安排好的barcode编号严格对应样品,具体加入体积如下表所示:The concentration of 72 samples to be tested was diluted to 10 ng/μL, and 20 μL per well was dispensed into a 96-well PCR plate. Then, according to the chip immobilization mode, PCR Mix, template DNA and external primer were separately added to the 384-well plate. The 70 holes, Outer Reverse Primer is connected with barcode. Pay attention to the sample according to the pre-arranged barcode number. The specific volume is shown in the following table:
试剂Reagent V/孔V/hole
PCR MixPCR Mix 9.3μL9.3μL
10ng/μL DNA10ng/μL DNA 3.11μL3.11μL
外引物反向(4μM)External primer reverse (4μM) 3.11μL3.11μL
c.准备引物板: c. Prepare the primer plate:
将引物干粉稀释至100μM配制成母液,再将母液稀释至0.25μM,每孔100μL分装至96孔板中,再根据芯片固定的模式,分别将PCR Mix、内引物对加入到384孔板的相应72个孔位,具体加入体积如下表所示:The primer dry powder was diluted to 100 μM to prepare a mother liquid, and the mother liquid was diluted to 0.25 μM, and 100 μL per well was dispensed into a 96-well plate, and then the PCR Mix and the inner primer pair were separately added to the 384-well plate according to the chip fixation mode. Corresponding 72 holes, the specific addition volume is shown in the following table:
试剂Reagent V/孔V/hole
PCR MixPCR Mix 9.3μL9.3μL
内引物对(0.25μM)Internal primer pair (0.25μM) 6.2μL6.2μL
d.上机点样:d. On the machine to spot:
分别将样品板和芯片放入点样仪的相应位置,选择72×72的模式,50nL体积,开始点样。约30min点样结束后,取下芯片,将芯片用特定封口膜封好,离心。取下样品板,封口膜封口存入-20℃冰柜备用。再将引物板和离心好的芯片放入点样仪的相应位置,选择72×72的模式,50nL体积,开始点样。约30min点样结束后,取下芯片,封口膜封口,离心,准备上PCR扩增。取下引物板,封口膜封口存入-20℃冰柜备用。Place the sample plate and chip into the corresponding positions of the spotter, select the 72×72 mode, 50nL volume, and start to spot. After the end of the sample for about 30 minutes, the chip was removed, and the chip was sealed with a specific sealing film and centrifuged. Remove the sample plate and seal the sealing film into a -20 ° C freezer for later use. Then put the primer plate and the centrifuged chip into the corresponding position of the spotter, select the 72×72 mode, 50nL volume, and start to spot. After about 30 minutes of sampling, the chip was removed, the parafilm was sealed, centrifuged, and ready for PCR amplification. Remove the primer plate and seal the sealing film into a -20 °C freezer for later use.
e.PCR仪扩增e. PCR instrument amplification
离心好的芯片,特定PCR仪运行如下程序:Centrifuged the chip, the specific PCR machine runs the following program:
Figure PCTCN2014087988-appb-000011
Figure PCTCN2014087988-appb-000011
Figure PCTCN2014087988-appb-000012
Figure PCTCN2014087988-appb-000012
(2)产物纯化(2) Product purification
将上述步骤得到的扩增产物应用板式离心机离心后收集到500ul的EP管中,取50ul加入1.5倍体积的磁珠进行产物纯化。The amplification product obtained in the above step was centrifuged in a plate centrifuge and collected into a 500 ul EP tube, and 50 ul of 1.5 volumes of magnetic beads was added to carry out product purification.
(3)质量检测(3) Quality inspection
a.应用Agilent 2100Bioanalyzer和荧光定量PCR(QPCR)进行文库质量检测,片段范围200-300bp,符合扩增目的片段大小,具体检测结果如下:a. Agilent 2100 Bioanalyzer and real-time PCR (QPCR) were used for library quality detection. The fragment range was 200-300 bp, which was consistent with the size of the amplified fragment. The specific detection results are as follows:
Figure PCTCN2014087988-appb-000013
Figure PCTCN2014087988-appb-000013
b上机检测 b on the machine detection
2.对实验以及测序部分的可重复性评估测和数据分型准确率的评估。2. Evaluation of repeatability assessment and data typing accuracy of the experimental and sequencing sections.
在此次实施例中,为证明流程的可重复型,加入了一个无关样本(样本2)的重复实验,70个位点基因分型后结果一致,但有一个位点深度太低未分型成功。位点以及测试的样本如表3。In this example, in order to prove the reproducible type of the procedure, a repetitive experiment of an irrelevant sample (sample 2) was added, and the results were consistent after 70 genotyping, but one site was too low and untyped. success. The sites and samples tested are shown in Table 3.
表3同一样本在本次实验的重复性评估Table 3 Repeatability evaluation of the same sample in this experiment
Figure PCTCN2014087988-appb-000014
Figure PCTCN2014087988-appb-000014
Figure PCTCN2014087988-appb-000015
Figure PCTCN2014087988-appb-000015
Figure PCTCN2014087988-appb-000016
Figure PCTCN2014087988-appb-000016
在本发明中,SNP位点来自炎黄项目的90个炎黄数据以及银屑病项目的1500个正常对照个体的数据。对于SNP的命名,NCBI里对所有提交的SNP进行分类考证之后,都会给出一个rs号,也可称作参考SNP,并给出SNP的具体信息,包括前后序列,位置信息,分布频率等,例如“rs11239930”是指编号为rs11239930的SNP位点。本领域技术人员可以在NCBI数据库中根据该编号确定该SNP位点的具体位置。In the present invention, the SNP site is derived from the data of 90 inflammatory yellow data of the Yanhuang project and 1500 normal control individuals of the psoriasis project. For the naming of SNPs, after NCBI classifies all submitted SNPs, it will give an rs number, which can also be called a reference SNP, and give specific information about the SNP, including the sequence before and after, location information, distribution frequency, etc. For example, "rs11239930" refers to a SNP site numbered rs11239930. One skilled in the art can determine the specific location of the SNP site based on the number in the NCBI database.
35个位点的准确性验证试验结果Accuracy test results of 35 sites
本案例在数据分型准确率评估的时候为验证此次分型的准确率,在此次实验中我们同时测序了之前含有分型结果金标准对照的11个样本(表2中前35个位点),将此次实验分析结果与金标准对照得到一致率的结果,现列出其中一个样本(样本1)的分型结果与金标准结果在35个位点的对照结果如下表4所示:In this case, the accuracy of the classification was verified during the data classification accuracy evaluation. In this experiment, we also sequenced 11 samples that previously contained the gold standard control of the classification results (the first 35 positions in Table 2). Point), the results of this experimental analysis are compared with the gold standard to obtain the results of the agreement rate. The results of the classification of one of the samples (sample 1) and the results of the gold standard at 35 sites are shown in Table 4 below. :
表4:样本1的分型结果与金标准的对照表Table 4: Comparison of the typing results of sample 1 with the gold standard
Figure PCTCN2014087988-appb-000017
Figure PCTCN2014087988-appb-000017
Figure PCTCN2014087988-appb-000018
Figure PCTCN2014087988-appb-000018
将此次实验分析结果与金标准对照得到一致率的结果,平均的准确率为98.2%,即错误率平均为1.8%,具体如表5所示(sanger测序验证的结果)。The results of this experimental analysis were compared with the gold standard to obtain a consistent rate. The average accuracy was 98.2%, that is, the error rate was 1.8% on average, as shown in Table 5 (the results of sanger sequencing verification).
表535个位点11个样本分型准确率统计Table 535 loci 11 sample classification accuracy statistics
样本编号Sample number 比较的位点数Number of sites compared 一致的个数Consistent number 一致率Consistency rate
11 3535 3535 11
22 3535 3535 11
33 3535 3535 11
44 3535 3535 11
55 3535 3434 0.97140.9714
66 3535 3434 0.97140.9714
77 3535 3333 0.94280.9428
88 3535 3535 11
99 3535 3434 0.97140.9714
1010 3535 3333 0.94280.9428
1111 3535 3535 11
3结果验证3 result verification
选取在70个SNP位点上有分型结果的47个无关个体。分别将父母子三者中的一个混在另外47个无关个体中,用此方法进行鉴定和排除,设置错误率为6%的阈值(错误率评估实验3%,保守性增加到6%),统计各自除错误以外产生的子代与父母不符合孟德尔遗传规律的位点个数。S:样本类型;S_ID:样本编号;T:70个位点中除去低位点之后的总位点数;N:不符合孟德尔遗传规律的位点数;WN:排除6%错误率之后不符合孟德尔遗传规律的位点数;家系1和家系2分别如表6和表7所示: 47 unrelated individuals with typing results at 70 SNP loci were selected. One of the parents and the other three were mixed in another 47 unrelated individuals, and this method was used for identification and elimination. The error rate was set at 6% (error rate evaluation experiment 3%, conservative increase to 6%), statistics The number of loci that are generated by the offspring other than the error and the parents do not conform to the Mendelian inheritance law. S: sample type; S_ID: sample number; T: total number of sites after removal of the low site from 70 sites; N: number of sites that do not conform to Mendelian inheritance; WN: does not conform to Mendel after excluding 6% error rate The number of loci of genetic laws; family 1 and family 2 are shown in Table 6 and Table 7, respectively:
表6-1家系1中父亲样本混入47个其他无关个体中的检出结果Table 6-1 Detection results of father samples mixed into 47 other unrelated individuals in family 1
Figure PCTCN2014087988-appb-000019
Figure PCTCN2014087988-appb-000019
Figure PCTCN2014087988-appb-000020
Figure PCTCN2014087988-appb-000020
表6-2家系1中母亲样本混入47个其他无关个体中的检出结果Table 6-2 Detection results of maternal samples in family 1 mixed into 47 other unrelated individuals
Figure PCTCN2014087988-appb-000021
Figure PCTCN2014087988-appb-000021
Figure PCTCN2014087988-appb-000022
Figure PCTCN2014087988-appb-000022
表6-3家系1中孩子样本混入47个其他无关个体中的检出结果Table 6-3 Detection results of children's samples in family 1 mixed into 47 other unrelated individuals
Figure PCTCN2014087988-appb-000023
Figure PCTCN2014087988-appb-000023
Figure PCTCN2014087988-appb-000024
Figure PCTCN2014087988-appb-000024
表7-1家系2中父亲样本混入47个其他无关个体中的检出结果 Table 7-1 Detection results of father samples mixed into 47 other unrelated individuals in Family 2
Figure PCTCN2014087988-appb-000025
Figure PCTCN2014087988-appb-000025
Figure PCTCN2014087988-appb-000026
Figure PCTCN2014087988-appb-000026
表7-2家系2中母亲样本混入47个其他无关个体中的检出结果Table 7-2 Detection results of mother samples mixed into 47 other unrelated individuals in Family 2
Figure PCTCN2014087988-appb-000027
Figure PCTCN2014087988-appb-000027
Figure PCTCN2014087988-appb-000028
Figure PCTCN2014087988-appb-000028
Figure PCTCN2014087988-appb-000029
Figure PCTCN2014087988-appb-000029
表7-3家系2中孩子样本混入47个其他无关个体中的检出结果Table 7-3 Detection results of children in family 2 mixed into 47 other unrelated individuals
Figure PCTCN2014087988-appb-000030
Figure PCTCN2014087988-appb-000030
Figure PCTCN2014087988-appb-000031
Figure PCTCN2014087988-appb-000031
Figure PCTCN2014087988-appb-000032
Figure PCTCN2014087988-appb-000032
通过对两个家系进一步的分析表明,此方法能够准确鉴定出混入随机个体中的双亲以及孩子,在高度容错的条件下(设置6%的错误率,实际在2%左右)非父排除概率最低在9.99999999,用本发法鉴定出亲子关系经过传统STR亲子鉴 定和ABO血型检测可以确认为亲子关系如表8。说明本方法在所用的两个实施例中能够得到了可靠的结果。Further analysis of the two families shows that this method can accurately identify the parents and children who are mixed into the random individuals. Under the condition of high tolerance (set 6% error rate, actually around 2%), the non-parent elimination probability is the lowest. At 9.99999999, use this method to identify parent-child relationships through traditional STR parent-child The determination of the ABO blood group can be confirmed as a parent-child relationship as shown in Table 8. It is indicated that the method yields reliable results in the two embodiments used.
表8两家系的ABO血型结果Table 8 ABO blood group results of the two families
家系1父亲Family 1 father AOAO
家系1母亲Family 1 mother OOOO
家系1孩子Family 1 child AOAO
家系2父亲Family 2 father AAAA
家系2母亲Family 2 mother AOAO
家系2孩子Family 2 children AAAA
在我们的两组平行实施例中,其中一个实施例家系1的样本做过传统的STR的亲子鉴定,经鉴定验证为是同一家系,结果在如下表1中显示:In our two parallel sets of examples, one of the samples of Family 1 was subjected to a paternity test of a conventional STR, which was verified to be the same family, and the results are shown in Table 1 below:
表1:Table 1:
STRSTR 父亲father 母亲mother 孩子child
D8S1179D8S1179 12,1412,14 11,1311,13 11,1211,12
D21S11D21S11 29,35.229,35.2 30,3130,31 30,35.230, 35.2
D7S820D7S820 1111 8,118,11 1111
CSF1POCSF1PO 10,1310,13 10,1110,11 10,1110,11
D3S1358D3S1358 16,1716,17 15,1815,18 15,1615,16
THO1THO1 7,97,9 99 7,97,9
D13S317D13S317 8,98,9 8,12,8,12, 9,129,12
D16S539D16S539 1111 11,1311,13 1111
D2S1338D2S1338 18,2318,23 19,2219,22 19,2319,23
D19S433D19S433 14,15.214,15.2 14,14, 14,15.214,15.2
VWAVWA 16,1716,17 1717 16,1716,17
TPOXTPOX 8,118,11 10,1110,11 1111
D18S51D18S51 14,1514,15 13,1713,17 15,1715,17
AMELAMEL x,yx,y xx x,yx,y
D5S818D5S818 10,1110,11 11,1211,12 10,1210,12
FGAFGA 22,2422,24 21,2421,24 24twenty four
本次实施例利用PGM平台测序,数据的平均深度958X。This example was sequenced using the PGM platform, and the average depth of the data was 958X.
上面用到的SNP分型方法以及参数意义:The SNP typing method used above and the meaning of the parameters:
表9:采用自己研发的个体定点的SNP分型方法,所设参数为Table 9: Using the SNP typing method of individual fixed-point developed by ourselves, the parameters are
Figure PCTCN2014087988-appb-000033
Figure PCTCN2014087988-appb-000033
在本实例的SNP分型方法里,不同深度的数据量对应不同的分型参数,本实施例中的样本数据量为958X,所用的参数为上表所示,参数为经验总结,能够达到较高的准确率。其分型思路为提取出所测SNP位点的碱基信息(碱基类型及数目),如果该点的主要碱基支持数目低于50X则判定为低覆盖度不足以准确分型,当主要碱基支持数目高于50X则按照如下判定:如果主要碱基与第二碱基所占比大于所有碱基的4/5,则进行如下杂合型的判定[如果第二碱基数目与第三碱基数目的比值超过上表中的阈值,则对杂合型判定给予一个高的宽容度(主要碱基与第二碱基比值在15倍之内),反之给予一个中等宽容度(主要碱基与第二碱基比值在10倍之内)],如果主要碱基与第二碱基所 占比不大于4/5,则对杂合型的判定给予一个低的宽容度(主要碱基与第二碱基比值在2倍之内)。In the SNP classification method of the present example, the data amounts of different depths correspond to different classification parameters, and the sample data amount in this embodiment is 958X, and the parameters used are as shown in the above table, and the parameters are empirical summaries, which can be compared. High accuracy. The classification idea is to extract the base information (base type and number) of the tested SNP locus. If the number of main base supports at this point is lower than 50X, it is judged that the low coverage is not enough for accurate typing. If the number of base supports is higher than 50X, it is determined as follows: If the ratio of the main base to the second base is greater than 4/5 of all bases, the following heterozygous type is determined [if the number of second bases and The ratio of the number of three bases exceeds the threshold in the above table, giving a high tolerance to the heterozygous determination (the primary base to the second base ratio is within 15 times), and vice versa giving a medium tolerance (mainly The base to the second base ratio is within 10 times)], if the main base and the second base are A ratio of no more than 4/5 gives a low tolerance to the determination of the heterozygous type (the ratio of the major base to the second base is within 2 times).
如上所述,多位点联合的高通量亲子方法,在鉴定的准确性方面相对于传统亲子鉴定方法有着有效的提高。尤其在加入更多的位点以及降低容错度后,在排除的可靠性方面有了进一步提升。本实施例仅是用以解释本方法的一种常规实施方案,并不用于限定本发明。凡在本发明的原则和精神至内所做的替换、修改和改进等均包含在本发明的权利要求范围之内。As described above, the multi-site combined high-throughput parent-child method has an effective improvement in the accuracy of identification relative to the conventional paternity test method. Especially after adding more sites and reducing the tolerance, the reliability of the exclusion has been further improved. This example is only intended to explain a conventional embodiment of the method and is not intended to limit the invention. All such changes, modifications, improvements, etc., made within the spirit and scope of the invention are intended to be included within the scope of the appended claims.
参考文献:references:
1.D.E.Reich,S.F.Schaffner,M.J.Daly,G.McVean,J.C.Mullikin,J.M.Higgins,D.J.Richter,E.S.Lander,D.Altshuler,Human genome sequence variation and the influence of gene history,mutation and recombination,Nat.Genet.32(2002)135-140.1. DEReich, SF Schaffner, MJ Daly, G. McVean, JCMullikin, JMHiggins, DJ Richter, ES Lander, D. Altshuler, Human genome sequence variation and the influence of gene history, mutation and recombination, Nat. Genet. 32 (2002) 135-140.
2.Q.Y.Huang,F.H.Xu,H.Shen,H.Y.Deng,Y.J.Liu,Y.Z.Liu,J.L.Li,R.R.Recker,H.W.Deng,Mutation patterns at dinucleotide microsatellite loci in humans,Am.J.Hum.Genet.70(2002)625-634.2.QYHuang, FHXu, H.Shen, HYDeng, YJLiu, YZLiu, JLLi, RRRecker, HWDeng, Mutation patterns at dinucleotide microsatellite loci in humans, Am.J.Hum.Genet.70 (2002 ) 625-634.
3.B.M.Dupuy,M.Stenersen,T.Egeland,B.Olai sen,Y-chromosomal microsatellite mutation rates:differences in mutation rate between and within loci,Hum.Mutat.23(2004)117-124. 3. B. M. Dupuy, M. Stenersen, T. Egeland, B. Olai sen, Y-chromosomal microsatellite mutation rates: differences in mutation rate between and within loci, Hum. Mutat. 23 (2004) 117-124.

Claims (13)

  1. 一种基于SNP分型结果进行亲子鉴定的方法,所述方法包括如下步骤:A method for paternity testing based on SNP typing results, the method comprising the following steps:
    1)选择SNP位点作为分子标记;1) selecting a SNP site as a molecular marker;
    2)对来自待测子代和父母的样品核酸构建文库;2) constructing a library of sample nucleic acids from the progeny and parents to be tested;
    3)对步骤2)建的库进行测序;3) Sequencing the library built in step 2);
    4)将去除外接头的数据在含有扩增目的序列的目的库中进行序列的比对,获得比对后的序列数据集;4) comparing the data of the outer linker in the target library containing the amplification target sequence, and obtaining the aligned sequence data set;
    5)对4)中比对后的序列,进行SNP基因型的分型;5) performing the typing of the SNP genotype on the sequence after the comparison in 4);
    6)对于5)中得到的分型,统计在父母子三者中所述子代与父母排除由于实验和测序错误后不一致的位点数n,若n等于0认定为亲子关系,否则有1-n*10-8的可行度确认无亲子关系。6) For the classification obtained in 5), the number of sites in the parent and child that the parent and the parent exclude from the experiment and the sequencing error are excluded. If n is equal to 0, it is considered as a parent-child relationship, otherwise there is 1- The feasibility of n*10 -8 confirms that there is no parent-child relationship.
  2. 权利要求1的方法,所述SNP位点满足以下标准:The method of claim 1 wherein said SNP site meets the following criteria:
    MAF>0.4的位点;a site with a MAF > 0.4;
    无连锁不平衡;No linkage imbalance;
    SNP位点之间符合Hardy-Weinberg平衡。The SNP sites are in harmony with the Hardy-Weinberg equilibrium.
  3. 权利要求2的方法,SNP位点的标准进一步包括:能通过设计引物进行扩增,扩增产物长度在150bp-1000bp,优选150-500,特别优选180-220bp;The method of claim 2, wherein the standard of the SNP site further comprises: amplification by designing a primer, the amplification product having a length of from 150 bp to 1000 bp, preferably from 150 to 500, particularly preferably from 180 to 220 bp;
  4. 权利要求1或2或3的方法,构建文库按如下进行:The method of claim 1 or 2 or 3, wherein the library is constructed as follows:
    对来自待测子代和父母的样品核酸,利用内外双引物扩增的方法,内引物扩增目的产物,外引物扩增加入建库接头使得扩增产物可以直接上机测序达到快速建库的目的。For the sample nucleic acid from the progeny and parents to be tested, the internal and external double primers are used for amplification, the inner primer is used to amplify the target product, and the external primer is amplified and added to the ligated adaptor so that the amplified product can be directly sequenced on the machine to achieve rapid establishment of the library. purpose.
  5. 权利要求1-4任一项的方法,所选择的SNP位点是如下SNP位点:The method of any one of claims 1 to 4, wherein the selected SNP site is the following SNP site:
    rs11239930、rs10801520、rs3899750、rs11714239、rs1397228、rs472728、rs7429010、rs4478233、rs2172651、rs325238、rs7715674、rs1337823、rs574202、rs7741536、rs4719491、rs13438255、rs7834428、rs6994603、rs10124916、rs4606122、rs7035090、rs2038597、rs1484443、rs518357、rs895648、rs1939904、rs991718、rs7306163、rs10860402、 rs11146962、rs1147437、rs4789817、rs8083190、rs2829066、rs2076039、rs4076086、rs1106201、rs3756050、rs11123823、rs2274212、rs3829868、rs2276967、rs9821880、rs1049500、rs3811474、rs2292564、rs2013162、rs1997660、rs14134、rs26821、rs7690296、rs5745448、rs1343469、rs1699798、rs1698647、rs2293195、rs3805392、rs227368、rs6909306、rs62431284、rs562381、rs10734685、rs929310、rs1355634、rs2356027、rs1657741、rs8076154、rs2235907、rs228104、rs5749426。Rs11239930, rs10801520, rs3899750, rs11714239, rs1397228, rs472728, rs7429010, rs4478233, rs2172651, rs325238, rs7715674, rs1337823, rs574202, rs7741536, rs4719491, rs13438255, rs7834428, rs6994603, rs10124916, rs4606122, rs7035090, rs2038597, rs1484443, rs518357, rs895648, Rs1939904, rs991718, rs7306163, rs10860402, Rs11146962, rs1147437, rs4789817, rs8083190, rs2829066, rs2076039, rs4076086, rs1106201, rs3756050, rs11123823, rs2274212, rs3829868, rs2276967, rs9821880, rs1049500, rs3811474, rs2292564, rs2013162, rs1997660, rs14134, rs26821, rs7690296, rs5745448, rs1343469, rs1699798, Rs1698647, rs2293195, rs3805392, rs227368, rs6909306, rs62431284, rs562381, rs10734685, rs929310, rs1355634, rs2356027, rs1657741, rs8076154, rs2235907, rs228104, rs5749426.
  6. 权利要求5的方法,扩增所述SNP位点的引物是SEQ ID NO.3-142,每两个引物依次扩增所述SNP位点。The method of claim 5, wherein the primer for amplifying the SNP site is SEQ ID NO. 3-142, and each of the two primers sequentially amplifies the SNP site.
  7. 权利要求1-6任一项的方法,所述构建建文库通过Wafergen平台进行。The method of any of claims 1-6, wherein the constructing the library is performed by a Wafergen platform.
  8. 权利要求1-7任一项的方法,所述子代和父母的样品是可以提取出核酸构建文库进行测序,优先二代测序平台(包括Hiseq、Miseq、Proton、PGM等相关二代测序机型)进行样本测序,但不限于二代测序,所述样本包括但不限于毛发、口腔细胞、血液、尿液、羊水、指甲、烟头等。The method according to any one of claims 1 to 7, wherein the progeny and parental samples are capable of extracting a nucleic acid construction library for sequencing, and a preferred second generation sequencing platform (including Hiseq, Miseq, Proton, PGM, etc.) Sample sequencing is performed, but is not limited to second generation sequencing, including but not limited to hair, oral cells, blood, urine, amniotic fluid, nails, butts, and the like.
  9. 权利要求1-8任一项的方法,对于SNP基因型的分型,提取出所测SNP位点的包括碱基类型及数目的碱基信息,如果该点的主要碱基支持数目低于50X则判定为低覆盖度不足以准确分型,当主要碱基支持数目高于50X则按照如下判定:The method according to any one of claims 1-8, wherein for the typing of the SNP genotype, base information including the base type and number of the tested SNP site is extracted, if the number of primary base supports at the point is less than 50X Then it is judged that the low coverage is not enough for accurate typing, and when the number of primary base supports is higher than 50X, it is determined as follows:
    如果主要碱基与第二碱基所占比大于所有碱基的4/5,则进行如下杂合型的判定:如果第二碱基数目与第三碱基数目的比值超过表9中的阈值,则对杂合型判定给予一个高的宽容度(主要碱基与第二碱基比值在15倍之内),反之给予一个中等宽容度(主要碱基与第二碱基比值在10倍之内),If the ratio of the primary base to the second base is greater than 4/5 of all bases, the following heterozygous type is determined: if the ratio of the number of second bases to the number of third bases exceeds the threshold in Table 9. , giving a high tolerance to the heterozygous determination (the primary base to the second base ratio is within 15 times), and vice versa giving a medium latitude (the ratio of the primary base to the second base is 10 times Inside),
    如果主要碱基与第二碱基所占比不大于4/5,则对杂合型的判定给予一个低的宽容度(主要碱基与第二碱基比值在2倍之内)。If the ratio of the primary base to the second base is not more than 4/5, the determination of the heterozygous type is given a low tolerance (the ratio of the primary base to the second base is within 2 times).
  10. 一种SNP基因型分型判定亲子关系方法: A method for determining parent-child relationship by SNP genotyping:
    1)选取测序数据不低于50X的目标SNP位点进行统计1) Select the target SNP locus with no less than 50X sequencing data for statistical analysis.
    2)根据SNP定点位置的主要碱基,第二碱基,第三碱基数量之间的关系确定该位点的基因型2) Determine the genotype of the site based on the relationship between the major base, the second base, and the number of third bases at the SNP site.
    3)根据步骤2)确定的基因型以及选取的错误率的阈值,在三联体家系中统计不一致的位点数进行亲子关系的判定3) According to the genotype determined in step 2) and the threshold of the selected error rate, the number of statistically inconsistent sites in the triplet family is used to determine the parent-child relationship.
  11. 权利要求10的方法,所述的主要碱基,第二碱基,第三碱基数量之间的关系确定基因型的方法为:如果主要碱基与第二碱基所占比大于所有碱基的4/5,则进行如下杂合型的判定:The method of claim 10, wherein the relationship between the major base, the second base, and the third base number determines the genotype: if the ratio of the primary base to the second base is greater than all bases For 4/5, the following heterozygous type is determined:
    如果第二碱基数目与第三碱基数目的比值超过表9中的阈值,则对杂合型判定给予一个高的宽容度(主要碱基与第二碱基比值在15倍之内),反之给予一个中等宽容度(主要碱基与第二碱基比值在10倍之内),If the ratio of the number of second bases to the number of third bases exceeds the threshold in Table 9, a high tolerance is given to the heterozygous determination (the primary base to the second base ratio is within 15 times), Conversely giving a moderate tolerance (the ratio of the primary base to the second base is within 10 times),
    如果主要碱基与第二碱基所占比不大于4/5,则对杂合型的判定给予一个低的宽容度(主要碱基与第二碱基比值在2倍之内)。If the ratio of the primary base to the second base is not more than 4/5, the determination of the heterozygous type is given a low tolerance (the ratio of the primary base to the second base is within 2 times).
  12. 权利要求10的方法,所述错误率阈值选择范围为:3-6%,优选6%The method of claim 10, wherein said error rate threshold is selected from the range of 3-6%, preferably 6%
  13. 权利要求10的方法,还包括:统计在父母子三者中所述子代与父母排除由于实验和测序错误后不一致的位点数n,若n等于0认定为亲子关系,否则有1-n*10-8的可行度确认无亲子关系。 The method of claim 10, further comprising: counting the number of sites n that are inconsistent between the parent and the parent in the parent and child due to experimental and sequencing errors, and if n is equal to 0, the parental relationship is determined, otherwise 1-n* The feasibility of 10 -8 confirms that there is no parent-child relationship.
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