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.     

Genetic polymorphisms for 11 Y-STRs haplotypes of Chinese Yi ethnic minority group

Eleven Y-STRs loci including minimal haplotypes (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, and DYS385a,b) and two additional loci, namely DYS438 and DYS439 have been co-amplified in 100 healthy unrelated males of Chinese Yi minority ethnic group using the Y-PLEX 5 and Y-PLEX 6 kit, in order to investigate allele and haplotype frequencies of Yi population, evaluate their usefulness in forensic paternity testing and human identification, and enrich Chinese population genetic informational resources. Out of a total of 100 individuals 85 showed different haplotypes, while 8 haplotypes occurred more than once. The overall haplotype diversity for 11 Y-STRs loci was 0.9945.     

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).     

Utility of the Y-STR typing systems Y-PLEX 6 and Y-PLEX 5 in forensic casework and 11 Y-STR haplotype database for three major population groups in the United States

The Y-PLEX 6 and Y-PLEX 5 systems enable analysis for 11 Y-STR loci. We present here the utility of these systems in forensic casework. A total of 188 samples, including 127 evidence samples, were analyzed using either or both of the systems. The evidence sample types included fingernail scrapings, sperm or seminal fluid, epithelial cells, blood and other tissues. The Y-STR typing systems provided useful probative results in difficult cases. A reference database for Caucasian (n = 517), African American (n = 535), and Hispanic (n = 245) population groups within the United States was generated for estimating the haplotype frequency in forensic casework. Among the individuals profiled, 311 Caucasians, 412 African Americans, and 194 Hispanics provided unique profiles in their respective population datasets. This is the first report describing the haplotype database for the set of 11 Y-STR loci recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Linkage analysis reveals that the frequencies from forensically important autosomal loci can be multiplied with the Y-STR haplotype frequency. The results from Y-PLEX systems have been accepted in courts in the United States.     

Y-STR polymorphism in Central Anatolian Region of Turkey

Eight Y-chromosome specific STR (Y-STR) loci including DYS19, DYS388, DYS389I, DYS389II, DYS390, DYS391, DYS392 and DYS393 were investigated in a group of males from Central Anatolian Region of Turkey. Healthy 59 males living in this region for at least three generations were included in the study. PCR analysis was carried out with Y-STR specific primers on genomic DNA obtained from peripheral blood samples and size determination of PCR products was performed by silver staining following 6% polyacrylamide gel electrophoresis (PAGE). DYS388 was found to be the locus with lowest diversity (D) whereas DYS389II was the locus with highest diversity. The current study presented a framework of variation for the eight Y-STR loci in Central Anatolian population.     

Analysis of human fecal material for autosomal and Y chromosome STRs

Human stool samples from eight volunteers were stored under various conditions and extracted by three different procedures. Fecal material and tissue paper soiled with fecal material obtained from a crime scene were also extracted. Extracted DNA was amplified using the AmpFlSTR Profiler Plus, AmpFlSTR COfiler, and the AmpFlSTR Identifiler PCR amplification kits for the detection of the autosomal STR allelic patterns. DNA extracted from the male volunteers and from the soiled tissue paper evidence sample was also amplified using the Y-PLEX 6 and Y-PLEX 5 amplification kits. Analysis of the amplified products was carried out by capillary electrophoresis on the ABI PRISM 310 Genetic Analyzer. Autosomal and Y-STR profiles obtained from the fecal material were concordant with the results from the donors' buccal swabs.     

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.     

Characterization and haplotype analysis of 10 novel Y-STR loci in Chinese Han population

In this study, we analyzed allelic sequences of 10 novel Y-specific STR loci, DYS454, DYS510, DYS513, DYS520, DYS542, DYS544, DYS552, DYS561, DYS587 and DYS593, surveyed the distribution of haplotypes in a Chinese Han population. Extracted DNA was amplified with PCR, followed by a horizontal non-denaturing polyacrylamide gel electrophoresis with discontinuous buffer system. Purified alleles were sequenced on DNA sequencer (ABI Model 377) to verify the number of motif repeats. The number of alleles observed at each locus ranged from 3 to 8, yielding 102 haplotypes in 103 unrelated males samples. The allele diversity values for each locus ranged from 0.2099 (DYS544) to 0.7523 (DYS552). The haplotype diversity using all these loci was 0.9998. Our study revealed that they were valuable Y-specific markers for forensic applications.     

Robustness of the Y STRs DYS19, DYS389 I and II, DYS390 and DYS393: optimization of a PCR pentaplex

Various technical methods were investigated with the aim of developing a multiplex system to amplify five Y-chromosome STR loci in the same PCR reaction: DYS393, DYS19, DYS390, DYS389 I and DYS389 II. A sequenced allelic ladder was constructed with previously sequenced alleles including the most common ones. A number of reamplification conditions of the allelic ladders were tested. The pentaplex was evaluated for typing using two different platforms (ABI and ALF) with promising results. However, in degraded samples non-specific artifacts were observed in the DYS393 system in the same range of sizes as the real alleles. This system can also be typed in females under relatively low stringency conditions in the PCR amplification, making this system prone to errors in critical samples. This lack of specificity can be reduced by increasing the stringency of the PCR conditions. The DYS19 ladder cannot be reamplified as stutters appear after a few reamplifications. These stutters are probably due to a 2 bp slippage induced by the presence of a TA repeat stretch in the PCR amplified fragments. Non-specific products were also noted in the DYS389 I and DYS389 II amplification, although out of the range of other alleles in this pentaplex. This newly constructed pentaplex has proved to be very useful in population genetic studies because all five Y STR markers can be loaded in the same lane of a gel with other Y STR singleplex or multiplexes. The usefulness of Y-chromosome STRs in criminal casework is especially evident in analyzing azoospermic individuals.     

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.     

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.     

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.     

Y-STRs in forensic medicine: DNA analysis in semen samples of azoospermic individuals

ABSTRACT: The incidence of rape has increased, especially in metropolitan areas, such as the city of São Paulo. In Brazil, studies about it have shown that the majority of this type of crime is committed by the relatives and persons close to the victim. This has made the crime more difficult to be denounced, as only 10% of the cases are reported to competent police authorities. Usually, cytological exams are carried out in sex crime investigations. The difficulty in showing the presence of spermatozoa is frequent, but it does not exclude the presence of male DNA. The absence of spermatozoa in material collected from rape victims can be due to several factors, including the fact that the agressor suffers from azoospermia. This condition can be the result of a successful vasectomy. As the majority of DNA in the ejaculation sample is from spermatozoa, there is much less DNA to be analyzed. This study presents the application of Y‐STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393) in DNA analysis of sperm samples from 105 vasectomized men. The study demonstrated a great variation in DNA concentration. DNA extraction and amplification was possible in all sperm samples even in the absence of spermatozoa. The same profile was observed, for each individual, from DNA extracted from blood, pre‐ and postvasectomy semen samples. The use of markers specific for Y chromosome in sex crime cases, especially in the absence of spermatozoa, is very important, mainly because in most situations there is a small quantity of the agressor's DNA in the medium and a large quantity of the victim's DNA.     

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。     

藏族群体7个Y-STR基因座及单倍型的遗传多态性

目的调查藏族Y染色体7个STR基因座及单倍型的遗传多态性。方法应用PCR、变性聚丙烯酰胺凝胶电泳结合银染显色分型技术,检测67例藏族男性DNA样品。结果在DYS393、DYS389Ⅰ、DYS19、DYS390、DYS389Ⅱ、DYS392等6个基因座中共检出25个等位基因,DYS385基因座检出22个等位基因组,频率分布在0.0149～0.5075之间,各基因座DP值分布在0.5075～0.8991之间,以DYS385基因座最高。由7个基因座组成的YH6系统单倍型有51种,DP值为0.9887,DYS393与DYS389Ⅱ基因座等位基因少,推测藏族群体在进化过程中可能发生过瓶颈事件。结论上述7个STR基因座属于高鉴别能力基因座,单倍型具有很高的遗传多态性。     

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.