Population genetic analyses of the Powerplex® Fusion kit in a cosmopolitan sample of Chubut Province (Patagonia Argentina)

Allele frequencies and forensic parameters for 22 autosomal STR loci and DYS391 locus included in the PowerPlex^® Fusion System kit were estimated in a sample of 770 unrelated individuals from Chubut Province, southern Patagonia. No significant deviations from Hardy–Weinberg equilibrium were observed after Bonferroni’s correction. The combined power of discrimination and the combined probability of exclusion were >0.999999 and 0.999984, respectively. Comparisons with other worldwide populations were performed. The MDS obtained show a close biological relation between Chubut and Chile. The estimated interethnic admixture supports a high Native American contribution (46%) in the population sample of Chubut. These results enlarge the Argentine databases of autosomal STR and would provide a valuable contribution for identification tests and population genetic studies.     

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.     

NGMSElect™ and Investigator® Argus X-12 analysis in population samples from Albania,Iraq, Lithuania,Slovenia, and Turkey

The analysis of STRs is the main tool when studying genetic diversity in populations or when addressing individual identification in forensic casework. Population data are needed to establish reference databases that can be used in the forensic context. To that end, this work investigated five population samples from Albania, Iraq, Lithuania, Slovenia, and Turkey. Individuals were typed for 16 autosomal STRs and 12 X-chromosomal STRs using the NGMSElect™ and Investigator^® Argus X-12 kits, respectively. The aim of the study was to characterize the diversity of both STR kits in these population samples and to expand our forensic database.The results showed that all markers were polymorphic in the five populations studied. No haplotype was shared between the males analysed for X-STRs. No statistically significant deviations from Hardy–Weinberg equilibrium were observed for any of the genetic markers included in both the kits. Pairwise LD was only detected in X-STRs between markers located in the same linkage group. Power of discrimination values for males and females and the probability of exclusion in duos and trios were high for the populations in this study.     

Population data and phylogenetic structure of 19 STR loci in Chinese Hui ethnic group residing in Yunnan province of China

Allele frequency distributions and statistical forensic parameters of 19 autosomal STR loci in a sample of 535 unrelated healthy Hui individuals from Yunnan province were estimated. A total of 236 alleles at these loci were identified and their corresponding allele frequencies ranged from 0.000935 to 0.527103. Penta E is the most informative in Hui population, whereas TPOX showed the lowest. All of the STR loci reached the Hardy–Weinberg equilibrium after Bonferroni correction. The combined discrimination power and probability of excluding paternity of the 19 STR loci were 0.999 999 999 999 999 999 999 984 47 16 and 0.999 999 988, respectively. Furthermore, the genetic relationship between the Yunnan Hui population and other 8 different Hui groups or 25 previously investigated groups residing in other areas of China were also estimated based on pairwise genetic distance. These results suggest that the 19 STR loci are highly polymorphic, which is suitable for forensic personal identification and paternity testing.     

Evaluation of the Precision ID Identity Panel for the Ion Torrent™ PGM™ sequencer

In cases where only a partial or incomplete STR profile is obtained from a sample, information contained in single nucleotide polymorphisms (SNPs) can prove informative for human identification. Thermo Fisher Scientific, which developed the high throughput Ion Torrent^™ PGM^™ sequencer, released the Precision ID Identity Panel, a multiplex SNP panel for human identity. We evaluated the reproducibility and sensitivity of this multiplex, which contains primers for the amplification of 90 autosomal SNPs and 34 Y-clade SNPs. The manufacturer’s protocol was tested using five commercially available pure native DNAs and six forensic type samples at a range of DNA input amounts (0.2–1.0 ng; n, 90). In addition to analyzing the data using the manufacturer’s software, HID SNP Genotyper (v4.3.1), we also used CLC Genomics Workbench (Qiagen). Although library yields and templating of ion sphere particles (ISPs) were low, downstream sequencing was still successful. Across all samples, only 1.5% of all possible quality control (QC) flags were raised by both the plugin QC filter and CLC; 85% of those flags were raised as the SNP had a major allele frequency outside the thresholds specified by the manufacturer. For the remaining SNPs, coverage of >1500 X and >780 X was obtained for autosomal and Y-clade SNPs respectively, and 100% congruence among genotype calls from both analysis programs was observed. Our results demonstrate that it is possible to obtain reliable and reproducible genotypes using the Precision ID Identity Panel, when using low quantities (≥0.2 ng) of either pure native DNA or forensic type DNA samples.     

Molecular characterization and forensic relevance of the autosomal STRs for the population of North Indian geographical province Himachal Pradesh,India

Keeping in view the diverse demography of India, present study was undertaken to explore the molecular characterization and forensic relevance of 20 autosomal STRs for the highly diverse population of north Indian state Himachal Pradesh. 724 unrelated individuals from the admixed population of Himachal Pradesh were undertaken for present study and 20 autosomal STRs used to explore genomic diversity of studied population. A total of 270 different alleles along with 13.5 alleles per locus were observed. The allele 8 of the locus TPOX was observed as the most frequent allele. Observed heterozygosity ranged from 0.677 to 0.898, which supported wide range of selection of the unrelated individuals for this study. Combined power of discrimination, power of exclusion, matching probability and paternity index were observed as 1, 0.9999999958, 3.9 × 10⁻²⁶ and 2.3 × 10⁸ respectively, across the studied loci. In the population differentiation test, studied population showed genetic relatedness with Indian population rather than the populations of West, North and North east countries. Present study deciphered the novel autosomal STR data, which could be useful for the forensic application and population genetic studies.     

Current next generation sequencing technology may not meet forensic standards

In a Nature paper of 2010, the concern was raised that intra-individual mtDNA variation may be more pronounced than previously believed, in that heteroplasmies are common and vary markedly from tissue to tissue. This claim taken at face value would have considerable impact on forensic casework. It turns out however that the employed technology detected the germ-line variation relative to the reference sequence only incompletely: on average at least five mutations were missed per sample, as an in silico reassessment of the data reveals. Before one can really set out to access to entire mtDNA genome data with relative ease for forensic purposes, one needs careful calibration studies under strict forensic conditions-or might have to wait for another generation.     

The STR locus D11S554: allele frequencies and sequence data in a Japanese population

A population study on the short tandem repeat (STR) locus D11S554 was carried out in a sample of 362 unrelated Japanese individuals living in the Gifu Prefecture. A total of 46 different alleles ranging from 180 bp to 340 bp and 135 genotypes were revealed. Sequence analysis of alleles was carried out for 185 samples. The sequence structures of the repeat regions of the alleles were found to be complex and the alleles were classified into nine sequence types, including four new sequence types. According to the system of Adams et al. (1993), we designated the new sequence types IA³, IA⁴, IA⁵ and IB³, respectively. Out of the 46 different alleles, 11 showed sequence heterogeneity. The results of this study demonstrated that the D11S554 locus is a powerful and useful genetic marker for forensic practice in the Japanese population. Received: 6 December 2000 / Accepted: 31 May 2001     

Assessment of application value of 19 autosomal short tandem repeat loci of GoldenEyeTM 20A kit in forensic paternity testing

This study was carried out to assess the application value of 19 autosomal short tandem repeat (STR) loci of GoldenEye^TM 20A kit, in which 13 combined DNA index system core STR loci and PentaE, PentaD, D2S1338, D19S433, D12S391, and D6S1043 of six STR loci could be used in forensic paternity testing in Chinese population. We amplified the genomic DNA from blood samples on FTA paper of 289 paternity testing cases by using the GoldenEye^TM 20A kit. The amplified products were detected by capillary electrophoresis, and then the genotypes of 20 genetic markers including 19 STR loci as well as Amelogenin for sex determination were analyzed by GeneMapper v3.2 and GeneMarker HID Software. The results of genotypes were compared to the three commonly used commercial kits including AmpF?STR Identifiler^TM, PowerPlex^TM16, and AmpF?STR Sinofiler^TM kits. Compared to the three other common commercial kits, the GoldenEye^TM 20A kit had higher value of combined paternity index in certainty of paternity or non-exclusion paternity cases, and more numbers of STR loci were excluded in exclusionary paternity cases. Our data in this study showed that the GoldenEye^TM 20A kit has a higher application value in forensic paternity testing and will be of help for kinship analysis.     

Mutation rate estimates for 13 STR loci in a large population from Rio Grande do Sul, Southern Brazil

Short tandem repeat (STR) polymorphisms have been extensively used in forensic genetics analysis. Knowledge about the locus-specific mutation rates of STRs improves forensic probability calculations and interpretations of diversity data. To incorporate single-locus diversity information into autosomal STR mutation rate estimations, 13 STR loci were studied during 2007–2009 in 10,959 paternity investigation cases from Rio Grande do Sul, the southernmost state of Brazil, covering an overall number of 284,934 allelic transfers. A total of 355 mutations were identified; 348 repeats were gains or losses of one step, three were gains or losses of two steps, and four were gains or losses of not stepwise mutation. The mutation rates ranged from 4.6?×?10^?5 to 2.3?×?10^?3, and the overall mutation rate estimate was 1.2?×?10^?3. The average of the paternal mutation rate (1.8?×?10^?3) was five times higher than the maternal rate (0.36?×?10^?3). The observed mutational features for STRs have important consequences for forensic applications, including the definition of criteria for exclusion in paternity testing and the interpretation of DNA profiles in identification analysis.     

D20S161 data for three ethnic populations and forensic validation

In order to evaluate the forensic applicability of the STR locus D20S161 and construct a preliminary database, the genotype distributions and allele frequencies in five populations from three main ethnic groups were investigated, including Germans, Slovakians, African Americans, Japanese and Chinese. A total of 512 samples from unrelated individuals and 85 confirmed father/mother/¶child triplets were analyzed by PCR and allele determination was carried out by comparison with a sequenced human allelic ladder. The results showed that D20S161 typing was both precise and reliable. A total of 7 alleles was found in these populations and no evidence of deviation from Hardy-Weinberg equilibrium was observed. Pairwise comparisons between populations showed that there were significant differences in the distributions of the allele frequencies among the three main ethnic groups. No mutation events were observed from the confirmed father/mother/¶child triplets. With a maximum likelihood method, the mutation rate was indirectly estimated as 2.5 × 10^–5. These results suggest that D20S161 is a useful marker for forensic casework and paternity analysis.     

Genetic analysis of 20 autosomal STR loci in the Miao ethnic group from Yunnan Province,Southwest China

The genetic polymorphisms of 20 autosomal short tandem repeat (STR) loci included in the PowerPlex^® 21 kit were evaluated from 748 unrelated healthy individuals of the Miao ethnic minority living in the Yunnan province in southwestern China. All of the loci reached Hardy–Weinberg equilibrium. These loci were examined to determine allele frequencies and forensic statistical parameters. The genetic relationship between the Miao population and other Chinese populations were also estimated. The combined discrimination power and probability of excluding paternity of the 20 STR loci were 0.999 999 999 999 999 999 999 991 26 and 0.999 999 975, respectively. The results suggested that the 20 STR loci were highly polymorphic, which makes them suitable for forensic personal identification and paternity testing.     

Evaluation of genetic parameters of 22 autosomal STR loci (PowerPlex® Fusion System) in a population sample from Northern Italy

The PowerPlex® Fusion System (Promega, Madison, WI) is a short tandem repeat (STR) multiplex that allows co-amplification of 22 autosomal STRs, including the CODIS core and the European Standard Set loci, plus amelogenin for gender determination and DYS391 male specific marker included in order to avoid errors in gender assignment when null Y-alleles or deletions of the Y-chromosome short arm involve the amelogenin locus. Allele frequencies and forensic efficiency parameters were estimated in a population sample of 303 unrelated healthy individuals living in Northern Italy. No significant deviations from Hardy–Weinberg expectations were observed after applying Bonferroni’s correction for multiple testing. The combined power of discrimination was 0.999999999999 and the combined power of exclusion was 0.9999956. A rare 28 allele at locus D12S391 was observed, while one tri-allelic pattern at Penta E locus was detected. Population differentiation test revealed significant genetic diversity between our population sample and other European populations considered. The results showed that the PowerPlex® Fusion System is one of the most informative kit available in forensic genetics and may prove useful in both human identification and kinship analysis.     

Genetic polymorphisms of twelve X-STRs of the investigator Argus X-12 kit and additional six X-STR centromere region loci in an Egyptian population sample

Recently, many researchers have focused on analysis of different X-chromosomal STRs as they bear the potential to efficiently complement the analysis of autosomal and Y-chromosomal STRs in solving special complex kinship deficiency cases. In the current study we examined a sample of 250 unrelated Egyptian males with the Investigator Argus X-12 kit (Qiagen GmbH, Hilden, Germany) which detects 12 X-STR markers distributed over the entire X-chromosome as four closely linked clusters. Microvariant off ladder alleles as well as null alleles have been detected in some loci. Furthermore, discordant results were observed between the Investigator Argus X-12 and the Mentype^® Argus X-8 kits (Biotype AG, Dresden, Germany). New primers were designed for loci DXS10101, DXS10146 and DXS10148 to correct the allele drop outs observed in these loci with the Investigator Argus X-12 kit. Additionally, DNA sequence analysis revealed the polymorphisms responsible for the allele drop outs. Furthermore, six additional X-STRs (DXS10161, DXS10159, DXS10162, DXS10163, DXS10164 and DXS10165) located in the centromere region at Xp11.21–Xq11.1 were examined in a single multiplex reaction. Allele and haplotype frequencies as well as different forensic statistical parameters of the 18 X-STR loci tested indicated that they are highly informative in different forensic applications in the Egyptian population. However, some modifications still need to be performed on the Investigator Argus X-12 kit before its use in forensic casework is validated.     

Allele frequency data for 17 short tandem repeats in a Czech population sample

Allele frequencies for 17 short tandem repeats (STRs) autosomal loci (D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, CSF1PO, FGA, PentaD, PentaE, TH01, TPOX, vWA) were studied in an extensive sample (max. N = 1411) of unrelated individuals originating from the Czech Republic. Population and forensic parameters were estimated. Except for FGA and Penta E loci, no deviations from the Hardy–Weinberg equilibrium were detected. A comparative analysis with published data revealed significant differences in allele frequencies for some loci from the Polish population and three Hungarian populations (Ashkenazim population and Romany populations from Debrecen and Baranya County, respectively). A combination of these 17 STR loci provides a powerful tool for forensic identification in the native Czech population.     

Population data of 24 STRs in Mexican-Mestizo population from Monterrey,Nuevo Leon (Northeast,Mexico) based on Powerplex® Fusion and GlobalFiler® kits

The STR loci included into new commercial human identification kits compels geneticists estimating forensic parameters for interpretation purposes in forensic casework. Therefore, we studied for the first time in Mexico the GlobalFiler^® and Powerplex^® Fusion systems in 326 and 682 unrelated individuals, respectively. These individuals are resident of the Monterrey City of the Nuevo Leon state (Northeast, Mexico). Population data from 23 autosomal STRs and the Y-STR locus DYS391 are reported and compared against available STR data from American ethnic groups and the unique Mexican population studied with Powerplex^® Fusion.     

Evaluation of the Genplex SNP typing system and a 49plex forensic marker panel

Using a 52 SNP marker set previously developed for forensic analysis, a novel 49plex assay has been developed based on the Genplex typing system, a modification of SNPlex™ chemistry (both Applied Biosystems) using oligo-ligation of pre-amplified DNA and dye-labeled, mobility modified detection probes. This gives highly predictable electrophoretic mobility of the allelic products generated from the assay to allow detection with standard capillary electrophoresis analyzers. The loci chosen comprise the 48 most informative autosomal SNPs from the SNPforID core discrimination set supplemented with the amelogenin gender marker. These SNPs are evenly distributed across all 22 autosomes, exhibit balanced polymorphisms in three major population groups and have been previously shown to be effective markers for forensic analysis. We tested the accuracy and reproducibility of the Genplex system in three SNPforID laboratories, each using a different Applied Biosystems Genetic Analyzer. Genotyping concordance was measured using replicates of 44 standardized DNA controls and by comparing genotypes for the same samples generated by the TaqMan^®, SNaPshot^® and Sequenom iPLEX^® SNP typing systems. The degree of informativeness of the 48 SNPs for forensic analysis was measured using previously estimated allele frequencies to derive the cumulative match probability and in paternity analysis using 24 trios previously typed with 18 STRs together with three CEPH families with extensive sibships typed with the 15 STRs in the Identifiler^® kit.     

Analysis of uni and bi-parental markers in mixture samples: Lessons from the 22nd GHEP-ISFG Intercomparison Exercise

Since 1992, the Spanish and Portuguese-Speaking Working Group of the ISFG (GHEP-ISFG) has been organizing annual Intercomparison Exercises (IEs) coordinated by the Quality Service at the National Institute of Toxicology and Forensic Sciences (INTCF) from Madrid, aiming to provide proficiency tests for forensic DNA laboratories. Each annual exercise comprises a Basic (recently accredited under ISO/IEC 17043: 2010) and an Advanced Level, both including a kinship and a forensic module. Here, we show the results for both autosomal and sex-chromosomal STRs, and for mitochondrial DNA (mtDNA) in two samples included in the forensic modules, namely a mixture 2:1 (v/v) saliva/blood (M4) and a mixture 4:1 (v/v) saliva/semen (M8) out of the five items provided in the 2014 GHEP-ISFG IE. Discrepancies, other than typos or nomenclature errors (over the total allele calls), represented 6.5% (M4) and 4.7% (M8) for autosomal STRs, 15.4% (M4) and 7.8% (M8) for X-STRs, and 1.2% (M4) and 0.0% (M8) for Y-STRs. Drop-out and drop-in alleles were the main cause of errors, with laboratories using different criteria regarding inclusion of minor peaks and stutter bands. Commonly used commercial kits yielded different results for a micro-variant detected at locus D12S391. In addition, the analysis of electropherograms revealed that the proportions of the contributors detected in the mixtures varied among the participants. In regards to mtDNA analysis, besides important discrepancies in reporting heteroplasmies, there was no agreement for the results of sample M4. Thus, while some laboratories documented a single control region haplotype, a few reported unexpected profiles (suggesting contamination problems). For M8, most laboratories detected only the haplotype corresponding to the saliva. Although the GHEP-ISFG has already a large experience in IEs, the present multi-centric study revealed challenges that still exist related to DNA mixtures interpretation. Overall, the results emphasize the need for further research and training actions in order to improve the analysis of mixtures among the forensic practitioners.     

Genetic variation of 15 autosomal short tandem repeat (STR) loci in the Palestinian population of Gaza Strip

Fifteen autosomal STR loci included in the PowerPlex^®16 System were typed in a population sample of 125 unrelated individuals from Palestinian population of Gaza Strip. Allele frequencies, Hardy–Weinberg equilibrium and forensic parameters were determined for the following loci: Penta E, D18S51, D21S11, TH01, D3S1358, FGA, TPOX, D8S1179, vWA, Penta D, CSF1PO, D16S539, D7S820, D13S317 and D5S818.     

Population database on 15 autosomal STR loci in 1000 unrelated individuals from the Lodz region of Poland

Allele frequency data and forensic efficiency parameters for 15 STR loci: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA, were estimated from the sample of 1000 unrelated individuals from the Lodz region of Poland. The combined MP and PE for all 15 loci are 4.8 × 10⁻¹⁸ and 0.9999989, respectively. The comparison of our data with other Polish populations revealed statistically significant differences in 6 out of 15 loci between Lodz and the Podlasie region of Poland.