Genetic profile characterization and population study of 21 autosomal STR in Chinese Kazak ethnic minority group

Short tandem repeat loci have been recognized as useful tools in the routine forensic application and in recent decades, more and more new short tandem repeat (STR) loci have been constantly discovered, studied, and applied in forensic caseworks. In this study, we investigated the genetic polymorphisms of 21 STR loci in the Kazak ethnic minority as well as the genetic relationships between the Kazak ethnic minority and other populations. Allelic frequencies of 21 STR loci were obtained from 114 unrelated healthy Kazak individuals in the Ili Kazak Autonomous Prefecture, Xinjiang Uigur Autonomous Region of China. We observed a total of 159 alleles in the group with the allelic diversity values ranging from 0.0044 to 0.5088. The highest polymorphism was found at D19S433 locus and the lowest was found at D1S1627. Statistical analysis of the generated data indicated no deviation from Hardy–Weinberg equilibriums at all 21 STR loci. In order to estimate the population differentiation, allelic frequencies of all STR loci of the Kazak were compared with those of other neighboring populations using analysis of molecular variance method. Statistically significant differences were found between the studied population and other populations at 2–7 STR loci. A neighbor‐joining tree was constructed based on allelic frequencies of the 21 STR loci and phylogenetic analysis indicates that the Kazak has a close genetic relationship with the Uigur ethnic group. The present results may provide useful information for forensic sciences and population genetics studies, and can also increase our understanding of the genetic background of this group. The present findings showed that all the 21 STR loci are highly genetically polymorphic in the Kazak group, which provided valuable population genetic data for the genetic information study, forensic human individual identification, and paternity tests.     

Structural polymorphism analysis of Chinese Mongolian ethnic group revealed by a new STR panel: Genetic relationship to other groups

Mongolian is the eighth largest ethnic minority on Chinese population data according to the 2010 census. In the present study, we presented the first report about the allelic frequencies and forensic statistical parameters at the 21 new STRs and analyzed linkage disequilibrium of pairwise loci in the Mongolian ethnic minority, China. Hardy–Weinberg equilibrium tests demonstrated no significant deviations except for the D1S1627 locus. The cumulative power of discrimination and power of exclusion of all the loci are 0.9999999999999999992576 and 0.9999997528, respectively. The results of analysis of molecular variance showed that significant differences between the Mongolian and the other eight populations were found at 1‐9 STR loci. In population genetics, the results of principal component analysis, structure analysis, and phylogenetic reconstruction analysis indicated shorter genetic distance between the Mongolian group and the Ningxia Han. All the results suggest that the 21 new STR loci will contribute to Chinese population genetics and forensic caseworks in the Mongolian group.     

Developmental validation of the Microreader™ 20A ID system

The Microreader? 20A ID system is designed for forensic applications such as personal identification, parentage testing, and research. It includes 13 combined DNA index system (CODIS) short tandem repeat (STR) loci (CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11), three expanded CODIS STR loci (D12S391, D19S433, and D2S1338), three non‐CODIS STR loci (D6S1043, Penta D, and Penta E), and the amelogenin locus in one reaction with a six‐dye fluorescent (FAM, HEX, TAMAR, ROX, PUR, and QD550) analysis system. In this study, the Microreader? 20A ID system was validated according to the Scientific Working Group on DNA Analysis Methods validation guidelines for forensic DNA Analysis methods and Chinese national standard, including PCR‐based studies, sensitivity study, precision, and accuracy evaluation, stutter calculation, inhibitor tests, species specificity, and DNA mixture studies. Our results suggest that the Microreader? 20A ID system is a useful tool for personal identification and parentage testing.     

Development and validation of a forensic six-dye multiplex assay with 29 STR loci

This paper describes the development and validation of a novel 31-locus, six-dye STR multiplex system, which is designed to meet the needs of the rapidly growing Chinese forensic database. This new assay combines 20 extended-CODIS core loci (D3S1358, D5S818, TPOX, CSF1PO, TH01, vWA, D7S820, D21S11, D8S1179, D18S51, D16S539, D13S317, FGA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045), nine highly polymorphic loci in Chinese Han population (D3S3045, D6S1043, D6S477, D8S1132, D10S1435, D15S659, D19S253, Penta D, and Penta E), and two gender determining markers, amelogenin and Y-Indel, which could amplify DNA from extracts, as well as direct amplification from substrates. To demonstrate the suitability for forensic applications, this system was validated by precision and accuracy evaluation, concordance tests, case sample tests, sensitivity, species specificity, stability, stutter calculation, and DNA mixtures, according to the guidelines described by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and regulations published by the China Ministry of Public Security. The validation results indicate the robustness and reliability of this new system, and it could be a potentially helpful tool for human identification and paternity testing in the Chinese population, as well as facilitating global forensic DNA data sharing.     

Allele frequency of 19 autosomal STR loci in the Bai population from the southwestern region of mainland China

The aim of this study was to investigate a 19 STR loci database using the Bai population from China. This multiplex amplification kit included 13 CODIS STR markers and six plus STR markers (D19S433, Penta E, D2S1338, Penta D, D6S1043, and D12S391) that were successfully analyzed by using 1158 DNA samples from the Bai population from the southwestern part of mainland China. These results indicate that this multiplex amplification kit may provide significant polymorphic information for kinship testing and relationship investigations.     

Genetic polymorphisms in 12 autosomal STRs in a Shanghai Han population from China

In this study, a total of 484 unrelated healthy individuals and 359 two‐generation families in the Han population in Shanghai, China were successfully analyzed with the Investigator HDplex Kit (Qiagen, Hilden, Germany). Hardy–Weinberg equilibrium tests demonstrated no significant deviation from expected values (p > 0.05) for the 12 autosomal STRs included in this kit. A total of 173 alleles were detected in 12 autosomal STR loci; the largest number of alleles (32) was detected for the SE33 locus. The combined power of discrimination was 0.99999999992. The combined mean exclusion chance in duo cases was 0.99982, whereas the combined mean exclusion chance in trio cases was 0.9999986. The average mutation rate across all loci was 0.0023 (95% confidence intervals: 0.0013–0.0039). These results suggest that the 12 autosomal STR loci can provide highly informative polymorphic data for paternity testing and forensic identification in the Han population in Shanghai, China.     

Development of a new 25plex STRs typing system for forensic application

We have developed a novel STR 25‐plex florescence multiplex‐STR kit (DNATyper25) to genotype 23 autosomal and two sex‐linked loci for forensic applications and paternity analysis. Of the 23 autosomal loci, 20 are non‐CODIS. The sex‐linked markers include a Y‐STR locus (DYS391) and the Amelogenin gene. We present developmental validation studies to show that the DNATyper25 kit is reproducible, accurate, sensitive, and robust. Sensitivity testing showed that full profiles were achieved with as low as 125 pg of human DNA. Specificity testing demonstrated a lack of cross reactivity with a variety of commonly encountered non‐human DNA contaminants. Stability testing showed that full profiles were obtained with humic acid concentration ≤60 ng/μL and hematin concentration <400 μM. For forensic evaluation, the 23 autosomal STRs followed the Hardy–Weinberg equilibrium. In an analysis of 509 Chinese (CN) Hans, we detected a combined total of 181 alleles at the 23 autosomal STR loci. Since these autosomal STRs are independent from one another, PM was 8.4528 × 10^?22, TDP was 0.999 999 999 999 999 999 999, CEP was 0.999 999 8395. The forensic efficiency parameters demonstrated that these autosomal STRs are highly polymorphic and informative in the Han population of China. We performed population comparisons and showed that the Northern CN Han has a close genetic relationship with the Luzhou Han, Tujia, and Bai populations. We propose that the DNATyper25 kit will be useful for cases where paternity analysis is difficult and for situations where DNA samples are limited in quantity and low in quality.     

Validation of the Microreader 28A ID System: A 6-dye multiplex amplification assay for forensic application

The Microreader 28A ID System is a new 28-plex genotyping system with 6-dye multiplex amplification, which allows the simultaneous amplification of all 20 Combined DNA Index System (CODIS) core loci (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, vWA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, D22S1045), plus five extended STRs loci (D6S1043, Penta D, Penta E, DYS391, SE33), 2 Y-Indels (Rs2032678, Rs771783753), and the amelogenin loci. This system can be used for forensic analyses, such as personal identification, kinship testing, scientific research, database applications, and other aspects of human genetic identification. The validation of the Microreader 28A ID System followed the “Validation Guidelines for DNA Analysis Methods (2016)” described by the Scientific Working Group on DNA Analysis Methods and the regulations published by the China Ministry of Public Security. Our tests included PCR-based studies, sensitivity study, precision and accuracy evaluation, stutter percentage and heterozygous peak height ratio, inhibitor tests, species specificity, and population studies. The validation results suggest that the Microreader 28A ID system is a robust and reliable amplification kit for personal identification, kinship testing, and forensic database applications.     

Allele and haplotype diversity of new multiplex of 19 ChrX‐STR loci in Han population from Guanzhong region (China)

X‐chromosomal short tandem repeats (X‐STRs) have been proved to be useful for some deficiency paternity cases in recent years. Here, we studied the genetic polymorphisms of 19 X‐STR loci (DXS10148‐DXS10135‐DXS8378, DXS10159‐DXS10162‐DXS10164, DXS7132‐DXS10079‐DXS10074‐DXS10075, DXS6809‐DXS6789, DXS7424‐DXS101, DXS10103‐HPRTB‐DXS10101 and DXS7423‐DXS10134) in 252 male and 222 female individuals from Guanzhong Han population, China. No deviation for all 19 loci was observed from the Hardy–Weinberg equilibrium. The polymorphism information content values of the panel of 19 loci were more than 0.5 with the exception of the locus DXS7423. The combined power of discrimination were 0.9999999999999999999994340 in females and 0.9999999999997662 in males, respectively; and the combined mean exclusion chances were 0.999999993764 in duos and 0.999999999997444 in trios, respectively. The haplotype diversities for all the seven clusters of linked loci were more than 0.9. The results showed that the panel of 19 X‐STR loci were powerful for forensic applications in Guanzhong Han population. Locus by locus population comparisons showed significant differences at more than seven loci between Guanzhong Han population and the groups from North America, Europe and Africa.     

"Paterniplex", a highly discriminative decaplex STR multiplex tailored for investigating special problems in paternity testing

The goal of the study was to develop a STR multiplex ("Paterniplex") that is--as supplement to commercially available multiplex kits like the Identifiler kit (Applied Biosystems, Foster City, CA)--suitable for solving complex paternity cases such as deficiency cases or cases with mutations. The Paterniplex comprises the nine highly polymorphic STRs D8S1132, D7S1517, D10S2325, D12S391, Se33, D17S976, Penta E, Penta D and FGA in addition to Amelogenin as sex determination marker. The loci were selected because of their high degree of polymorphism (higher than that of the widely used TH01 marker). Only one locus, FGA, is shared with the Identifiler kit to avoid sample mix up. The study further gives details on the population genetics of the loci in a German Caucasian population (allelic distribution, Hardy-Weinberg Equilibrium and forensic efficiency markers such as the Discriminating Power) and three examples for cases that could not be solved using commercially available kits alone, but using the Paterniplex in addition to a commercial kit.     

Development and validation of a novel SiFaSTRTM 23‐plex system

Human identification and paternity testing are usually based on the study of STRs depending on their particular characteristics in the forensic investigation. In this paper, we developed a sensitive genotyping system, SiFaSTR^? 23‐plex, which is able to characterize 18 expanded Combined DNA Index System STRs (D3S1358, D5S818, D2S1338, TPOX, CSF1PO, TH01, vWA, D7S820, D21S11, D10S1248, D8S1179, D1S1656, D18S51, D12S391, D19S433, D16S539, D13S317, and FGA), three highly polymorphic STRs among Chinese people (Penta D, Penta E, and D6S1043), one Y‐chromosome Indel and amelogenin using a multiplex PCR; the PCR amplified products were analyzed using the Applied Biosystems 3500 Genetic Analyzer. Full genotyping profiles were obtained using only 31.25 pg of control DNA; various case‐type specimens, as well as ten‐year‐old samples were also successfully genotyped. Additionally, in the validation studies, this multiplex was demonstrated to be human‐specific and compatible with the interference of multiple PCR inhibitors. The system also enabled the detection of mixtures, and complete profiles could be obtained at the mixed ratios of 1:1, 1:3, and 3:1. The development and validation study here illustrated that the SiFaSTR^? 23‐plex system is accurate, powerful, and more sensitive than many other systems. What's more, the population data in our study not only illustrated that this 23‐plex typing system was straightforward and efficient but also expanded the Chinese STR database, which could facilitate the appropriate application of the 23 genetic markers in forensic genetics, especially in the Chinese population.     

Development of the 16 X‐STR loci typing system and genetic analysis in a Shanghai Han population from China

This study developed a new multiplex PCR system that simultaneously amplifies 16 X‐STR loci in the same PCR reaction, and the polymorphism and mutation rates of these 16 X‐STR loci were explored in a Shanghai Han population from China. These loci included DXS10134, DXS10159, DXS6789, DXS6795, DXS6800, DXS6803, DXS6807, DXS6810, DXS7132, DXS7424, DXS8378, DXS9902, GATA165B12, GATA172D05, GATA31E08, and HPRTB. Samples from 591 unrelated individuals (293 males and 298 females) and 400 two‐generation families were successfully analyzed using this multiplex system. Allele frequencies and mutation rates of the 16 loci were investigated, with the comparison of allele frequency distributions among different populations performed. Polymorphism information contents of these loci were all >0.6440 except the locus DXS6800 (0.4706). Nine cases of mutations were detected in the 16 loci from the investigation of 9232 meioses. Pairwise comparisons of allele frequency distributions showed significant differences for most loci among populations from different countries and ethnic groups but not among the Han population living in other areas of China. These results suggest that the 16 X‐STR loci system provides highly informative polymorphic data for paternity testing and forensic identification in the Han population in Shanghai, China, as a complementary tool.     

Construction of two fluorescence‐labeled non‐combined DNA index system miniSTR multiplex systems to analyze degraded DNA samples in the Chinese Han Population

MiniSTR loci have been demonstrated to be an effective approach in recovering genetic information from degraded specimens, because of the reduced PCR amplicon sizes which improved the PCR efficiency. Eight non‐combined DNA index system miniSTR loci suitable for the Chinese Han Population were analyzed in 300 unrelated Chinese Han individuals using two novel five fluorescence‐labeled miniSTR multiplex systems(multiplex I: D10S1248, D2S441, D1S1677 and D9S2157; multiplex II: D9S1122, D10S1435, D12ATA63, D2S1776 and Amelogenin). The allele frequency distribution and forensic parameters in the Chinese Han Population were reported in this article. The Exact Test demonstrated that all loci surveyed here were found to be no deviation from Hardy–Weinberg equilibrium. The accumulated power of discrimination and power of exclusion for the eight loci were 0.999999992 and 0.98, respectively. The highly degraded DNA from artificially degraded samples and the degraded forensic case work samples was assessed with the two miniSTR multiplex systems, and the results showed that the systems were quite effective.     

Massively parallel sequencing of STRs using a 29-plex panel reveals stutter sequence characteristics

Massively parallel sequencing of forensic STRs simultaneously provides length-based genotypes and core repeat sequences as well as flanking sequence variations. Here, we report primer sequences and concentrations of a next-generation sequencing (NGS)-based in-house panel covering 28 autosomal STR loci (CSF1PO, D1GATA113, D1S1627, D1S1656, D1S1677, D2S441, D2S1776, D3S3053, D5S818, D6S474, D6S1017, D6S1043, D8S1179, D9S2157, D10S1435, D11S4463, D13S317, D14S1434, D16S539, D18S51, D18S853, D20S482, D20S1082, D22S1045, FGA, TH01, TPOX, and vWA) and the sex determinant locus Amelogenin. Preliminary evaluation experiments showed that the panel yielded intralocus- and interlocus-balanced sequencing data with a sensitivity as low as 62.5 pg input DNA. A total of 203 individuals from Yunnan Bai population were sequenced with this panel. Comparative forensic genetic analyses showed that sequence-based matching probability of this 29-plex panel reached 2.37 × 10⁻²⁹, which was 23 times lower than the length-based data. Compound stutter sequences of eight STRs were compared with parental alleles. For seven loci, repeat motif insertions or deletions occurred in the longest uninterrupted repeat sequences (LUS). However, LUS and non-LUS stutters co-existed in the locus D6S474 with different sequencing depth ratios. These results supplemented our current knowledge of forensic STR stutters, and provided a sound basis for DNA mixture deconvolution.     

A powerful,novel, multiplex typing system for six short tandem repeat loci and the allele frequency distributions in two Japanese regional populations

A new multiplex system for six tetranucleotide short tandem repeat (STR) loci was devised based on multicolor dye technology. Six loci (D20S480, D6S2439, D6S1056, D9S1118, D4S2639, and D17S1290), each with high discriminating power (each unbiased estimates of expected heterozygosity, Exp. Hz, > 0.80 in a preliminary test), were selected from more than 100 tetranucleotide STRs included in a commercially available primer set. These loci were also selected so as not to link with general STRs in commercially released kits including the combined DNA index system (CODIS) 13 core STRs. The primers were newly designed in the present study, in which each amplicon size had a range of less than 200 base pairs (bp), in order to genotype from highly degraded template DNA. Using this system, we genotyped 270 Honshu (mainland)-Japanese and 187 Okinawa-Japanese. From these allele frequencies, we performed three tests, a homozygosity test, a likelihood ratio test and an exact test for Hardy-Weinberg equilibrium (HWE), and no significant deviations (p < 0.05) from HWE were observed. We also compared the allele distributions at six STRs between both populations, and they were significantly different (p < 0.05) at three loci (D6S2439, D9S1118 and D4S2639). Furthermore, the Exp. Hz and the power of discrimination (PD) at all loci in the Honshu-Japanese population were higher than 0.80 and 0.93, respectively. These statistical values for discriminating power in the Honshu-Japanese were almost the same as in the Okinawa-Japanese. This novel, multiplex polymerase chain reaction (PCR) amplification and typing system for six STR loci thus promises to be a convenient and informative new DNA profiling system in the forensic field.     

Diversity study of 12 X‐chromosomal STR loci in Hui ethnic from China

X‐chromosomal STRs (X‐STRs) have been used as complements of autosomal STR application in recent years. In this work, we present population genetic data of 12 X‐STRs including DXS101, DXS10159, DXS10162, DXS10164, DXS6789, DXS7133, DXS7423, DXS7424, DXS8378, DXS981, GATA165B12, and GATA31E08 loci in a sample of 231 unrelated healthy individuals from the Hui ethnic group in Ningxia Hui Autonomous Region, China. Allelic frequencies of the 12 X‐STR loci and haplotypic frequencies of the reported linkage groups (DXS7424‐DXS101 and DXS10159‐DXS10164‐DXS10162) were investigated in the group, respectively. No STR loci showed significant deviations from the Hardy–Weinberg equilibriums and no linkage disequilibriums of pairwise loci were found after Bonferroni correction, respectively. A combined power of discrimination in female individuals was 0.999999999985 and that in male individuals was 0.99999967, respectively. The combined mean exclusion chance in deficiency cases, normal trios and duo cases were 0.999934, 0.995754, and 0.999796, respectively. Significant differences were observed from 0 to 8 loci, when making comparisons between the data of Hui ethnic group and previously reported data from other 16 populations. The results indicated the new panel of 12 X‐STR loci might be useful for forensic science application.     

24 Y‐chromosomal STR haplotypic polymorphisms for Chinese Uygur ethnic group and its phylogenic analysis with other Chinese groups

The Uygur ethnic minority is the largest ethnic group in the Xinjiang Uygur Autonomous Region of China, and is a precious resource for the study of ethnogeny and forensic biology. Previous studies have focused on the genetic background of the Uygur group, however, the patrilineal descent of the group is still unclear. In this study, we investigated the genetic diversity of 24 Y‐STR loci in the Uygur group and analyzed the population differentiations as well as the genetic relationships between the Uygur group and other previously reported populations using 17 Y‐filer loci. According to haplotypic analysis of the 24 Y‐STR loci in 109 Uygur individuals, 104 different haplotypes were obtained, 99 of which were unique. The haplotypic diversity and discrimination capacity of these 24 Y‐STR loci in Uygur group were 0.9992 and 0.9541, respectively. An additional 7 loci (DYS388, DYS444, DYS447, DYS449, DYS522, and DYS527a,b) showed high genetic diversity and improved the overall discrimination capacity of the 24 Y‐STR system. Pairwise Fst and neighbor‐joining analysis showed that the Uygur group was genetically close to the Han populations from different regions.     

Genetic diversity and haplotype structure of 24 Y‐chromosomal STR in Chinese Hui ethnic group and its genetic relationships with other populations

In the present study, 24 Y‐chromosomal short tandem repeat (Y‐STR) loci were analyzed in 115 unrelated Hui male individuals from Haiyuan county or Tongxin county, Ningxia Hui Autonomous Region, China, to evaluate the forensic application of the 24 STR loci and to analyze interpopulation differentiations by making comparisons between the Hui group data and previously published data of other 13 populations. A total of 115 different haplotypes were observed on these 24 Y‐STR loci. The gene diversities ranged from 0.4049 (DYS437) to 0.9729 (DYS385a, b). The overall haplotype diversity was 1 at AGCU 24 Y‐STR loci level, while the values were reduced to 0.999237, 0.996949, and 0.996644 at the Y‐filer 17 loci, 11 Y‐STR loci of extended haplotype and 9 Y‐STR loci of minimal haplotype levels, respectively; whereas, haplotype diversity for additional 7 loci (not included in Y‐filer 17 loci) was 0.995271. The pairwise F_ST, multidimensional scaling plot and neighbor‐joining tree indicated the Hui group had the closest genetic relationship with Sala in the paternal lineage in the present study. In summary, the results in our study indicated the 24 Y‐STRs had a high level of polymorphism in Hui group and hence could be a powerful tool for forensic application and population genetic study.     

Population genetics and forensic efficiency of twenty‐one novel microsatellite loci of Chinese Yi ethnic group

In this study, we investigated polymorphic distributions of allelic frequencies and forensic genetic parameters of 21 novel autosomal microsatellite loci from 110 unrelated healthy individuals of Chinese Yi ethnic group. Expected heterozygosity, power of discrimination, and polymorphic information content ranged from 0.617 to 0.812, 0.777 to 0.936 and 0.560 to 0.790. The microsatellite loci showed high forensic efficiency. The total discrimination power and cumulate probability of exclusion were 0.99999999999999999986902 and 0.999998818, respectively. Locus‐by‐locus allelic frequencies were compared using analysis of molecular variance (AMOVA) method, and the statistically significant differences were observed between Yi group and Russian, Tujia, Kazak, Bai, Ningxia Han, Salar, Tibetan, and Uigur groups at 5, 6, 7, 7, 7, 8, 12, and 13 loci, respectively. The results of genetic distance comparisons, genetic structure analyses, and principal component analysis all indicated that the Yi group showed relatively short genetic relationships with Russian, Salar, and Bai group. The experimental results showed that the 21 loci in the multiplex system provided highly polymorphic information and forensic efficiency for forensic individual identification and paternity testing, also basic population data for population genetics and anthropological research.     

Slippage mutation rates in 15 autosomal short tandem repeat loci for forensic purposes in a Southeastern Brazilian population

Well‐defined estimates of mutation rates in highly polymorphic tetranucleotide STR loci are a prerequisite for human identification in genetics laboratory routines useful for civil and criminal investigations. Studying 15 autosomal STR loci of forensic interest (CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX, and vWA), we detected 193 slippage mutations (189 one‐step and four two‐step mutations) in 148 875 parent‐child allelic transfers from 5171 paternity cases with true biological relationship (15 096 individuals; 4754 trios and 417 duos; 9925 meiosis) from the state of São Paulo, a very representative population of Brazil. The overall mutation rate was 1.3 × 10^?3 and the highest rates were observed at loci vWA (2.8 × 10^?3), FGA and D18S51 (2.7 × 10^?3 for both), while loci TH01 and TPOX did not present any mutations. The mean slippage mutation rate of paternal origin (1.8 × 10^?3) was six times higher than that observed for maternal origin (0.3 × 10^?3).