A DYS438 null allele observed in two generations of a large family

Typing of Y-chromosomal STR loci using the AmpFISTR^® Yfiler kit showed a DNA profile lacking the DYS438 allele in an Austrian Caucasoid brother pair.Buccal swabs were collected from additional males of two generations and different branches of the family. All family members investigated did not show a DYS438 allele in their Yfiler profile. Using the Powerplex^® Y system an allele with dramatically reduced peak height was amplified. Subsequent sequencing of the DYS438 locus exhibited a transition upstream of the repetitive region.     

Identification of a novel polymorphism in the X-chromosome region homologous to the DYS456 locus

During an extensive multipopulation study with Y-short tandem repeat (STR) loci, amplified using the AmpFlSTR Yfiler PCR amplification kit, amplification of a 71 bp fragment was observed in 2.32% of the male samples analyzed (N = 3141). By direct sequencing of this fragment, it was determined that the primer binding sequences were identical to those of the DYS456 locus. A T to G single-nucleotide polymorphism (SNP) enabled amplification of the 71 bp fragment. The SNP is located within an X-Y homologous region at Xq21.31 and was observed with the highest frequency within the African American and Sub-Saharan African populations in our study. Presence of SNP on the X chromosome did not interfere with the reliability of typing the DYS456 locus and the other Y-STR loci typeable using the AmpFlSTR Yfiler PCR amplification kit. Full profiles in a mixture of male:female at 1:4000 were obtained using the current configuration of the AmpFlSTR Yfiler kit even in the presence of female DNA containing the G variant.     

Development and validation of the AmpFlSTR Yfiler PCR amplification kit: a male specific, single amplification 17 Y-STR multiplex system

In the past 5 years, there has been a substantial increase in the use of Y-short tandem repeat loci (Y-STRs) in forensic laboratories, especially in cases where typing autosomal STRs has met with limited success. The AmpFlSTR Yfiler PCR amplification kit simultaneously amplifies 17 Y-STR loci including the loci in the "European minimal haplotype" (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393), the Scientific Working Group on DNA Analysis Methods (SWGDAM) recommended Y-STR loci (DYS438 and DYS439), and the highly polymorphic loci DYS437, DYS448, DYS456, DYS458, Y GATA H4, and DYS635 (formerly known as Y GATA C4). The Yfiler kit was validated according to the FBI/National Standards and SWGDAM guidelines. Our results showed that full profiles are attainable with low levels of male DNA (below 125 pg) and that under optimized conditions, no detectable cross-reactive products were obtained on human female DNA, bacteria, and commonly encountered animal species. Additionally, we demonstrated the ability to detect male specific profiles in admixed male and female blood samples at a ratio of 1:1000.     

A Y‐Chromosomal Haplotype with Two Short Tandem Repeat Mutations*

Abstract:  The male‐specific Y‐chromosomal short tandem repeat (STR) is a useful tool in forensic casework. The Y haplotype comprised of 16 loci, which is amplified simultaneously by AmpFlSTR^® Yfiler^TM PCR kit and provides strong exculpatory evidence in individual identification. We reported a rare Y‐STR profile with a null allele at the DYS448 locus and an off‐ladder allele at the DYS456 locus, when genotyping material from a vaginal swab in an alleged rape case. Sequence analysis revealed that the DYS448 null allele was a true type of null allele because of a total deletion of 11 upstream repeats and 9 bp of the N₄₂ region, and there were numerous primer binding site mutations as well. The amplicon of the DYS456 locus was a small 92‐bp fragment that was off‐ladder, and sequencing analysis showed that there were only 10 repeats (AGAT)₁₀. This Y chromosome haplotype that was comprised of two variations provided helpful evidence for personal identification.     

Internal validation of the AmpFlSTR Yfiler amplification kit for use in forensic casework.

Y-chromosomal short-tandem repeat (Y-STR) amplification has been used in forensic casework at the Bureau of Criminal Apprehension (BCA) Forensic Science Laboratory since 2003. At that time, two separate amplifications were required to type the SWGDAM recommended loci (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS438, and DYS439). The Yfiler kit coamplifies these loci as well as DYS437, DYS448, DYS456, DYS458, DYS635, and Y GATA H4. The Yfiler kit was validated following the internal validations outlined in the SWGDAM revised validation guidelines. Our studies show that 0.125 ng of male DNA will generate a complete 17 locus profile and that as little as 0.06 ng of male DNA yields an average of nine loci. In the male-male mixtures, a complete profile from the minor component was detected up to 1:5 ratio; most of the alleles of the minor component were detected at a 1:10 ratio and more than half the alleles of the minor component were detected at a 1:20 ratio. Complete YSTR profiles were obtained when 500 pg male DNA was mixed with female DNA at ratios up to 1:1000. At ratios of 1:5000 and 1:10,000 (male DNA to female DNA) inhibition of the YSTR amplification was evident. The YSTR results obtained for the adjudicated case samples gave significantly more information than the autosomal results. Our studies demonstrate that the Yfiler kit is extremely sensitive, does not exhibit cross-reactivity with female DNA, successfully types male DNA in the presence of overwhelming amounts of female DNA and is successful in typing actual forensic samples from adjudicated cases.     

AMELY无效扩增个体Yp11.2区域STS位点检测

目的通过Y-STR复合扩增技术检测AMELY缺失的男性个体Y染色体的完整性,并对数据库中的STS位点进行筛选并设计引物,检测Y染色体AMEL区域的STS位点缺失情况,为进一步研究中国人口中AMELY的缺失提供数据和理论依据。方法应用Goldeneye 20A PCR扩增试剂盒、华夏TM白金PCR扩增试剂、Y filer plus试剂盒和Goldeneye 27YB试剂盒进行PCR扩增、通过ABI 3500遗传分析仪进行毛细管电泳以及STR分型。通过UniSTS database和UCSC对Y染色体p11.2区域的多个STS位点进行筛选,部分STS位点自行设计引物,梯度PCR和常规PCR扩增结合琼脂糖凝胶电泳进行STS位点的分析。结果 Goldeneye 20A和华夏TM白金两种试剂盒分型结果均显示两父子AMELY等位基因无效扩增。Y filer plus和27YB试剂盒分型结果显示两父子均在DYS570和DYS576位点无效扩增。STS位点扩增显示两父子的Y染色体缺失的STS位点有BV703904、BV703923、SY2137、SY716、DYS256、DYS267、SY2232、SY2233、DYS261和DYS260。结论 AMELY无效扩增的父子Yp11.2缺失片段包含了BV703904-DYS260之间的区域,长度为2.63~2.74 Mb。     

Developmental validation of the Microreader™ Y Prime Plus ID System: An advanced Y-STR 38-plex system for forensic applications

Y-STR is widely used in sexual assaults and familial searches of suspects. Here, we reported a novel 38-plex STR genotyping system designed for forensic applications. Microreader? Y Prime Plus ID System (YPP) amplifies 38 loci in one reaction, including 29 loci from commonly used Yfiler® Plus PCR Amplification Kit & PowerPlex® Y23 System (DYS393, DYS570, DYS19, DYS392, DYS549, Y GATA H4, DYS460, DYS458, DYS481, DYS635, DYS448, DYS533, DYS449, DYS456, DYS389I, DYS390, DYS389Ⅱ, DYS438, DYS391, DYS439, DYS437, DYS385a/b, DYS643, DYS518, DYS576, DYF387S1a/b, and DYS627), 6 commonly used loci for the Y-STR database (DYS444, DYS447, DYS596, DYF404a/b, DYS527a/b, DYS557) and one Y-indel specific for the Chinese population. YPP is designed for different types of samples, such as blood card and swabs. In this work, YPP was validated following SWGDAM guidelines (2016) and guidelines from Ministry of Public Security of the People’s Republic of China, including PCR-based, sensitivity, accuracy and precision, mixture, stability and inhibitor, and species specificity. The results indicate that the Microreader? Y Prime Plus ID System is a powerful identification kit designed for forensic databases.     

Autosomal short tandem repeat analysis of ancient DNA by coupled use of mini- and conventional STR kits

Abstract: Multiplex autosomal short tandem repeat (STR) genotyping enables researchers to obtain genetic information from ancient human samples. In this study, we tested newly developed AmpF?STR^® MiniFiler? kit for autosomal STR analysis of ancient DNA (aDNA), using human femurs (n = 8) collected from medieval Korean tombs. After extracting aDNA from the bones, autosomal STR analyses were repeated for each sample using the AmpF?STR^® MiniFiler? and Identifiler? kits. Whereas only 21.87% of larger‐sized loci profiles could be obtained with the Identifiler? kit, 75% of the same loci profiles were determined by MiniFiler? kit analysis. This very successful amplification of large‐sized STR markers from highly degraded aDNA suggests that the MiniFiler? kit could be a useful complement to conventional STR kit analysis of ancient samples.     

4个YSTRs基因座的复合扩增

目的 建立DYS19、DYS389 Ⅰ、DYS389 Ⅱ、DYS385复合扩增体系。方法 遴选Y STRs基因座的引物，分别用FAM、TAMRA、TET标记DYS19、DYS385、DYS389 Ⅰ、DYS389 Ⅱ，优化扩增条件，考察扩增体系的个体识别能力、灵敏度、种属特异性及突变情况。结果 所建立的4基因座Y STRs复合扩增体系分型清晰，单倍型多样性达0.989，且特异性好，灵敏度高(1ng DNA)，未观察到突变。结论 所建立的4个Y STRs基因座复合扩增方法适合法医学应用。     

16 Y chromosomal STR haplotypes in Japanese

A total of 1079 Japanese males were typed for the following 16 Y chromosomal short tandem repeat (Y-STR) markers: DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438 and DYS448 using an AmpFlSTR(R) Yfiler PCR Amplification kit (Applied Biosystems). A total of 950 haplotypes for the 16 Y-STR markers were detected and, of these, 886 haplotypes were unique. The most frequent haplotype was found in 22 Japanese males. The haplotype diversity was 0.9992, indicating a high potential for differentiating between male individuals. There were 10 haplotypes with no allele detected at the DYS448 marker. Thus, the presence of such atypical haplotypes should be noted, when DNA typing results obtained from degraded DNA samples and/or DNA mixture samples from more than one male individual are being interpreted.     

Identification of the human Y-chromosomal microsatellite locus DYS19 from degraded DNA

STR DYS19 seems to be one of the most useful markers for population genetic, evolutionary, and forensic applications. However, the authors have noticed that the amplification of the DYS19 polymorphism fails when highly degraded DNA is used as a template. The authors designed a new pair of primers that reduce the DYS19 fragment sizes compared with those of the known protocol. Using these primers, an improved success rate can be achieved, particularly when putrefied samples are under investigation.     

Reduction of Powerplex® Y23 Reaction Volume for Genotyping Buccal Cell Samples on FTATM Cards

PowerPlex^® Y23 is a novel kit for Y‐STR typing that includes new highly discriminating loci. The Israel DNA Database laboratory has recently adopted it for routine Y‐STR analysis. This study examined PCR amplification from 1.2‐mm FTA punch in reduced volumes of 5 and 10 μL. Direct amplification and washing of the FTA punches were examined in different PCR cycle numbers. One short robotically performed wash was found to improve the quality and the percent of profiles obtained. The optimal PCR cycle number was determined for 5 and 10 μL reaction volumes. The percent of obtained profiles, color balance, and reproducibility were examined. High‐quality profiles were achieved in 90% and 88% of the samples amplified in 5 and 10 μL, respectively, in the first attempt. Volume reduction to 5 μL has a vast economic impact especially for DNA database laboratories.     

Alternative primers for DYS391 typing: advantages of their application to forensic genetics

The amplification of the STR DYS391, using the primers described in the Genome Data Base (GDB: G00-365-251), shows not only an additional band to the Y-specific one in males with a size range of 26 bp less than those of DYS391 locus alleles, but also a polymorphic pattern in females in the same size range as the additional band observed in males. The DYS391 pattern in families reflects a Y-specific linked locus and also a polymorphic X locus with an X-linked pattern of inheritance. A first screening in the X homologous locus allowed the identification of five different alleles. Allele frequencies were explored in different population groups for both the Y locus and the homologous locus in the X chromosome showing a similar allele distribution pattern in the X and Y homologous loci. An alternative reverse primer was designed to amplify the Y-chromosome specific STR in order to improve the specificity and applicability of this system to forensic genetics. Comparative results of the amplification with the new and the previously described primers proved that with this new primer there is a substantial increase in the specificity of the amplification. Moreover, a smaller fragment is amplified with a size out of the range of the alleles of the other Y-STRs usually used in forensic applications, therefore simplifying its inclusion in multiplex systems.     

9个Y-STR基因座荧光复合扩增系统的法医学应用

目的建立9个Y-STR基因座的复合扩增系统,提高Y-STR的法医学检测效能。方法6-FAM标记DYS434、Y-GATA-A10、DYS438、DYS439,HEX标记DYS531、DYS557、DYS448,TAMRA标记DYS456、DYS444引物,PCR复合扩增,毛细管电泳得到结果,考察扩增系统的个体识别能力、灵敏度、特异性、组织同一性。结果所建立的9个Y-STR复合扩增系统分型清晰,单倍型多样性达0.9968,特异性好,灵敏度高(0.5ngDNA),并且在男女混合斑检验上较常染色体STR分型更有优势。结论9个Y-STR复合扩增系统具有较高的识别能力,对建立Y染色体STR数据库,研究群体遗传学和进行法医学混合斑物证鉴定有重要意义。     

Efficiency Analysis of EX16 +10Y Kit on Detection of the Uygur Population in Xinjiang ProvinceCSCD

Objective: To analyse the efficiency of EX16+10Y kit on the forensic detection of the Uygur in Xinjiang province. Methods: The blood samples were extracted from 4 620 male individuals of Uygur in Xinjiang province, and amplified by EX16+10Y kit. The typing of amplification products was performed by 3130xl genetic analyzer. Results: The genotyping graphs of 15 autosomal STR loci and 10 Y -chromosomal STR loci from 4 620 male individuals of Uygur in Xinjiang province were acquired completely. The genotype distribution of 15 autosomal STR loci was consistent with Hardy-Weinberg equilibrium. The heterozygosity, polymorphism information content and discrimination power of STR loci were 0.637-0.838, 0.580-0.860 and 0.811-0.978, respectively. There were 766 haplotypes in 10 Y -chromosomal STR loci. Conclusion: The test results of EX16+10Y kit is accurate and trustworthy, which can simultaneously be used for the individual identification and the screening of paternal pedigree in practical work. © 2018 by the Editorial Department of Journal of Forensic Medicine.     

2个Y-STR基因座在成都汉族群体中的遗传学研究

目的　为了寻求新的适合于法医学应用的Y染色体STR基因座 ,我们调查了基因座DYS44 2和DYS44 6在成都群体中的分布。　方法　样本来自于成都地区汉族无血缘关系的个体 ,通过Chelex法提取样本DNA ,利用PCR扩增硝酸银染色方法进行分型。　结果　DYS44 2是一个四核苷酸简单重复基因座 ,而DYS44 6则为五核苷酸简单重复基因座。男性样本都出现了谱带 ,而女性样本则无PCR产物。DYS44 2基因座和DYS44 6基因座变异度分别为 :0 .6 86 7、0 .75 5 2。　结论　DYS44 2和DYS44 6是非常适合于法医学应用的STR基因座。     

A 27‐Locus STR Assay to Meet All United States and European Law Enforcement Agency Standards,

Different national and international agencies have selected specific STR sets for forensic database use. To enhance database comparison across national and international borders, a 27‐locus multiplex system was developed comprising all 15 STR loci of the European standard set, the current 13 STR loci of the CODIS core, the proposed 22 STR loci of the expanded CODIS core, 4 additional commonly used STR loci, and the amelogenin locus. Development required iterative primer design to resolve primer‐related artifacts, amplicon sizing, and locus‐to‐locus balance issues. The 19.5‐min assay incorporated newly developed six‐dye chemistry analyzed using a novel microfluidic electrophoresis instrument capable of simultaneous detection and discrimination of 8 or more fluorescent dyes. The 27‐locus multiplex offers the potential for a new international STR standard permitting laboratories in any jurisdiction to use a single reaction to determine profiles for loci they typically generate plus an expanded common STR profiling set of global interest.     

Haplotype frequencies of 16 Y-chromosome STR loci in the Barcelona metropolitan area population using Y-Filer kit

Haplotype frequencies for 16 Y-chromosomal short tandem repeat (STR) loci, included in the Y-Filer kit, were determined in 247 unrelated healthy individuals from the Barcelona metropolitan area (Catalonia, NE Spain). After PCR amplification and denaturing PAGE electrophoresis, DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4.1, DYS437, DYS438 and DYS448 loci were typed. The aim of this study is to evaluate the performance in our population of the 16 loci of the Y-chromosome present in the new Y-Filer commercial identification kit, and acquire haplotype frequencies for mathematic processing of the forensic diagnosis in our geographical working area. In this sample, all haplotypes were unique. From the forensic point of view, the combined polymorphisms of the Y-Filer kit provide a high diagnostic efficiency.     

11个Y—STR基因座遗传多态性研究

目的 调查11个Y染色体STR基因座的遗传多态性。方法 利用PCR扩增和聚丙烯酰胺凝胶电泳技术对广州地区汉族群体无关男性血样进行11个Y—STR基因座的分型。结果 11个Y—STR基因座在广州地区汉族群体分别发现3～5个等位基因,GD值最低为0.3037(DYS434),最高为0.8455(DYS390)。结论 11个Y—STR基因座在广州地区汉族群体均具有较高的遗传多态性,可应用于法科学个体识别和亲子鉴定。