Internal Validation of the AmpFlSTR Yfiler™ Amplification Kit for Use in Forensic Casework

Abstract:  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 probative 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.     

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.     

Validation of a male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex

Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 microL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5-2x primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2-4 degrees C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 degrees C and significant locus dropout with a 4 degrees C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with >or = 125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of < or = 500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with < or =1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.     

Validation of male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex

Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 microL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5-2x primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2-4 degrees C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 degrees C and significant locus dropout with a 4 degrees C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with >or = 125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of < or = 500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with < or =1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.     

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.     

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.     

湖北地区汉族人群24个 Y-STR基因座遗传多态性研究

目的 对湖北汉族人群24个Y-STR基因座多态性进行调查,并获得相关的基础遗传学数据.方法 应用AGCU Y24 STR荧光标记复合直接扩增系统及3130XL型DNA测序仪,对湖北地区320对已确定父子关系的640个男性个体血样进行24个Y-STR检测分型.结果 在320名父亲男性个体中,在DYS391、DYS389工、DYS439、DYS389Ⅱ、DYS438、DYS449、DYS456、DYS458、DYS437、DYS635、DYS448、Y-GATA-H4、DYS447、DYS19、DYS392、DYS522、DYS393、DYS388、DYS390、DYS444基因座在湖北地区汉族人群分别检出4～17个等位基因,DYS527a/b检出45个等位基因组,DYS385a/b检出57个等位基因组,各基因座基因多样性最低为0.3838,最高为0.9650;并检出320种单倍型.比对320对父子Y-STR分型,在7680次基因遗传传递中,在DYS449、DYS527、DYS444、DYS389Ⅱ、DYS447、DYS522、DYS385、Y-GATA-H4等10个基因座中检出16个突变,突变率为1.5625‰～1.5653％,平均突变率为2.0833‰;等位基因增加突变与等位基因减少突变比为1∶1.结论 24个基因座单倍型在湖北地区汉族人群中具有丰富的遗传多态性,其数据对法医学应用、Y-STR数据库建设和群体遗传学等研究应用具有重要意义.     

Population data for Y-chromosome haplotypes defined by 17 STRs (AmpFlSTR YFiler) in Portugal

The 17 Y-chromosomal short tandem repeats (STRs) included in the AmpFlSTR YFiler Amplification Kit (AB Applied Biosystems) (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 and GATA H4.1) were typed in 250 samples from Portugal. A total of 231 different haplotypes were found, where 17 haplotypes were shared by two individuals and one haplotype by three. The overall haplotype diversity (HD) was 0.9994. DYS458 non-consensus alleles found in 5 samples (out of 85) are all associated with paragroup J*(xJ1,2). Population comparisons with available Yfiler loci data in European samples were undertaken, namely with Northern Portuguese data (N=174) where no significant differences were observed with our sample (Rst=0.0000; P=0.8649+/-0.0310). Since both Portuguese databases can be joined (N=424; HD=0.9997; 394 distinct haplotypes), a study on the best loci for HD increment in this sample was also undertaken: by fixing the haplotypes generated from the minimal haplotype and SWGDAM core set (www.yhrd.org) and adding the other Yfiler loci one by one, the order in which the loci contribute more is DYS458, DYS456, GATA H4.1, DYS437 or DYS635, and finally DYS448. Therefore, at least in this population sample, all Yfiler loci are contributing for haplotype discrimination.     

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.     

DYS448基因座分型缺失分析

目的调查分析DYS448基因座分型缺失,为法医学提供有意义的数据。方法收集中国汉族5487名无关男性个体血样,其中4479份样本用Y-filerTM试剂盒,1008份样本应用Yfiler PlusTM试剂盒进行复合扩增,所有样本应用AGCU Y-24试剂盒复核;统计DYS448基因座出现基因分型缺失的概率。结果在5487名无关个体的Y-STR数据中,观察到35个个体的35种单倍型中DYS448基因座分型出现缺失,其中2个样本在其它基因座位同时出现多谱带。结论 DYS448基因座分型缺失率为0.637%,在Y-STR数据库与父系鉴定应用中应予以关注。     

Effectiveness of Coupled Application of AmpFℓSTR Yfiler Kit and Reduced Size Y‐chromosomal Short Tandem Repeat Analysis for Archeological Human Bones

The AmpF?STR Yfiler PCR Amplification (Yfiler) kit continues to be improved for a better analytical efficiency in cases of highly degraded DNA. The authors endeavored to determine whether coupling of the Yfiler kit with supplemental multiplex amplification of some Y‐STR loci is a more efficient analytical mode for poorly preserved human femurs (n = 15) discovered at Korean archeological sites. To reveal locus profiles not easily obtained by Yfiler analysis, custom‐designed primers were adopted for the DYS390, DYS391, DYS392, DYS438, DYS439, and DYS635 loci. The success rate for 16 Y‐STR locus profiles obtained from the 15 femurs was improved from 18.33% (in the use of Yfiler kit only) to 49.17% (the coupled use of Yfiler and custom‐designed primers). In this study, the authors established that the custom‐designed primers offer a markedly improved success rate for obtainment of Y‐STR profiles from degraded aDNA not easily identified by sole use of the Yfiler assay.     

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.     

珠海地区汉族人群10个Y-STR基因座的多态性

目的 调查珠海地区汉族人群10个Y-STR基因座及其单倍型的遗传多态性，探讨其法医学应用价值。方法 应用Y-PLEX荧光标记复合扩增系统，对珠海地区汉族200名无关男性个体进行10个Y-STR基因座的复合扩增，用ABI310型基因分析仪对扩增产物进行检测，统计10个Y-STR基因座的群体遗传学参数。结果 9个Y-STR基因座分别检出5、6、6、5、4、5、5、5、7个等位基因，DYS385基因座检出44种单倍型；GD值最低为0.3904(DYS391)，最高为0.9497(DYS385)；10个Y-STR基因座共同构成的单倍型161种，其中134种单倍型只出现1次，20种单倍型出现2次，3种单倍型出现3次，3种单倍型出现4次，1种单倍型出现5次，累计GD值为0.9948。结论 10个Y-STR基因座具有较高的个体识别能力，可应用于法庭科学中的个体识别与亲权鉴定。     

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.     

云南汉族人群11个Y-STR基因座遗传多态性调查及法医学应用

目的调查11个Y-STR基因座及其单倍型在云南汉族人群中的遗传多态性分布,探讨其法医学应用价值,为法医学应用提供基础数据。方法应用Powerplex!Y系统对云南汉族201名无关男性个体进行11个Y-STR基因座的复合扩增,用ABI310型基因分析仪对扩增产物进行检测,统计其群体遗传学参数。结果Powerplex!Y系统前10个Y-STR基因座分别检出3、5、6、8、5、4、5、8、4、7个等位基因,DYS385a/b基因座检出56种单倍型;GD值最低为0.4273(DYS438),最高为0.9747(DYS385a/b);观察到11个Y-STR基因座共同构成的单倍型175种,其中有154种单倍型只出现1次,16种出现2次,5种出现3次,累计GD值为0.9984。结论11个Y-STR基因座具有较强的个体识别能力,可应用于云南地区汉族人群的个体识别与亲权鉴定。     

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。     

24个Y-STR基因座荧光标记复合扩增体系的建立

目的构建包含24个Y-STR基因座的荧光标记复合扩增体系。方法选择DYS531、DYS630、DYS622、DYS552、DYS510、DYS449、DYS459a/b、DYS446、DYS443、DYS635、DYS587、DYS527a/b、DYS460、Y-GATA-A10、DYS520、DYS557、DYS522、DYS481、DYS570、DYS385a/b、DYS444 24个Y-STR基因座,构建荧光复合扩增体系。检测该体系的特异性、同一性、灵敏度、扩增均衡性、抗干扰性和准确性,调查其在广东地区人群的基因多样性。结果建立的复合扩增体系在检测的非人类及女性样本中未发现谱带,同一个体不同组织检测结果一致,0.1 ng以上标准品9948可检测获得完整分型结果。常见抑制物中体系内加入120~200μmol/L血红素、1.5~2.0 mmol/L钙离子时出现等位基因丢失,对靛蓝、腐殖酸、EDTA具有较强抗干扰性。通过146例无关个体与Yfiler系统平行检测比对,24 Y-STR检测体系分型准确。广东地区人群单倍型多样性(HD)为0.999 72,优于Yfiler系统(HD=0.998 58)。结论本研究建立的24个Y-STR基因座荧光标记复合扩增体系法医学应用前景广阔,可用于案件检验、亲权鉴定与Y-STR数据库建设。     

Validation of a multiplex amplification system of 19 autosomal STRs and 27 Y-STRs

This article describes a newly devised autosomal short tandem repeat (STR) multiplex polymerase chain reaction (PCR) system for 19 autosomal loci (D12S391, D13S317, D16S539, D18S51, D19S433, D2S1338, D21S11, D3S1358, D5S818, D6S1043, D7S820, D8S1179, CSF1PO, FGA, TH01, TPOX, vWA, Penta D and Penta E), 27 Y-chromosome STR loci (DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS518, DYS533, DYS570, DYS576, DYS635, DYS627, YGATAH4 and DYF387S1) and amelogenin with six-colour fluorescent labelling. Various parameters were evaluated, such as its accuracy, sensitivity, specificity, stability, ability to analysis of mixtures and effects of changes in the PCR-based procedures. All of the 47 selected STR loci were accurately and robustly amplified from 282 bloodstain samples. The species-specificity was high and some ability to inhibit Hematin was identified. The lowest detectable DNA amount was ≥0.125 ng. All of the male loci of the secondary component were revealed precisely when the control DNA was mixed at male/female and male/male ratios of 1:4 or more. We conclude that the present 19-plex autosomal STR and 27 Y-STR assay is both accurate and sensitive. It constitutes an additional powerful tool for forensic applications.