Y-chromosome STR system, Y-PLEX 12, for forensic casework: development and validation

The Y-PLEX 12 system, developed for use in human identification, enables simultaneous amplification of eleven polymorphic short tandem repeat (STR) loci, namely DYS392, DYS390, DYS385 a/b, DYS393, DYS389I, DYS391, DYS389II, DYS 19, DYS439 and DYS438, residing on the Y chromosome and Amelogenin. Amelogenin provides results for gender identification and serves as internal control for PCR. The validation studies were performed according to the DNA Advisory Board's (DAB) Quality Assurance Standards. The minimal sensitivity of the Y-PLEX 12 system was 0.1 ng of male DNA. The mean stutter values ranged between 3.76-15.72%. A full male profile was observed in mixture samples containing 0.5 ng of male DNA and up to 400 ng of female DNA. Amelogenin did not adversely affect the amplification of Y-STRs in mixture samples containing male and female DNA. The primers for the Y-STR loci present in Y-PLEX 12 are specific for human DNA and some higher primates. None of the primate samples tested provided a complete profile at all 11 Y-STR loci amplified with the Y-PLEX 12 system. Y-PLEX 12 is a sensitive, valid, reliable, and robust multiplex system for forensic analysis, and it can be used in human forensic and male lineage identification cases.     

Y-chromosome STR haplotype diversity in Brazilian populations

A sample of 198 unrelated males distributed among the five geopolitical regions in Brazil were typed for the minimal Y-STR haplotype composed of microsatellite loci DYS19, DYS385I/II, DYS389I, DYS389II, DYS390, DYS391, DYS392 and DYS393. Gene frequency data, gene diversity, haplotype diversity and power of discrimination were estimated. An AMOVA indicated that 99.97% of the haplotypic variation is found within regions and only a small 0.03% and non significant variation is found among the five regions (Fst=0.00031, P-value=0.43). This result suggests that a single national database of Y-STR haplotypes can be used in the quantitative assessment of matches in forensic casework in the Brazilian population. A significant haplotype diversity of 99.8% was found and 172 different haplotypes were observed in 198 chromosomes. Haplotype (14-11, 14-13-29-24-11-13-13) with five occurrences in 198 chromosomes was the most frequent in Brazil.     

Properties of novel and widely studied Y-STR loci in three South African populations

The objective of the present study was to examine the properties of a set of single-copy Y-STR loci to assess their suitability for forensic casework in three South African populations. Three criteria were used to select markers for assessment. Firstly, the single-copy markers of the minimal haplotype were selected based on their established use in forensic studies. Secondly, 8 markers were selected on the basis of high gene diversity values reported for several population studies, and thirdly 19 markers were chosen from a survey of Y-chromosome sequence with selections made primarily on the basis of the number of repeated elements present. Samples were typed from 101 English-speaking Caucasians, 88 Xhosa individuals and 77 Asian Indians. Gene diversity values, the number of alleles identified and the average stutter was determined for each locus.     

Haplotype data for the 12 RM Y-STR loci in a Syrian population

Researches with RM Y-STRs have shown that these loci provide substantially higher haplotype diversity and haplotype discrimination capacity in worldwide populations when compared with the YSTRs commonly used in genetic forensics. The aim of this study was to develop an allelic frequency database for the Syrian population living in Turkey in order to obtain population data of 12 RM Y-STRs. A total of 80 unrelated males from the Syrian population living in Turkey were typed with 12 RM Y-STRs loci: DYF387S1, DYF399S1, DYF404S1, DYS449, DYS518, DYS526a/b, DYS547, DYS570, DYS576, DYS612, DYS626 and DYS627. The highest GD was observed for the locus DYF399S1 (0.91), followed by loci DYS449 (0.86) and DYS518 (0.83). RM Y-STR haplotype diversity was found 1.00 in these samples. Based on the results of this study, the RM YSTR loci showed remarkable haplotype resolution power in the Syrian population, high genetic diversity and, therefore, demonstrating their usefulness in forensic identification cases.     

复合扩增20个Y-STR基因座及其法医学的应用

目的建立20个Y-STR基因座的复合扩增体系,进行遗传多态性调查,并评价其法医学应用价值。方法采用五色荧光素标记技术,对20个Y-STR基因座(DYS391、DYS389Ⅰ、DYS390、DYS389Ⅱ、DYS438、DYS460、Y GATA H4、DYS456、DYS439、DYS635、DYS448、DYS393、DYS388、DYS437、DYS19、DYS392、DYS458、DYS447、DYS385 a/b)进行复合扩增和毛细管电泳检测;调查辽宁汉族376名无关男性个体20个Y-STR基因座的遗传多态性数据;并对系统性能进行检测。结果本文方法同时检测20个Y-STR基因座,在376名个体中共检出376种单倍型,基因多样性在0.371 1～0.969 8之间;方法特异性好,分型结果准确稳定,灵敏度达0.062 5ng,实际案例常见生物检材的检验结果良好。结论20个Y-STR基因座复合扩增检测法可以用于实际案例检验,调查所获数据对建立Y-STR数据库和相关研究和应用具有重要意义。     

Haplotypes for 13 Y-chromosomal STR loci in South Tunisian population (Sfax region)

Nine Y-STR loci from the "minimal haplotype" (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393) included in Y-STR Haplotype Reference Databases (YHRD) with 4 additional Y-STRs (DYS436, DYS437, DYS438, DYS439) were analyzed by PCR using duplex and Y-PLEX 12 kit, followed by automatic genotyping in a sample of 105 Tunisian males originating from Sfax region (south Tunisia). Allelic frequencies and gene diversities for each Y-STR locus were determined. The high haplotype diversity (0.9932) and discrimination capacity (0.7714) show the usefulness of these loci for human identification in forensic studies and paternity tests in Tunisia. The most common haplotype was shared by 4.7% (5 individuals) of the sample was only found in samples from the Tunisian population reported in YHRD. One private allele for DYS392 (allele 17) was discovered and duplications were observed for five loci (DYS19, DYS389I, DYS393, DYS437 and DYS439).     

A new method for the evaluation of matches in non-recombining genomes: application to Y-chromosomal short tandem repeat (STR) haplotypes in European males

A 9-locus microsatellite framework (minimal haplotype), previously developed for forensic purposes so as to facilitate stain analysis, personal identification and kinship testing, has been adopted for the establishment of a large reference database of male European Y-chromosomal haplotypes. The extent of population stratification pertaining to this database, an issue crucial for its practical forensic application, was assessed through analysis of molecular variance (AMOVA) of the 20 regional samples included. Despite the notion of some significant haplotype frequency differences, which were found to correlate with known demographic and historic features of Europeans, AMOVA generally revealed a high level of genetic homogeneity among the populations analyzed. Owing to their high diversity, however, accurate frequency estimation is difficult for Y-STR haplotypes when realistic (i.e. moderately sized) datasets are being used. As expected, strong pair-wise and higher order allelic associations were found to exist between all markers studied, implying that haplotype frequencies cannot be estimated as products of allele frequencies. A new extrapolation method was therefore developed which treats haplotype frequencies as random variables and generates estimates of the underlying distribution functions on the basis of closely related haplotypes. This approach, termed frequency 'surveying', is based upon standard population genetics theory and can in principle be applied to any combination of markers located on the Y-chromosome or in the mitochondrial genome. Application of the method to the quality assured reference Y-STR haplotype database described herein will prove very useful for the evaluation of positive trace-donor matches in forensic casework.     

High-throughput Y-STR typing of U.S. populations with 27 regions of the Y chromosome using two multiplex PCR assays

Two Y-chromosome short tandem repeat (STR) multiplex polymerase chain reaction (PCR) assays were used to generate haplotypes for 19 single copy and 3 multi-copy Y-STRs. A total of 27 PCR products were examined in each sample using the following loci: DYS19, DYS385 a/b, DYS388, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS426, DYS437, DYS438, DYS439, DYS447, DYS448, DYS450, DYS456, DYS458, DYS460, DYS464 a/b/c/d, H4, and YCAII a/b. The first multiplex is the Y-STR 20plex previously described by Butler et al. [Forensic Sci. Int. 129 (2002) 10]. The second multiplex is a novel Y-STR 11plex and includes DYS385 a/b, DYS447, DYS448 and the new markers DYS450, DYS456, DYS458, and DYS464 a/b/c/d. These two multiplexes were tested on 647 males from three United States population sample sets: 260 African Americans, 244 Caucasians, and 143 Hispanics. Haplotype comparisons between common loci included in the 20plex and 11plex assays as well as commercially available kits found excellent agreement across a sampling of the population samples. The multi-copy loci DYS464, DYS385, and YCAII were the most polymorphic followed by the following single copy Y-STRs: DYS458, DYS390, DYS447, DYS389II, DYS448, and DYS456. Samples containing the most common type in the European database could be well resolved with additional markers beyond the minimal haplotype loci.     

DNA profiling of azoospermic semen samples from vasectomized males by using Y-PLEX 6 amplification kit

Post-vasectomized azoospermic semen samples (N = 6) were analyzed for short tandem repeats (STR) on the Y-chromosome by using Y-PLEX 6 and the 310 Genetic Analyzer. We have observed a wide variation in the yield of extracted DNA from 12.5-1,000 ng. This variation was attributed to the number of epithelial and/or white blood cells that are present in these azoospermic samples. DNA profiles of these vasectomized males were obtained for all six Y-STR loci, namely DYS393, DYS 19, DYS389II, DYS390, DYS391, and DYS385 amplified by using the Y-PLEX 6.     

Y染色体STR的银染复合扩增

目的建立一套Y染色体STR的复合扩增体系,检测中国藏族人群的单倍型分布。方法利用复合扩增的方法扩增DYS434、DYS443和DYS456三个基因座,利用聚丙烯酰胺凝胶电泳银染进行分型,检测西藏藏族101名无关男性个体单倍型分布。结果三个基因座在藏族样本中分别检测出4、4、6个等位基因,共检测出31种单倍型,其单倍型的变异度是0.9481,标准误为0.0049。结论Y-STR的复合扩增在法医学的亲权鉴定和个人识别中有重要的作用。     

荧光复合扩增检测3个Y—STR基因座单倍型

目的建立检测3个Y-STR基因座Y-GATA-A7.1、DYS456和DYS443的荧光复合扩增体系,并获取中国汉族人群单倍型频率分布。方法用荧光标记引物对郑州地区203名汉族男性无关个体进行3个基因座复合扩增,ABI3100型遗传分析仪检测、分型。结果Y-GATA-A7.1、DYS456和DYS443基因座分别检出5、6和6个等位基因,其基因多样性(GD值)分别为0.6692、0.5839和0.7053。三个基因座构成的单倍型共有44种,单倍型多样性(HD值)为0.9523。结论建立的3个Y-STR基因座荧光标记复合扩增系统具有很高的识别能力,可应用于法医学实践。     

Y-STR基因座应用于刑事案件的独特作用

目的探讨Y-STR基因座在刑事案件中的应用价值。方法采用Y-STR荧光标记复合扩增技术,结合案例应用。结果Y-STR基因座对于涉及男女混合、多名男性混合样本、性别鉴定、父权鉴定等案例中具有特有应用价值。结论Y-STR基因座可应用于法庭科学中的个体识别与同一认定,但在应用中要注意各种特例的发生。     

用复合扩增方法检测4个Y-STR基因座单倍型

目的建立复合扩增Y-STR基因座的体系,获得中国汉族人的单倍型频率。方法复合扩增DYS439、DYS390、GATA-A7.2和DYS3934个基因座,用聚丙烯酰胺凝胶电泳和银染法进行基因分型。结果调查中国汉族558名无关男性个体,4个基因座分别检出7、7、7和6个等位基因,共180种单倍型,其单倍型的个体识别率为0.9853。结论该复合扩增体系在建立Y染色体STR数据库、在群体遗传研究和法医学鉴定中有应用意义。     

SWGDAM developmental validation of a 19-locus Y-STR system for forensic casework

A Scientific Working Group on DNA Analysis Methods (SWGDAM) developmental validation study was carried out on two Y-STR multiplex systems (MPI and MPII) that collectively permit the co-amplification of 19 Y-STR markers, including DYS393, DYS392, DYS391, DYS389I, DYS389II, Y-GATA-A7.2 (DYS461), DYS438, DYS385a and DYS385b (MPI); DYS425, DYS388, DYS390, DYS439, DYS434, DYS437, Y-GATA-C.4, Y-GATA-A7.1 (DYS460), Y-GATA-H.4, and DYS 19 (MPII). Performance checks subsequent to PCR parameter optimization indicated that MPI and MPII were suitably reproducible, precise and accurate for forensic use. The sensitivity of the systems was such that a full 19-locus Y-STR profile was obtainable with 150-200 pg of male DNA, and some loci were detectable even with as little as 20-30 pg of input DNA. Primate specificity was demonstrated by the lack of cross-reactivity with a variety of commonly encountered bacterial and animal species, with the single exception of a monomorphic canine product that was outside of the size range of human alleles from any of the 19 loci. Not surprisingly, cross-reactivity was observed with a number of male and female nonhuman primates. Environmentally compromised samples produced full or partial Y-STR profiles. For example, a semen stain exposed to the outdoor elements for six months still gave a 13-locus Y-STR profile. Although a limited number of female DNA artifacts were observed in mixed stains in which the male DNA comprised 1/300 of the total, the full 19-locus male profile was easily discernible. Even at a 1500-to-2000-fold dilution of male DNA with female DNA partial Y-STR profiles were obtained. Furthermore, the potential utility of MPI and MPII for forensic casework is exemplified by their ability to dissect out the male haplotype in a variety of case-type samples, including, inter alia, post-coital vaginal swabs, admixed male and female bloodstains, the nonsperm fraction from a differentially extracted semen stain, and determination of the number of male donors in mixed semen stains.     

24个Y-STR基因座在云南苗族人群中的序列多态性

目的研究法医学常用Y-STR基因座在中国云南苗族男性个体中的序列多态性。方法采用M48磁珠提取纯化试剂盒提取样本DNA,使用ForenSeqTM DNA Signature Prep试剂盒制备文库,Miseq FGx平台进行测序,ForenSeq Universal Analysis v1.2.1软件进行数据分析,用Arlequin v3.5软件计算各Y-STR基因座相关的统计学参数,将Y-STR基因座长度多态性与序列多态性进行比较。结果108名云南苗族个体中共检出106种单倍型,总体单倍型多样性(HD)和Y-STR分型系统的分辨能力(DC)分别为0.9993和0.9815。24个Y-STR基因座共检出204个基因,基因多样性(gene diversity,GD)值为0.2177~0.9481,15个Y-STR基因座的GD值大于0.6。DYF387S1、DYS390、DYS389II、DYS437、DYS438、DYS448、DYS612基因座存在长度相同的基因核心序列不同的情况。结论该24个Y-STR基因座在云南苗族男性人群中具有丰富的遗传多态性。研究结果可为Y-STR数据库的建立、群体遗传学和法医学实践提供参考。     

6个Y-STR荧光复合扩增系统的建立及应用

目的建立6个Y-STR荧光复合扩增体系,评价其法医学应用价值。方法设计DYS444,GATA-A7.2,GATA-A10,DYS390,GATA-A7.1,DYS443荧光复合扩增引物,扩增总体积20μL,内含模板DNA0.5～10ng,PCR产物用3130遗传分析仪电泳,GenemapperID v 3.2分析结果,根据等位基因标准命名各等位基因,并评价该系统的特异性、准确性、均衡性、灵敏度及对混合血样的分析能力。结果当dNTP、Mg2+分别为200μmol/L、1.5mmol/L时扩增效果最佳,0.5～10ngDNA模板量均能获得较好的扩增效果,各基因座分型结果清晰,扩增均衡,特异性强,重现性好,基本满足实际应用的性能要求。对湖北汉族群体进行遗传学调查,结果GD值0.580 3～0.722 3,共检出158种单倍型,其多样性为0.995 8。结论本文6个Y-STR扩增系统分型可靠,配合常用Y-STR分型试剂盒,可进一步提高个体识别能力。     

南通汉族人群17个Y-STR基因座的遗传多态性

目的分析南通汉族人群的基因表型,评测17个Y-STR基因座在南通人群中的应用价值。方法采集343名南通汉族男性无关个体的外周血样本,通过Chelex-100法提取基因组DNA,用Amp FlSTR Yfiler~(TM)试剂盒进行基因分型,并与12个汉族人群[安徽、江苏、江西、山东、上海、浙江(1)、兰州、南阳、泸州、牡丹江、山西和浙江(2)]以及9个少数民族人群(蒙古族、锡伯族、拉萨藏族、青海藏族、哈萨克族、维吾尔族、满族、台湾排湾族和土家族)进行比较。结果南通汉族群体在17个Y-STR基因座共检出327种单倍型,单倍型多样性(haplotype diversity,HD)值为0.999 7,与其他人群间的R_(st)值范围为-0.000 6~0.263 5。多维尺度图结果显示南通汉族人群与大多数汉族人群之间差异无统计学意义,但明显有别于其他少数民族人群。结论 17个Y-STR基因座在南通汉族人群中的群体多态性高,具有法医学应用价值。