AmpFlSTR Yfiler PCR Amplification Kit Research Articles

Short tandem repeat typing technologies used in paternity testing: a case study

Background: In forensic field, for establishing the paternity of disputed offspring, STR typing (autosomal, and Y typing) are commonly used to dissolve the cases. Short tandem repeat (STR) technology is used to evaluate specific regions (loci) within nuclear DNA. Variability in STR regions can be used to distinguish one DNA profile from another. The Federal Bureau of Investigation (FBI) uses a standard set of 13 specific STR regions for CODIS. Aim of the study: The present work was done to find out the biological father of child in a case where mother was killed by her husband for the doubt of her pregnancy. Methods: After taking blood samples from the murdered mother, the baby and the killer husband, Autosomal STRs Using AmpFlSTR Identifiler Plus PCR Amplification Kit and Y-STR analysis using AmpFlSTR Yfiler PCR Amplification Kit were done to solve the disputed paternity case. Results: After the analysis of DNA STRs profiles of autosomal and Y-chromosome markers, the dead fetus was proved to be the son of the killer father. Conclusions: Autosomal STR multiplex analysis combined with Y-STR analysis help to reach a conclusion in disputed paternity cases with greater confidence. Keywords: Disputed; Paternity; Multiplex; Short tandem reprats; Y-STR; Analysis

Importance of Autosomal and Y-STR Markers in Establishing Sibling Relationship by DNA Genotyping.

The aim of the present study was the identification of an unknown person found in an advanced decomposed state using DNA samples provided by two alleged brothers as reference samples. To obtain an increased reliability of the test, we used autosomal and Y-STR markers. Tissue fragments were obtained for the DNA isolation during the autopsy examination from the unidentified person. DNA was isolated from the reference samples obtained from buccal swabs of the two alleged brothers. The DNA was isolated from the biological samples using PureLink Genomic DNA (Invitrogen, USA). The quantification of the DNA samples was done on an ABI 7500 real-time PCR system with HID Analysis software v1.2 incorporated. For DNA amplification we used the multiplex PCR kit AmpFlSTR Identifiler Plus Kit for autosomal STR markers and AmpFlSTR Y-filer PCR Amplification Kit for the Y-STR markers. Further, we separated the DNA products on an ABI 3500 genetic analyzer. Gene Mapper ID-X version 1.4 software was used to visualize the DNA fragments. Data interpretation was done using the Kinship Examination of GenoProof-3 (qualitype, Dresden, Germany). We obtained genetic profiles for the three alleged brothers on autosomal and Y-STR markers and, thus, could establish a full sibling relationship between them. Since the introduction of DNA in human identification, it represents a useful tool in establishing sibling relationship from different biological samples.

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

The AmpFℓSTR 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.

Comparison of DNA typing using AmpFlSTR Yfiler and PowerPlex Y System, for specimens subject to very long storage

We performed DNA typing from blood stains stored at room temperature for 22–30 years, using AmpFlSTR Yfiler PCR amplification Kit and PowerPlex Y System to extract and amplify the DNA and performing electrophoresis with an ABI 310 Genetic Analyzer. We identified alleles using GeneMapper ID v3.2.1 software. We found that long-term storage tended to reduce the number of detectable loci in the blood stains and that it was easier to detect the loci of smaller amplicons than larger amplicons.

Population data for 17 Y-chromosome STRs in a sample from Apulia (Southern Italy)

The 17 Y-STR loci included in the AmpFLSTR Yfiler PCR Amplification Kit were analyzed in 98 unrelated healthy males from Apulia (Southern Italy). A total of 97 different haplotypes were identified, of which 96 haplotypes were unique and 1 occurred twice. Allele frequencies for each Y-STR locus in pooled sample and estimated value of gene diversity (GD) were evaluated. The lowest value of GD was observed for DYS392 (0.126) and the highest one (0.936) for DYS385. The HD (haplotype diversity) for the studied Y-STR set showed a value of 0.9994, with an HMP (haplotype match probability) value of 0.0006, while the overall DC was 98.98%. Microvariant alleles were found for the DYS458 and DYS385 markers and sequenced. Furthermore, Φ(st)-based genetic distance computation and pair-wise analysis of molecular variance (AMOVA) test were carried out. When comparing our population with the Apulia sample previously investigated, the AMOVA analysis detected no evidence for significant differentiation. The comparison with all Italian populations submitted to the YHRD website showed no relevant differences with all Southern Italian populations (San Giorgio La Molara, Belvedere, Trapani and Catania) and significant genetic deviation with all Northern Italian populations (Udine, Biella, La Spezia, Modena, Ravenna, Marche and North Sardinia). Moreover, the other populations and meta-populations belonging to the whole Mediterranean area (Croatia, Macedonia, Albania, Greece, Turkey, Israel, Libya, Tunisia, Algeria, Morocco and Spain) were different from our Apulia sample. The data were submitted to YHRD.

Y-chromosomal haplotypes for the AmpFlSTR Yfiler PCR amplification kit in a population sample of Bedouins residing in the area of the Fourth Nile Cataract

The Bedouin are a part of a predominantly desert-dwelling Arab ethnic group traditionally divided into tribes or clans. The term ‘Bedouin’ means, ‘those who live in bādiyah’ or ‘those who live in the desert’. Altogether, the Bedouin population in Africa and in the Middle East numbers about 4 million. The contemporary Bedouin tribes have originated from the area of Arabian Peninsula and overlapped older Nubian populations of Christian Era that had developed after Marawy culture which was an extension to the Pharaonic culture. Anthropological studies on the native inhabitants dwelling in region of the 4th Nile Cataract revealed significant morphologic variation based on cephalometric and somatoscopic data. The skin color ranges from very dark, similar to that seen in inhabitants of Central Africa, to olive – typical for the Mediterranean race (prof. Jan Czekanowski – personal communication). Examples of facial types among individuals dwelling in the same village are presented in Supplementary Fig. 1. This dissimilarity results obviously from history of the tribes involved in banditry and trade of slaves from southern Sudan and other parts of Africa, since many of the slaves assimilated into the local population. Additionally, islamisation and arabisation of Sudan caused admixture to the pool of resident Christian inhabitants originating from the ancient Meroe and Nubia. Currently detailed genetic investigations are performed on these populations as well as on archeological sites (burial grounds) to learn more about the origin of the inhabitants.

Haplotype analysis of the polymorphic 17 YSTR markers in Kerala nontribal populations

The origin of the Kerala non tribal population has been a matter of contention for centuries. While some claim that Negritos were the first inhabitants, some historians suggest a Dravidian origin for all Keralites. The aim of our study has been to provide sufficient scientific evidence based on Y chromosome short tandem repeat (Y STR) analysis for tracing the paternal lineage and also to create a database of the Y STR haplotype of the male population for future forensic analysis. Whole blood samples (n = 168) were collected from unrelated healthy men of the Kerala non-tribal population over a period of 2 years from October 2009. Genomic DNA was extracted by salting out method. All samples were genotyped for the 17 Y STR loci by the AmpFLSTR Y-filer PCR Amplification Kit. The haplotype and allele frequencies were determined by direct counting and analyzed using Arlequin 3.1 software, and molecular variance was calculated with the Y chromosome haplotype reference database online analysis tool, www.yhrd.org . Haplotype diversity was calculated using HaPYDive ( http://portugene.com/hapydive.html ). The majority of haplotypes were unique (149/168). The variant allele 17.1 was observed in DYS 385 loci in three samples. Fifteen samples (8.93%) showed the presence of alleles that are not within the established marker range denoted as outside marker range (OMR). The allele frequency of Kerala non tribal population ranged from 0.00003 to 0.5809. The most polymorphic single locus marker was DYS 458. The haplotype diversity value for Kerala non tribal population was 0.9978. The pairwise difference value ranged from 0.0531 to 0.0854 on comparison of the haplotypes of the Kerala non tribals with other Indian populations. The multi dimensional scaling plot depicted the proximity of Kerala non tribal population with Vasterbotten population (Swedish) and Paiwan, Patyal population of Taiwan, Thailand, and Zhuang population of China. The results of the study indicate towards a European paternal lineage in the non tribal Kerala population.

Y-STR genetic diversity in autochthonous Andalusians from Huelva and Granada provinces (Spain)

Seventeen Y-chromosomal short tandem repeats (STRs) were analyzed in 347 healthy, unrelated, autochthonous males from the Andalusian provinces of Huelva (N=167) and Granada (N=180). AmpFlSTR Y-filer PCR Amplification kit (Applied Biosystems) was used to type the Y-STR markers. A total of 156 and 166 different haplotypes for the 17 Y-STR set were detected in Huelva, and Granada, respectively. The same haplotype diversity was found for both samples (0.998±0.001), and the overall discrimination capacity was 0.904. The most common minimal haplotype (DYS19, DYS389 I, DYS389 II, DYS390, DYS391, DYS392, DYS393) in both subpopulations was 14-13-16-24-11-13-13, which is also the most frequent haplotype among Atlantic European populations. Comparison analysis using pairwise RST values and Analysis of Molecular Variance (AMOVA) revealed a significant genetic distance between our Andalusian samples and other ones from the northern Iberian fringe (including Basque and Pyrenean populations). However, results from the multi-dimensional scaling analysis (MDS) yielded a well-defined group of Iberian populations separated from the other Mediterranean clusters observed.

Population genetics of 17 Y-chromosomal STR loci in Yakutia

Haplotype and allele frequencies of 17 Y-chromosomal short tandem repeat (Y-STR) markers in a population sample of 133 Yakut male volunteers from two regions: Central ( n = 41) and Western Yakutia ( n = 92) were determined using the AmpFlSTR Yfiler PCR Amplification Kit (Applied Biosystems). A total of 65 haplotypes were identified in the Yakut population, with 15 haplotypes in Central sample and 54 haplotypes in Western sample. Haplotype diversity values of 0.79 and 0.96, and average gene diversity values of 0.14 and 0.41 were calculated for Central and Western samples, respectively. The Fst distances between both our Yakut populations with other Russian, Siberian and Chinese populations were represented by MDS plot. The graphical view demonstrated close distances between most Yakut populations and differences with other Siberian populations.

Genetic polymorphism of 17 Y-STR loci in four minority populations in Guangxi of China

To investigate the genetic polymorphism of Y-chromosomal short tandem repeats (STR) loci in Jing, Yi, Yao, and Zhuang minority populations from Guangxi Province, China. 17 Y-STR loci were co-amplified using AmpFlSTR(R) Yfiler PCR Amplification Kit System, and the PCR products were analyzed by genetic analyzer. Cluster and phylogenic tree analyses were conducted to show the genetic distance among the populations. There were 61 different haplotypes in 100 unrelated Yao males, 67 in 105 unrelated Yi males, 79 in 103 unrelated Jing males, and 91 in 107 unrelated Zhuang males. The haplotype diversities of Jing, Yi, Yao and Zhuang were determined as 0.9784, 0.9866, 0.9911, and 0.9956, respectively. Among these 4 minority populations, the genetic distance between Jing and Zhuang was the smallest (0.0391), while the genetic distance between Yi and Yao was the largest (0.3376). The 17 Y-STR loci in the 4 minority populations from Guangxi Province revealed a highly polymorphic genetic distribution, which show a high potential for population genetics and forensic practice.

Evaluation of Modified Yfiler™ Amplification Strategy for Compromised Samples

To characterize the data produced using a modified amplification protocol for the AmpFlSTR Yfiler PCR Amplification Kit (Applied Biosystems) and explore the potential of Y-chromosomal short tandem repeat (Y-STR) recovery from severely degraded skeletal remains encountered at the Armed Forces DNA Identification Laboratory. Experiments were performed using two sets of Yfiler amplification parameters. One set of parameters reflected the manufacturer's recommendations. The second set of parameters included twice the recommended Taq concentration and 6 additional cycles. Recovery of authentic alleles and the incidence of drop-in alleles were assessed for 3 data sets: 8 different quantities of pristine DNA, 8 artificially-degraded samples, and 31 non-probative case samples. Samples tested with both protocols from all 3 data sets yielded twice as many authentic alleles under the modified parameters than under the standard parameters (62% vs 31%), with only a nominal associated increase in the occurrence of non-authentic alleles (1.36% of all alleles detected). When applied to a range of representative casework samples, the modified protocol leveraged 9 or more reproducible alleles from over half of the specimens tested. Reproducible and informative Y-STR profiles can be recovered from a broad range of degraded and inhibited skeletal remains extracts when a commercially available kit is employed under modified amplification parameters.

DNA typing using AmpFlSTR Y-filer for very long-term stored specimens

Aware that long-term storage bloodstain generally reduces numbers of detectable loci, we performed DNA typing with bloodstains stored at room temperature for 22–30 years, then extracted DNA from the bloodstains using the AmpFlSTR Y-filer PCR Amplification Kit. We performed electrophoresis with an ABI 310 Genetic Analyzer and determined alleles using GeneMapper ID v3.2 software. Our results suggest that Y-filer finds certain loci more difficult to anneal than others.

Y-chromosome haplotypes defined by 17 STRs included in AmpFlSTR Yfiler PCR Amplification Kit in a multi ethnical population from El Beni Department (North Bolivia)

Seventeen Y-STRs included in AmpFlSTR Yfiler™ PCR Amplification Kit (Applied Biosystems, USA) were studied in males from a multi ethnical population from El Beni Department (North Bolivia). Haplotypic and allelic frequencies were reported. Comparison of El Beni population with other samples from the region was carried out through Multidimensional Scalling over Rst distances matrix.

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

The 17 Y-chromosome STR loci (DYS19, DYS389I, DYS389II, DYS390, DYS456, DYS391, DYS392, DYS393 and DYS385 a/b, DYS458, DYS439, DYS635, GATA H4.1, DYS437, DYS438 and DYS448) were determined for 100 unrelated males, living in Central Portugal, using the AmpFlSTR YFiler PCR Amplification kit (Applied Biosystems). A total of 99 different haplotypes were found, with only two individuals sharing the same haplotype. The overall haplotype diversity (HD) was determined as 0.9998, a value similar to other Y Filer data sets. Y-STR polymorphisms in Central Portugal population, using YFiler, provide a powerful discrimination tool for routine forensic applications.

Population genetics of 17 Y-chromosomal STR loci in Japanese

Haplotypes and allele frequencies of 17 Y-chromosomal short tandem repeat (Y-STR) markers were examined using the AmpFlSTR Yfiler PCR Amplification Kit (Applied Biosystems) in a population sample of 1166 Japanese male volunteers in 6 prefectures: Miyagi, Yamagata, Osaka, Tottori, Fukuoka, and Okinawa. A total of 1058 haplotypes were observed from 1166 males, and the most common haplotype detected in 12 males had a frequency of 1.03% and the discrimination capacity was 0.907. The R ST analysis showed statistically significant differences between Okinawa and the other subpopulations.

Validation of AmpFlSTR® YfilerTM PCR Amplification Kit for Forensic Applications

A validation study was performed for the AmpFlSTR Yfiler PCR Amplification Kit (the Yfiler kit) to evaluate its potential applicability to Y-STR analysis in forensic casework. The optimal DNA input for PCR amplification was approximately 1 ng of male DNA. A marker-specific stutter ratio to filter stutter peaks higher than that defined by the kit manufacturer has been adopted for some markers to more readily and efficiently type Japanese male samples. No non-specific amplification was observed using 2000 ng female DNA as the PCR template. A full or partial DNA profile was detected from DNA mixture samples containing 1 ng of male DNA and 2000 ng of female DNA. From a mixture of two male DNA samples, all alleles possessed by the minor DNA contributor were detected when DNA derived from the minor contributor was present at 20% of the total DNA and above. Several loci were amplified when analyzing 23-year-old bloodstain samples derived from male individuals. Since they had not yet been defined by the kit manufacturer, we sequenced new alleles observed in Japanese males to assign accurate allele names. Analyses were also carried out of unusual alleles (an allele with poor amplification at DYS390 and apparent null alleles at DYS437 and DYS448) we observed in Japanese males. It is thought important that forensic biologists are aware of new and unusual alleles for DNA profile interpretation. Our validation study indicated the usefulness of the Yfiler kit for analyzing forensic biological samples containing male DNA.

Probability distribution of sibship determination with ABI Identifiler multiplex system using different software

Forensic laboratories may be asked to provide genetic evidence that two persons are related or not, when no other relatives are available for study. Sibship analysis of autosome polymorphisms are more complicated since there are no obligatory alleles between siblings that make it possible to exclude a biological relationship with absolute certainty.In this preliminary study, seventy full-sib pairs were genotyped using the AmpFLSTR Identifiler PCR Amplification kit. All subjects belonged to families that included mother, two or three children, and an alleged father, in which neither the mother nor the alleged father were excluded as biological parents, and no mutational event was observed. In addition, the Y chromosome was investigated with AmpFLSTR Yfiler PCR Amplification kit to provide further support for the relationship. The probability that each pair was composed of full sibs rather than non-relatives was calculated by standard formulas, and was verified using different published software. The distribution of these probability values was used to ascertain the statistical power of the Identifiler kit to resolve sibship relationships to forensic purposes.

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.

Y-chromosomal haplotypes for the AmpFlSTR Yfiler PCR Amplification Kit in a population sample from Central Poland

Haplotype and allele frequencies for 17 Y-STR loci (DYS456, DYS389I/II, DYS390, DYS458, DYS19, DYS385 I/II, Y GATA H4, DYS437, DYS438, DYS448, DYS393, DYS391, DYS439, Y GATA C4, DYS392) were determined in 255 unrelated males from central Poland using AmpFlSTR Yfiler PCR Amplification Kit. Two hundred and fifty-two different haplotypes were observed. The most common three haplotypes were shared by 0.8% of the sample, respectively. Two hundred and forty-nine haplotypes were encountered only once.

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.