Chromosome Short Tandem Repeat Loci Research Articles

Validation of the AGCU Expressmarker 20 + 20Y kit: A 6-dye multiplex assay for forensic application

The AGCU Expressmarker 20 + 20Y Kit is a newly devised short tandem repeat (STR) multiplex system that simultaneously analyses a set of 19 autosomal STR loci, 20 Y chromosomal STR loci and the amelogenin locus with six-dye fluorescent labelling. Here, the AGCU Expressmarker 20 + 20Y system was validated following the guidelines published by the Scientific Working Group on DNA Analysis Methods (SWGDAM), including PCR-condition, sensitivity, mixtures, species specificity, models of inhibition, precision, stutter percentage, concordance, population genetic studies, performance on three kinds of degraded DNA and a type of casework samples. The results indicated that the kit had high sensitivity when there was a small amount of DNA (0.0625 ng), more than one male (minor: major=1:19), or a mixture of males and females (male: female=1:32), models of inhibition (250 μM hematin, 1500 ng/μL humic acid and more than 100 ng/μL tannic acid) and degraded samples. The kit showed high precision level with standard deviation of allele size no more than 0.0930. Furthermore, this system was also tested in 444 random male samples of Chinese Han and Hui population, showing its high discrimination capability in Han and Hui population. Meanwhile, the system was applicable to the case of the AMELY abnormality. In short, the kit was verified and proved to be a robust, reliable and suitable tool for human identification and casework samples.

Costal cartilage ensures low degradation of DNA needed for genetic identification of human remains retrieved at different decomposition stages and different postmortem intervals

Abstract Introduction The study aimed to evaluate if costal cartilage is a good source of DNA for genetic individual identification tests performed in forensic autopsies. Materials and Methods The study included samples of costal cartilage collected from 80 cadavers retrieved from different environments: indoors (flat/hospital), outdoors (primarily in the forest), a coal mine, a fire site, uninhabited buildings, a basement, bodies of fresh water, exhumation sites, and unknown locations. After isolation of DNA chondrocytes, T. Large autosomal chromosome (214 bp), T. Small autosomal chromosome (80 bp), and the Y chromosome (75 bp; for male cadavers), sequences were amplified using real-time PCR. Additionally, 23 autosomal short tandem repeat (STR) loci and 16 Y chromosome STR loci were amplified using multiplex PCR. Forensic DNA typing was done using capillary electrophoresis and all results were analyzed. Results There was no statistically significant difference in DNA concentration after T. Large, T. Small autosomal chromosome and the Y chromosome amplification between samples collected from cadavers retrieved from different environments. The DNA degradation index was the same regardless of the postmortem interval. The results show that it is possible to generate a full genetic profile from costal cartilage samples collected from cadavers retrieved from different environments and at different times elapsed after death. Conclusions The results suggest that costal cartilage can be routinely collected during forensic autopsies, especially from cadavers at the advanced decomposition stage.

Identification of sequence polymorphisms at 58 STRs and 94 iiSNPs in a Tibetan population using massively parallel sequencing

Massively parallel sequencing (MPS) has rapidly become a promising method for forensic DNA typing, due to its ability to detect a large number of markers and samples simultaneously in a single reaction, and sequence information can be obtained directly. In the present study, two kinds of forensic genetic markers, short tandem repeat (STR) and identity-informative single nucleotide polymorphism (iiSNP) were analyzed simultaneously using ForenSeq DNA Signature Prep Kit, a commercially available kit on MPS platform. A total of 152 DNA markers, including 27 autosomal STR (A-STR) loci, 24 Y chromosomal STR (Y-STR) loci, 7 X chromosomal STR (X-STR) loci and 94 iiSNP loci were genotyped for 107 Tibetan individuals (53 males and 54 females). Compared with length-based STR typing methods, 112 more A-STR alleles, 41 more Y-STR alleles, and 24 more X-STR alleles were observed at 17 A-STRs, 9 Y-STRs, and 5 X-STRs using sequence-based approaches. Thirty-nine novel sequence variations were observed at 20 STR loci. When the flanking regions were also analyzed in addition to target SNPs at the 94 iiSNPs, 38 more alleles were identified. Our study provided an adequate genotype and frequencies data of the two types of genetic markers for forensic practice. Moreover, we also proved that this panel is highly polymorphic and informative in Tibetan population, and should be efficient in forensic kinship testing and personal identification cases.

Genetic polymorphism analyses of three novel X chromosomal short tandem repeat loci in the Xp22.3 region.

X chromosomal short tandem repeats (X-STRs) can be useful for haplotype analysis in DNA testing, particularly for complex kinship testing or when one parent is absent. We searched downstream of four previously detected loci in the Xp22.3 region (LC149476, LC149479, LC149480, and LC149484) and detected and analyzed three novel short tandem repeats (STRs), LC317283, LC317284, and LC317285, with the repeat sequences TATAA, TTTA, and TATC, respectively. The forensic statistical values in Japanese subjects were confirmed to be noninferior to existing loci, with values for polymorphism information content, the power of discrimination in males (PDm), and the power of discrimination in females (PDf) of 0.5606-0.7448, 0.6078-0.7774, and 0.7990-0.9178, respectively. Haplotype analysis also revealed linkage disequilibrium between LC317283 and the four known loci (LC149476, LC149479, LC149480, and LC149484) and between two other novel loci (LC317284 and LC317285). Analysis of three family samples suggested that these STRs could be useful in complex kinship testing, so we developed an X-STR multiplex polymerase chain reaction (PCR) system for the seven loci and confirmed its ability to provide favorable amplification. We anticipate that the identified loci and developed multiplex PCR system will be beneficial to the field of forensic medicine.

Population data of 23 Y chromosome STR loci for the five major human subpopulations of Ghana.

In this study, 268 samples for unrelated males belonging to the five major human subpopulation groups in Ghana (Akan, Ewe, Mole-Dagbon, Ga-Dangme and Guang) were genetically characterised for 23 Y chromosome short tandem repeat (STR) loci using the Powerplex® Y23 STR kit. A total of 263 complete haplotypes were recorded of which 258 were unique. The haplotype diversity, discriminating capacity and match probability for the pooled population data were 0.9998, 0.9627 and 0.0039, respectively. The pairwise genetic distance (RST) for the Ghanaian datasets and other reference populations deposited in the Y-STR Haplotype Reference Database (YHRD) were estimated and mapped using multidimensional scaling (MDS) plot. The Guang and Ewe were significantly different from the Akan, Mole-Dagbon and Ga-Dangme. However, the five Ghanaian datasets were all plotted close together with other African populations in the MDS data mapping.

Genetic portrait and phylogenetic analysis of an Aksu Uyghur population based on the 19 X-STR system.

The X chromosome has a special mode of inheritance, and is thus a rich resource for population studies. In this study, the allele frequencies and forensic statistics of the 19 X chromosomal short tandem repeat loci were evaluated in 500 Uyghur individuals from Aksu Prefecture in northwest China. We further aimed to study whether the Uyghur populations located in various regions of Xinjiang share similar allele and haplotype frequency distributions, as they have experienced genetic exchanges. Population comparisons, PCA and MDS were performed for the Uyghurs and 27 populations and the results indicate that the Uyghur minority in Aksu has a relatively close phylogenetic relationship with East Asians, especially the Kazakh minority.

The association of 22 Y chromosome short tandem repeat loci with initiative–aggressive behavior

Aggressive behavior represents an important public concern and a clinical challenge to behaviorists and psychiatrists. Aggression in humans is known to have an important genetic basis, so to investigate the association of Y chromosome short tandem repeat (Y-STR) loci with initiative–aggressive behavior, we compared allelic and haplotypic distributions of 22 Y-STRs in a group of Chinese males convicted of premeditated extremely violent crimes (n = 271) with a normal control group (n = 492). Allelic distributions of DYS533 and DYS437 loci differed significantly between the two groups (P < 0.05). The case group had higher frequencies of DYS533 allele 14, DYS437 allele 14, and haplotypes 11–14 of DYS533–DYS437 compared with the control group. Additionally, the DYS437 allele 15 frequency was significantly lower in cases than controls. No frequency differences were observed in the other 20 Y-STR loci between these two groups. Our results indicate a genetic role for Y-STR loci in the development of initiative aggression in non-psychiatric subjects.

Genetic analysis of 19 X chromosome STR loci for forensic purposes in four Chinese ethnic groups.

A new 19 X- short tandem repeat (STR) multiplex PCR system has recently been developed, though its applicability in forensic studies has not been thoroughly assessed. In this study, 932 unrelated individuals from four Chinese ethnic groups (Han, Tibet, Uighur and Hui) were successfully genotyped using this new multiplex PCR system. Our results showed significant linkage disequilibrium between markers DXS10103 and DXS10101 in all four ethnic groups; markers DXS10159 and DXS10162, DXS6809 and DXS6789, and HPRTB and DXS10101 in Tibetan populations; and markers DXS10074 and DXS10075 in Uighur populations. The combined powers of discrimination in males and females were calculated according to haplotype frequencies from allele distributions rather than haplotype counts in the relevant population and were high in four ethnic groups. The cumulative powers of discrimination of the tested X-STR loci were 1.000000000000000 and 0.999999999997940 in females and males, respectively. All 19 X-STR loci are highly polymorphic. The highest Reynolds genetic distances were observed for the Tibet-Uighur pairwise comparisons. This study represents an extensive report on X-STR marker variation in minor Chinese populations and a comprehensive analysis of the diversity of these 19 X STR markers in four Chinese ethnic groups.

Performance and concordance of the ForenSeq™ system for autosomal and Y chromosome short tandem repeat sequencing of reference-type specimens

Though the utility of next-generation sequencing (NGS) technologies for forensic short tandem repeat (STR) typing has been evident for several years, commercially available assays and software solutions developed specifically to meet forensic needs have only recently become available. One of these, the ForenSeq™ DNA Signature Prep Kit (Illumina, Inc.) sequences 27 autosomal STR (aSTR) and 24 Y chromosome STR (Y-STR) loci (concurrent with additional nuclear markers) per multiplexed sample, with automated secondary and tertiary analyses of the data accomplished via the associated ForenSeq™ Universal Analysis Software (UAS). In this study we investigated the performance of the ForenSeq system for aSTR and Y-STR typing by examination of 151 sample libraries developed from high quality DNAs amplified at the target 1ng template. Utilizing PCR Primer Mix B, greater than 99.5% of aSTR loci and 97.0% of Y-STR loci were recovered when 42 or fewer sample libraries were pooled for sequencing. A direct comparison of UAS developed fragment length results to capillary electrophoresis (CE) based data identified only two allele call discrepancies when no UAS quality flag was triggered. Review of the ForenSeq data indicated that most samples with total sequence read counts exceeding 40,000 could be interpreted to develop nearly complete aSTR genotypes or Y-STR haplotypes. However, markers D22S1045 and DYS392 produced poor or inconsistent results even when sample read counts were greater than 85,000. Excluding these two loci, analyst-interpreted aSTR and Y-STR ForenSeq profiles were 99.96% and 100% concordant, respectively, with CE data. In addition to demonstrating concordance on par with other CE kit to kit comparisons, the results from this study will assist laboratories seeking to develop workflows for high volume processing and analysis of aSTRs and Y-STRs from reference-type specimens using the ForenSeq system.

Developmental validation of the HomyGene19+14Y System.

The HomyGene19+14Y System (HG19+14Y) is a PCR-based amplification kit that enables typing of 18 autosomal short tandem repeat (STR) loci (i.e., CSF1PO, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, Penta E, TPOX, TH01, vWA), 14 widely used Y chromosome STR (Y-STR) loci (Y_GATA_H4, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS438, DYS439, DYS456, DYS458, DYS635), and amelogenin. This multiplex system was designed for the simultaneous analysis of amelogenin-Y allele mutation, single-source searches, kinship (including familial searching), mixture profiles, international data sharing, and other forensic applications. In this study, the multiplex system was validated for sensitivity, specificity, DNA mixtures, stability, precision, stutter, reproducibility, parallel tests, PCR-based conditions, and population analysis according to the Scientific Working Group on DNA Analysis Methods (SWGDAM) developmental validation guidelines. A total of 212 alleles were detected for the 18 autosomal STR loci among 528 Guangdong Han individuals, and 431 haplotypes were found for 14 Y-STRs among 452 unrelated males. The combined match probability (CMP) of the HG19+14Y System was calculated as 2.39×10-29. All the validation results showed that the HG19+14Y System would be a robust, reliable, highly polymorphic, and informative forensic kit.

Identification of human remains from the Second World War mass graves uncovered in Bosnia and Herzegovina.

AimTo present the results obtained in the identification of human remains from World War II found in two mass graves in Ljubuški, Bosnia and Herzegovina.MethodsSamples from 10 skeletal remains were collected. Teeth and femoral fragments were collected from 9 skeletons and only a femoral fragment from 1 skeleton. DNA was isolated from bone and teeth samples using an optimized phenol/chloroform DNA extraction procedure. All samples required a pre-extraction decalcification with EDTA and additional post-extraction DNA purification using filter columns. Additionally, DNA from 12 reference samples (buccal swabs from potential living relatives) was extracted using the Qiagen DNA extraction method. QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. PowerPlex ESI kit was used to simultaneously amplify 15 autosomal short tandem repeat (STR) loci, and PowerPlex Y23 was used to amplify 23 Y chromosomal STR loci. Matching probabilities were estimated using a standard statistical approach.ResultsA total of 10 samples were processed, 9 teeth and 1 femoral fragment. Nine of 10 samples were profiled using autosomal STR loci, which resulted in useful DNA profiles for 9 skeletal remains. A comparison of established victims' profiles against a reference sample database yielded 6 positive identifications.ConclusionDNA analysis may efficiently contribute to the identification of remains even seven decades after the end of the World War II. The significant percentage of positively identified remains (60%), even when the number of the examined possible living relatives was relatively small (only 12), proved the importance of cooperation with the members of the local community, who helped to identify the closest missing persons’ relatives and collect referent samples from them.

Genetic portrait of Tamil non-tribal and Irula tribal population using Y chromosome STR markers.

The 17 Y chromosomal short tandem repeat loci included in the AmpFlSTR® Yfiler™ PCR Amplification Kit were used to analyse the genetic diversity of 517 unrelated males representing the non-tribal and Irula tribal population of Tamil Nadu. A total of 392 unique haplotypes were identified among the 400 non-tribal samples whereas 111 were observed among the 117 Irula tribal samples. Rare alleles for the loci DYS458, DYS635 and YGATAH4.1 were also observed in both population. The haplotype diversity for the non-tribal and Irula tribal population were found to be 0.9999, and the gene diversity ranged from 0.2041 (DYS391) to 0.9612 (DYS385). Comparison of the test population with 26 national and global population using principal coordinate analysis (PCoA) and determination of the genetic distance matrix using phylogenetic molecular analysis indicate a clustering of the Tamil Nadu non-tribal and Irula tribal population away from other unrelated population and proximity towards some Indo-European (IE) and Asian population. Data are available in the Y chromosome haplotype reference database (YHRD) under accession number YA004055 for Tamil non-tribal and YA004056 for the Irula tribal group.

Genetic population study of 11 Y chromosome STR loci in Greece

Statistical properties of eleven Y chromosome Short Tandem Repeat (STR) markers were analyzed (DYS391, DYS389I, DYS439, DYS389II, DYS438, DYS437, DYS19, DYS392, DYS393, DYS390 and DYS385) in a Greek population sample. The 200 subjects where distributed across Greece, from various Peripheries. 182 distinct haplotypes were found. To validate our results gene diversity has been calculated for the whole population, as well as for each locus individually. Genetic distance has been estimated between this population and Albanian, Egyptian, Italian and Turkish populations. The results indicate that all Y loci are useful for forensic sciences.

Comparison of southern Chinese Han and Brazilian Caucasian mutation rates at autosomal short tandem repeat loci used in human forensic genetics

The short tandem repeat (STR) loci used in human genetic studies are characterized by having relatively high mutation rates. In particular, to ensure an appropriate evaluation of genetic evidence in parentage and forensic analyses, it is essential to have accurate estimates of the mutation rates associated with the commonly used autosomal and sex chromosome STR loci. Differences in STR mutation rates between different ethnic groups should also be determined. Mutation data from two laboratories working with different ethnic groups were extracted from many meiotic transmissions ascertained for 15 autosomal STR loci currently used in forensic routine. Forty-five thousand and eighty-five trios were checked for the biological consistency of maternity and paternity through the analysis of a minimum of 15 loci. Mutations were scored as paternal, maternal, or ambiguous according to the most parsimonious explanation for the inconsistency, using always the least requiring hypothesis in terms of number of repeat differences. The main findings are: (a) the overall mutation rate across the 15 loci was 9.78 × 10(-4) per gamete per generation (95% CI = 9.30 × 10(-4)-1.03 × 10(-3)), and with just 48 (out of 1,587) exceptions, all of the mutations were single-step; (b) repeat gains were more frequent than losses; (c) longer alleles were found to be more mutable; and (d) the mutation rates differ at some loci between the two ethnic groups. Large worldwide meiotic transmission datasets are still needed to measure allele-specific mutation rates at the STR loci consensually used in forensic genetics.

Genetic polymorphisms of six Y chromosome short tandem repeat loci in Chinese Korean ethnic group

To determine the allelic sequences and genetic polymorphism of six short tandem repeats (STRs) loci on Y chromosome, including DYS441, DYS442, DYS443, DYS444, DYS445 and DYS446 in Chinese Korean ethnic males from Yanbian region of Jilin province, China, and to construct a preliminary database. Allele frequencies of the six STR loci in 205 Chinese Korean ethnic individuals were analyzed by multiplex polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE). Respectively, 8, 7, 7, 5, 6 and 9 alleles were detected for each of the locus. Together they have formed 151 haplotypes, with a diversity of 0.9937. The six STR loci included in this study were found to be highly polymorphic, and may provide useful markers for genetic analysis.

Decreased Rate of Evolution in Y Chromosome STR Loci of Increased Size of the Repeat Unit

BackgroundPolymorphic Y chromosome short tandem repeats (STRs) have been widely used in population genetic and evolutionary studies. Compared to di-, tri-, and tetranucleotide repeats, STRs with longer repeat units occur more rarely and are far less commonly used.Principal FindingsIn order to study the evolutionary dynamics of STRs according to repeat unit size, we analysed variation at 24 Y chromosome repeat loci: 1 tri-, 14 tetra-, 7 penta-, and 2 hexanucleotide loci. According to our results, penta- and hexanucleotide repeats have approximately two times lower repeat variance and diversity than tri- and tetranucleotide repeats, indicating that their mutation rate is about half of that of tri- and tetranucleotide repeats. Thus, STR markers with longer repeat units are more robust in distinguishing Y chromosome haplogroups and, in some cases, phylogenetic splits within established haplogroups.ConclusionsOur findings suggest that Y chromosome STRs of increased repeat unit size have a lower rate of evolution, which has significant relevance in population genetic and evolutionary studies.

Y chromosome microsatellite genetic variation in two Native American populations from Argentina: Population stratification and mutation data

Two Native American populations from North and northwest regions of Argentina (Toba and Colla) were analyzed for 17 Y chromosome short tandem repeat loci (Y-STRs), namely, DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 and GATA H4.1. Over 357 allele transfers, two one-step mutations could be detected at DYS456 and GATA H4.1 loci. A new 16.1 ‘micro-variant’ allele was observed for DYS385, characterized by an insertion at the fifth GAAA repeat. We also observed two alleles at the DYS448 locus in three samples (two from Toba and one from Colla). A total of 34 and 16 different haplotypes were detected for Toba and Colla, respectively, the former with a haplotype diversity value of 0.9769 ± 0.01, whereas 0.9497 ± 0.02 for the latter. Significant population differences were observed between Colla and Toba, at least in part, due to a more prevalent European input in the Colla. In agreement with this observation is the fact that the genetic distances between Colla and Iberian populations are lower than those observed between Iberian and any other Native American population. The results of multiscaling dimensional analysis and genetic distances (Rst) among Native American population samples also reflect this fact. The data show the existence of clear population stratification in the Argentina, a fact that should be taken into account in forensic casework.

Direct STR Amplification from Whole Blood and Blood‐ or Saliva‐Spotted FTA® without DNA Purification*

The DNA purification step has been thought to be essential for typing of STR DNA. However, this process is time-consuming, and there is a risk of unexpected cross-contamination during purification. We report a new method for direct short tandem repeat (STR) amplification using a newly developed direct PCR buffer, AnyDirect, which can amplify STR loci from whole blood and blood- or saliva-spotted FTA cards without DNA purification. The autosomal and Y chromosomal STR loci were analyzed for whole blood and blood or saliva spots of random individuals, followed by comparison of the results with those of corresponding purified DNA. The results from whole blood and blood spots showed perfect concordance with those from purified DNA without allele or locus drop-out. However, in the case of saliva spots, no amplification or locus drop-out was observed in some of the samples, which offers a topic for further study. Additionally, some commercial hot-start DNA polymerases other than AmpliTaq Gold DNA polymerase were also found to be compatible with this buffer system. Therefore, this direct PCR buffer was demonstrated to be useful for fast forensic DNA analysis or criminal DNA databases for which there is no need to store DNA samples.

Microsatellite mutation in the maternally/paternally transmitted D18S51 locus: Two cases of allele mismatch in the child

Background We investigated 2 cases of paternity dispute with 17 autosomal short tandem repeats (STR), that indicated a mismatch to the maternally and paternally inherited allele at D18S51 locus in children under inquiry. Methods 17 autosomal and Y STR loci were analyzed using AmpF lSTR Identifiler, PowerPlex 16, AmpFℓSTR®Y-filer™ kits. The mitochondrial DNA hypervariable regions HV1 and HV2 and 6 STR markers on X chromosome were amplified and sequenced. Results In case M1, allelic representation in the mother, questioned child and suspected father was 14/19, 12/20 and 12/14 respectively. A complete match with the mother at 6 X STR loci and mitochondrial hypervariable regions was observed. In case F1, allelic representation was 13/14, 14/20 and 16/18 respectively. A complete match with the father at 17 Y chromosome STR loci was observed. D18S51 sequence analysis indicates the expansion of 1 repeat in M1 and 2 repeats in F1 leading to allele mismatch in the child. Conclusion The probability of maternity and paternity were 0.999999 and 0.999999 respectively. This is the first report of a maternally/paternally transmitted D18S51 mutations in the paternity DNA testing. These results conclusively determined that the mother and suspected father are the biological parents of the questioned children in both the cases.

Constituting a Y chromosome short tandem repeats loci database in Sicily

Many Y chromosome short tandem repeats (STRs) have been studied and characterized over the past years. A few Y-STRs multiplex kits have been placed on the market for forensic and population study purposes. The aim of our work was to analyse a sample of Sicilian male individuals to evaluate the allelic frequency and, thus, the possibility to implement a genetic database. An 11 loci Y-STR Typing kit was used to yield haplotype profiles from male DNA; amplification products were detected on ABI PRISM 310 Genetic Analyzer and examined by Genemapper v 3.2 (Applied Biosystems). The population sample subjected to the screening resulted to be very different with regards to certain loci whereas for other loci the allelic profile was less variable. Despite a minor discrimination power within the entire population, Y-STRs represent a valid tool to simplify male/female DNA mixture interpretation which is a major challenge when biological traces are found in case of sexual assault.