Polymorphic Short Tandem Repeat Loci Research Articles

Using short tandem repeat profiling to validate cell lines in biobanks

Aim. To approve the COrDIS kit (Gordiz, Russia) for the authenticity of cell lines from the Bioresource Collection of the N.N. Blokhin National Medical Research Center of Oncology by the short tandem repeat (STR) profiling.Material and methods. The chosen method proved to be a reliable and reproducible option. With this approach, a number of polymorphic STR loci are amplified using commercially available primer sets. Polymerase chain reaction (PCR) products are analyzed simultaneously with size standards using automated fluorescent detection methods. The results are presented as a simple number code corresponding to the lengths of the PCR products amplified at each locus. By applying this method to cell lines, the laboratory can both authenticate commercial cell lines and build a database of their lines. In the work, we used the COrDIS EXPERT 26 kit (Gordiz, Russia), validated for molecular genetic identification of personality based on multiplex PCR analysis of 26 highly polymorphic loci of human genomic deoxyribonucleic acid. PCR results were analyzed by capillary electrophoresis using an automatic genetic analyzer with laser-induced fluorescence detection (Applied Biosystems 3500xL).Results. When testing the method, profiling of 37 cell lines was carried out, of which 18 were announced in international databases and 19 were unique, obtained at the N. N. Blokhin National Medical Research Center of Oncology, as well as a cell line mixture in order to determine the limits of contamination detection. The obtained results showed the correspondence of commercial cell lines with the data in international databases. Within the framework of this work, profiles of unique lines were obtained and the foundation of own genetic database was laid. Studies to identify the limit of contamination detection by another line have shown that even 4% of the contaminant culture in the total pool can be used to identify its individual alleles.Conclusion. The results obtained indicate the possibility of using the method to identify samples of the collection and detect intraspecific contamination.

Genetic analysis of hog deer (Axis porcinus) in Victoria, Australia, and its applications to invasive species and game management

Hog deer were introduced to Australia in the 1860s, where they have spread across the Gippsland region of Victoria. Due to its status as an introduced species and an important game animal within Victoria, management of the species is complex. Given this complexity, genetic studies can provide important information regarding population structure and diversity which can assist in controlling problematic populations of hog deer, while also ensuring viable game stock in sites managed as game reserves. The aim of this study was to investigate the population genetic structure and diversity of the Victorian hog deer 150 years after introduction using short tandem repeats (STRs). Hog deer samples were collected across 15 sites of differing management regimes in the Gippsland region of Victoria and genotyped for 13 polymorphic STR loci. Up to four distinct genetic clusters were identified across the sites sampled, suggesting that despite low observed genetic diversity, population structure is present across their range. It was also possible to detect evidence of recent translocations among populations. This study suggests that the presence of distinct genetic clusters may enable management of separate genetic units, considering invasive species and game management objectives.

Exploring new short tandem repeat markers for DNA mixture deconvolution

AbstractRelying on their polymorphic nature, short tandem repeats (STRs) have been extensively studied and routinely utilized as human identity markers in forensic genetics. However, even the most comprehensive STR multiplexes in use today are limited in their ability to determine the number and appropriation of component contributor alleles in DNA mixtures. The difficulty in parsing out individuals in DNA mixtures is a consequence of overlapping length‐based alleles genotyped using the polymerase chain reaction (PCR) coupled with capillary electrophoresis (CE). Many challenges exist in the resolution of minor alleles (i.e., the alleles originating from a minor contributor) from stutter and stochastic effects (e.g., inherent heterozygote peak imbalance, undetected alleles [drop out]) in a given DNA profile, in addition to the complex statistical models and algorithms necessary to render DNA mixtures interpretable from a forensic casework standpoint. Therefore, we can either adapt to complex interpretation methods, or pursue new bench science approaches to DNA mixture deconvolution. One promising area of research includes the incorporation of additional highly polymorphic STR loci to compliment current marker multiplexes and offer great potential to improve the forensic genetic analysis of DNA mixtures.This article is categorized under: Forensic Biology > Forensic DNA Technologies Forensic Biology > Interpretation of Biological Evidence Forensic Biology > Ethical and Social Implications

Investigation of the forensic GlobalFiler loci in the genetically isolated Circassian subpopulation in Jordan

Circassians are a Caucasian ethnic group who make up a significant minority in Jordan. Although other ethnic groups have been the subject of forensic genetic analysis, no published study has investigated the forensic genetic efficiency of short tandem repeats (STRs) in Circassians, neither in Jordan nor in any other country. The main objective of the current study is to determine the allelic frequencies and evaluate the forensic efficiency parameters of 21 highly polymorphic autosomal STR loci among the Circassian subpopulation in Jordan. The GlobalFiler loci were amplified using DNA extracted from the whole blood samples of 150 Jordanian Circassians. The SE33 locus was found to be the most informative and polymorphic STR marker while TPOX was the least informative. However, allele 8 of TPOX was the most common across all of the investigated 21 loci in Jordanian Circassians. The combined matching probability (CMP) and combined power of discrimination (CPD) were 5.02E-24 and 0.9999999, respectively.

Characterization of Duck (Anas platyrhynchos) Short Tandem Repeat Variation by Population-Scale Genome Resequencing.

Short tandem repeats (STRs) are usually associated with genetic diseases and gene regulatory functions, and are also important genetic markers for analysis of evolutionary, genetic diversity and forensic. However, for the majority of STRs in the duck genome, their population genetic properties and functional impacts remain poorly defined. Recent advent of next generation sequencing (NGS) has offered an opportunity for profiling large numbers of polymorphic STRs. Here, we reported a population-scale analysis of STR variation using genome resequencing in mallard and Pekin duck. Our analysis provided the first genome-wide duck STR reference including 198,022 STR loci with motif size of 2–6 base pairs. We observed a relatively uneven distribution of STRs in different genomic regions, which indicates that the occurrence of STRs in duck genome is not random, but undergoes a directional selection pressure. Using genome resequencing data of 23 mallard and 26 Pekin ducks, we successfully identified 89,891 polymorphic STR loci. Intensive analysis of this dataset suggested that shorter repeat motif, longer reference tract length, higher purity, and residing outside of a coding region are all associated with an increase in STR variability. STR genotypes were utilized for population genetic analysis, and the results showed that population structure and divergence patterns among population groups can be efficiently captured. In addition, comparison between Pekin duck and mallard identified 3,122 STRs with extremely divergent allele frequency, which overlapped with a set of genes related to nervous system, energy metabolism and behavior. The evolutionary analysis revealed that the genes containing divergent STRs may play important roles in phenotypic changes during duck domestication. The variation analysis of STRs in population scale provides valuable resource for future study of genetic diversity and genome evolution in duck.

CHIIMP: An automated high-throughput microsatellite genotyping platform reveals greater allelic diversity in wild chimpanzees.

Short tandem repeats (STRs), also known as microsatellites, are commonly used to noninvasively genotype wild‐living endangered species, including African apes. Until recently, capillary electrophoresis has been the method of choice to determine the length of polymorphic STR loci. However, this technique is labor intensive, difficult to compare across platforms, and notoriously imprecise. Here we developed a MiSeq‐based approach and tested its performance using previously genotyped fecal samples from long‐term studied chimpanzees in Gombe National Park, Tanzania. Using data from eight microsatellite loci as a reference, we designed a bioinformatics platform that converts raw MiSeq reads into locus‐specific files and automatically calls alleles after filtering stutter sequences and other PCR artifacts. Applying this method to the entire Gombe population, we confirmed previously reported genotypes, but also identified 31 new alleles that had been missed due to sequence differences and size homoplasy. The new genotypes, which increased the allelic diversity and heterozygosity in Gombe by 61% and 8%, respectively, were validated by replicate amplification and pedigree analyses. This demonstrated inheritance and resolved one case of an ambiguous paternity. Using both singleplex and multiplex locus amplification, we also genotyped fecal samples from chimpanzees in the Greater Mahale Ecosystem in Tanzania, demonstrating the utility of the MiSeq‐based approach for genotyping nonhabituated populations and performing comparative analyses across field sites. The new automated high‐throughput analysis platform (available at https://github.com/ShawHahnLab/chiimp) will allow biologists to more accurately and effectively determine wildlife population size and structure, and thus obtain information critical for conservation efforts.

Population-scale whole genome sequencing identifies 271 highly polymorphic short tandem repeats from Japanese population

Forensic DNA typing is widely used to identify missing persons and plays a central role in forensic profiling. DNA typing usually uses capillary electrophoresis fragment analysis of PCR amplification products to detect the length of short tandem repeat (STR) markers. Here, we analyzed whole genome data from 1,070 Japanese individuals generated using massively parallel short-read sequencing of 162 paired-end bases. We have analyzed 843,473 STR loci with two to six basepair repeat units and cataloged highly polymorphic STR loci in the Japanese population. To evaluate the performance of the cataloged STR loci, we compared 23 STR loci, widely used in forensic DNA typing, with capillary electrophoresis based STR genotyping results in the Japanese population. Seventeen loci had high correlations and high call rates. The other six loci had low call rates or low correlations due to either the limitations of short-read sequencing technology, the bioinformatics tool used, or the complexity of repeat patterns. With these analyses, we have also purified the suitable 218 STR loci with four basepair repeat units and 53 loci with five basepair repeat units both for short read sequencing and PCR based technologies, which would be candidates to the actual forensic DNA typing in Japanese population.

Identification and characterization of short tandem repeats in the Tibetan macaque genome based on resequencing data.

The Tibetan macaque, which is endemic to China, is currently listed as a Near Endangered primate species by the International Union for Conservation of Nature (IUCN)(2017). Short tandem repeats (STRs) refer to repetitive elements of genome sequence that range in length from 1–6 bp. They are found in many organisms and are widely applied in population genetic studies. To clarify the distribution characteristics of genome-wide STRs and understand their variation among Tibetan macaques, we conducted a genome-wide survey of STRs with next-generation sequencing of five macaque samples. A total of 1 077 790 perfect STRs were mined from our assembly, with an N50 of 4 966 bp. Mono-nucleotide repeats were the most abundant, followed by tetra- and di-nucleotide repeats. Analysis of GC content and repeats showed consistent results with other macaques. Furthermore, using STR analysis software (lobSTR), we found that the proportion of base pair deletions in the STRs was greater than that of insertions in the five Tibetan macaque individuals (P<0.05, t-test). We also found a greater number of homozygous STRs than heterozygous STRs (P<0.05, t-test), with the Emei and Jianyang Tibetan macaques showing more heterozygous loci than Huangshan Tibetan macaques. The proportion of insertions and mean variation of alleles in the Emei and Jianyang individuals were slightly higher than those in the Huangshan individuals, thus revealing differences in STR allele size between the two populations. The polymorphic STR loci identified based on the reference genome showed good amplification efficiency and could be used to study population genetics in Tibetan macaques. The neighbor-joining tree classified the five macaques into two different branches according to their geographical origin, indicating high genetic differentiation between the Huangshan and Sichuan populations. We elucidated the distribution characteristics of STRs in the Tibetan macaque genome and provided an effective method for screening polymorphic STRs. Our results also lay a foundation for future genetic variation studies of macaques.

Allele frequencies of 15 STR loci in Bosnian and Herzegovinian population.

AimTo determine newest the most accurate allele frequencies for 15 short tandem repeat (STR) loci in the Bosnian and Herzegovinian population, calculate statistical parameters, and compare them with the relevant data for seven neighboring populations.MethodsGenomic DNA was obtained from buccal swabs of 1000 unrelated individuals from all regions of Bosnia and Herzegovina. Genotyping was performed using PowerPlex® 16 System to obtain allele frequencies for 15 polymorphic STR loci including D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, and FGA. The calculated allele frequencies were also compared with the data from neighboring populations.ResultsThe highest detected value of polymorphism information content (PIC) was detected at the PentaE locus, whereas the lowest value was detected at the TPOX locus. The power of discrimination (PD) values had similar distribution, with Penta E showing the highest PD of 0.9788. While D18S51 had the highest value of power of exclusion (PE), the lowest PE value was detected at the TPOX locus.ConclusionUpon comparison of Bosnian and Herzegovinian population data with those of seven neighboring populations, the highest allele frequency differentiation was noticed between Bosnian and Herzegovinian and Turkish population at 5 loci, the most informative of which was Penta E. The neighbor-joining dendrogram constructed on the basis of genetic distance showed grouping of Slovenian, Austrian, Hungarian, and Croatian populations. Bosnian and Herzegovinian population was between the mentioned cluster and Serbian population. To determine more accurate distribution of allelic frequencies and forensic parameters, our study included 1000 unrelated individuals from all regions of Bosnia and Herzegovina, and our findings demonstrated the applicability of these markers in both forensics and future population genetic studies.

Development of a polymorphic short tandem repeat locus multiplex system for efficient human identification.

This study aimed to develop a short tandem repeat (STR) multiplex system, made up of 22 highly informative STR loci, for application in forensic genetics. The system comprised 21 polymorphic autosomal loci (D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, FGA, D2S441, D17S1301, D19S433, D18S853, D20S482, and D14S1434) and the amelogenin gene locus. Strategies were developed to overcome the challenges involved in creating a multiplex system. Based on the literature and available databases, the STR loci were selected with the aim to obtain discriminatory markers, and followed specific criteria for this purpose. Primers were projected using the Primer3 software, and AutoDimer was used to evaluate possible interactions between them. The 22 selected STR loci were validated individually and jointly, both to assess their sensitivity and to test the efficiency of the multiplex system. Statistical analyses were based on the genetic data of 450 unrelated individuals living in the State of Goiás, thus allowing the establishment of the parameters necessary to use this system. A total of 239 alleles were detected for the 22 loci in the set, allowing for a probability of identity of 4.23 x 10-25 to be obtained. The combined power of discrimination was 0.999999999999999999999999 and the combined power of exclusion was 0.99999. Upon complete validation of the entire system, this multiplex assay was considered to be a powerful tool for application in human identification by DNA analysis.

Genetic variability and forensic efficiency of 39 microsatellite loci in the Li ethnic group from Hainan Island in the South China Sea

Background: Investigation of allele and genotype frequencies of microsatellite loci in various populations is an essential pre-requisite in forensic application.Aim: The present study obtained population genetic data and forensic parameters of 39 autosomal Short Tandem Repeats (STRs) loci from a Chinese Li ethnic group and estimated the genetic relationships between Li and other reference populations.Subjects and methods: Thirty-nine STR loci, which include D19S433, D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSF1PO, Penta D, D2S441, vWA, D8S1179, TPOX, Penta E, TH01, D12S391, D2S1338, FGA, D6S477, D18S535, D19S253, D15S659, D11S2368, D20S470, D1S1656, D22-GATA198B05, D8S1132, D4S2366, D21S1270, D13S325, D9S925, D3S3045, D14S608, D10S1435, D7S3048, D17S1290 and D5S2500, were amplified in two multiplex DNA-STR fluorescence detection systems for 189 unrelated healthy individuals of the Chinese Li ethnic group. The allele frequency distribution and several parameters commonly used in forensic science were statistically analysed.Results: A total of 378 alleles were observed with corresponding allelic frequencies ranging from 0.0026–0.5899. The power of discrimination and power of exclusion ranged from 0.7569–0.9672 and 0.2513–0.7355, respectively. The power of exclusion (PE) ranged from 0.2580–0.7943 for trio paternity cases and 0.1693–0.5940 for duo paternity cases. The polymorphism information content (PIC) ranged from 0.5001–0.8611. The cumulative match probability across these 39 loci was 2.4242 × 10−38.Conclusion: The results indicate that 39 STR loci are polymorphic among the Li ethnic group in Hainan Island in the South China Sea. This set of polymorphic STR loci provide highly polymorphic information and forensic efficiency for forensic individual identification and paternity testing, as well as basic population data for population genetics and anthropological research.

Investigator® HDplex (Qiagen) reference population database for forensic use in Argentina

Currently, autosomal Short Tandem Repeat (STR) markers represent the method of election in forensic human identification. Commercial kits of most common use nowadays –e.g. PowerPlex®Fusion, Promega Corp.; AmpFlSTR GlobalFiler, Thermofisher scientific; Investigator 24Plex QS,Qiagen-, allow the co-amplification of 23 highly polymorphic STR loci providing a high discrimination power in human identity testing. However, in complex kinship analysis and familial database searches involving distant relationships, additional DNA typing is often required in order to achieve well-founded conclusions. The recently developed kit Investigator® HDplex (Qiagen) co-amplify twelve autosomal STRs markers (D7S1517, D3S1744, D12S391, D2S1360, D6S474, D4S2366, D8S1132, D5S2500, D18S51, D21S2055, D10S2325, SE33), nine of which are not present in the above mentioned kits, providing a set of efficient supplementary markers for human identification purposes. In this study we genotyped a sample of 980 individuals from urban areas of ten Argentinean provinces using the Investigator® HDplex kit, aiming to provide forensic estimates for use in forensic casework and parentage testing in Argentina. We report reference allelic frequency databases for each of the provinces studied as well as for the combined samples. No deviation of Hardy-Weinberg equilibrium was observed. A reasonable discrimination capacity and power of exclusion was estimated which allowed predicting an acceptable forensic behavior of this kit, either to be used as the main STR panel for simple cases or as an auxiliary tool in complex cases. Additionally, population comparison tests showed that the studied samples are relatively homogeneous across the country for these STR set.

Immunological Profile of Patients after HSCT Using the FCC Conditioning Regimen for Treatment of Severe Aplastic Anemia Shows Sustained Mixed T-Cell Chimerism Is Due to Persistence of Recipient CD8 T Cells and Indicates Potential Basis for Tolerance and Extremely Low Incidence of Graft Versus Host Disease

Mixed T-cell chimerism is frequent and persists despite discontinuation of immunosuppression after hematopoietic stem cell transplantation (HSCT) for severe aplastic anemia (SAA) using the fludarabine, cyclophosphamide and alemtuzumab/campath (FCC) conditioning regimen. To investigate the basis for long-term co-existence of donor and patient T cells and remarkably low rates of graft versus host disease (GVHD), the lymphocyte composition and lymphocyte subset chimerism of adult patients transplanted for SAA using FCC was assessed.PatientsWe have transplanted 42 patients (pts) for acquired idiopathic SAA using FCC conditioning from 2007- March 2015 at King's College Hospital. Median age was 33 years (range 15-63). BM was stem cell source in 7(17%) pts and PBSC in 30 (83%). Median cell dose was 6.85 (1.9-12.4) CD34+ cells x106/Kg. Donor was matched sibling in 11(26%) and unrelated in 31 (74%) pts, 6 of whom also received 2 Gy TBI for 9/10 match. Post graft immunosuppression comprised ciclosporin (CSA) alone. Primary graft failure occurred in 1 pt (2%). Acute GVHD was seen in 6 (14%) pts (all grade I-II), and chronic GVHD in 5 (12%) patients (all skin, only 1 severe in a 9/10 match patient). Factors associated with cGVHD were previous aGVHD (p=0.035) and CD3+ chimerism at day+100 ³ 90% (p=0.006). Median follow-up of survivors was 41 months (4.3-96), with 93% overall survival and 90% event-free survival. At 1, 2 and 3 years post HSCT, 4/30, 20/23 and 16/16 patients, respectively, had discontinued CSA.ResultsComparison to 11 healthy age-matched individuals showed prolonged lymphopenia and abnormal proportions of T, B and NK cells. T cells were profoundly deficient comprising only 1.76% of lymphocytes at day 30, rising to 43.8% by day 360 but still significantly below normal (66%, p=0.018). CD8 T cells recovered faster than CD4 T cells producing inversion of the normal CD4 to CD8 T cell ratio. Composition of na•ve, memory and effector subsets within the CD4 T cell population was near normal by day 360 and na•ve CD4 T cells expressed CD31 indicative of renewed thymopoiesis. In contrast, CD8 T-cell subset composition remained abnormal at day 360 due to a high proportion of effector T cells (57.2% of CD8 T cells compared to 9.3% for healthy individuals, p=<0.005). T-cell subsets from five patients were isolated by fluorescence-activated cell sorting, and chimerism determined by analysis of informative alleles from polymorphic short tandem repeat loci. Results shown in the figure below revealed that mixed T-cell chimerism at day 360 was principally due to persistence of patient CD8 T cells, with notable contribution of the effector subset. Follow-up analysis of three patients at >2 years after HSCT showed the mixed chimerism profile within T-cell subsets was stable. A significant correlation was observed between lower donor T-cell chimerism at day 360 and CMV reactivation or EBV viremia early after HSCT (p=0.036), suggesting that antigen-driven expansion of virus-specific patient CD8 effector T cells may substantially contribute to persistent mixed T-cell chimerism.Sustained co-existence of donor and patient T cells and low rates of GVHD indicates conditions favor mutual tolerance. Findings suggested a potential role for cells with immunomodulatory properties. B cell recovery was robust, with normal numbers by day 100. Notably, the B cell population present early after HSCT contained a significantly increased proportion of cells with an immature transitional phenotype (CD24+ CD38+, CD27- IgMhigh IgDhigh 17.1% compared to 2.7% for healthy individuals, p=<0.005) and significantly more B cells produced IL-10 after in vitro stimulation with CD40L (14.6% compared to 6.3% for healthy individuals, p=0.013), consistent with the known immunosuppressive activity of immature B cells. Furthermore, proportions of regulatory T cells (CD4+ CD25high CD27+ FoxP3+) within the CD4 T-cell population were normal (range 4.2-7.4% compared to 5.3% in healthy controls).ConclusionsPatient-derived effector CD8 T cells shape mixed T-cell chimerism after FCC conditioning and presence of regulatory B and T cells may contribute to long-term tolerance of mixed T-cell chimerism with associated low incidence of GVHD in the absence of post graft immunosuppression.Note: Marsh JCWand Barber LD joint last authors [Display omitted] DisclosuresKulasekararaj:Alexion: Consultancy.

Evaluating the genetic impact of South and Southeast Asia on the peopling of Bangladesh.

Despite rapidly growing understandings and dependency on single nucleotide polymorphisms (SNPs), highly variable autosomal short tandem repeats (STRs) are still regarded as the most established method to differentiate individuals at forensic level. Here with large number of various ethnic groups we undertook this study to reveal the genetic structure of the most densely populated part of South Asia i.e. the Bangladesh. The purpose of this work was to estimate population parameters based on the allele frequencies obtained for 15 polymorphic autosomal STR loci investigated in caste and tribal populations from Bangladesh (n=706). We compared the results in a broader context by merging 24 different populations of Asia to pertain their affinity. Various statistical analyses suggested a clear cut demarcation of tribal and non-tribal in Bangladesh. Moreover, beside the phylogenetic structure of the studied populations, it is found that the mean heterozygosity value was highest among the populations of Bangladesh, likely because of gene flow from different directions. However, Tonchangya, Adi and Khumi showed sign of genetic isolation and reduced diversity, possibly as a result of genetic drift and/or strong founder effects working on small endogamous populations.

PSTR Finder: a rapid method to discover polymorphic short tandem repeat markers from whole-genome sequences.

BackgroundWhole-genome sequencing is performed routinely as a means to identify polymorphic genetic loci such as short tandem repeat loci. We have developed a simple tool, called pSTR Finder, which is freely available as a means of identifying putative polymorphic short tandem repeat (STR) loci from data generated from genome-wide sequences. The program performs cross comparisons on the STR sequences generated using the Tandem Repeats Finder based on multiple-genome samples in a FASTA format. These comparisons generate reports listing identical, polymorphic, and different STR loci when comparing two samples.MethodsThe web site http://forensic.mc.ntu.edu.tw:9000/PSTRWeb/Default has been developed as a means to identify polymorphic STR loci within complex mass genome sequences. The program was developed to generate a series of user-friendly reports.ResultsAs proof of concept for the program, four FASTA genome sequence samples of human chromosome X (AC_000155.1, CM000685.1, NC_018934.2, and CM000274.1) were obtained from GenBank and were analyzed for the presence of putative STR regions. The sequences within AC-000155.1 were used as an initial reference sequence from which there were 5443 identical and 4305 polymorphic STR loci identified using a repeat unit of 1–6 and 10 bp as the flanking sequence either side of the putative STR loci. A reliability test was used to compare five FASTA samples, which had sections of DNA sequence removed to mimic partial or fragmented DNA sequences, to determine whether pSTR Finder can efficiently and consistently find identical, polymorphic, and different STR loci.ConclusionsFrom the mass of DNA sequence data, the project was found to reproducibly identify polymorphic STR loci and generate user-friendly reports detailing the number and location of these potential polymorphic loci. This freely available program was found to be a useful tool to find polymorphic STR within whole-genome sequence data in forensic genetic studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13323-015-0027-x) contains supplementary material, which is available to authorized users.

Genetic Polymorphism of Five STR Markers among Four Groups of Punjabi Population in North-West Punjab, India

Historically, Punjab has served a major passage way for human migration to Indian subcontinent and has influenced the genetic structure of extant populations in Punjab. Therefore, Punjabi population possesses an exclusive genetic profile primarily due to the many migratory events in this region which caused an extensive range of genetic diversity. Hence, the present study is an attempt to understand the effect of these influences on genetic differentiation, diversity a nd popula tion structure of Punjab. Genetic polymorphism at five highly polymorphic short tandem repeat loci (STR) is studied in four endogamous population groups of Punjab, India. The studied groups included Jat Sikh, Majbi Sikh, Brahmin and Ramdasia to evaluate their significance in human identification and genetic study. All selected groups practice endogamy and a total of 358 individuals belonging to these four endogamous groups were studied for five highly polymorphic with greater power of exclusion STR loci: THO1, TPOX, D7S820, CSF1PO and vWA. The highest observed heterozygosity was found in Ramdasia population for almost all the markers except vWA which had highest observed heterozygosity among Majbi Sikh population group. In this study, the average sub-ethnic differentiation (Fst) among the four populations of north-west Punjab was 0.0821. The marker with the highest contribution to interpopulation genetic difference was obser ved to be vWA.

Automated analysis of sequence polymorphism in STR alleles by PCR and direct electrospray ionization mass spectrometry

Short tandem repeats (STRs) are the primary genetic markers used for the analysis of biological samples in forensic and human identity testing. The discrimination power of a combination of STRs is sufficient in many human identity testing comparisons unless the evidence is substantially compromised and/or there are insufficient relatives or a potential mutation may have arisen in kinship analyses. An automated STR assay system that is based on electrospray ionization mass spectrometry (ESI-MS) has been developed that can increase the discrimination power of some of the CODIS core STR loci and thus provide more information in typical and challenged samples and cases. Data from the ESI-MS STR system is fully backwards compatible with existing STR typing results generated by capillary electrophoresis. In contrast, however, the ESI-MS analytical system also reveals nucleotide polymorphisms residing within the STR alleles. The presence of these polymorphisms expands the number of alleles at a locus. Population studies were performed on the 13 core CODIS STR loci from African Americans, Caucasians and Hispanics capturing both the length of the allele, as well as nucleotide variations contained within repeat motifs or flanking regions. Such additional polymorphisms were identified in 11 of the 13 loci examined whereby several nominal length alleles were subdivided. A substantial increase in heterozygosity was observed, with close to or greater than 5% of samples analyzed being heterozygous with equal-length alleles in at least one of five of the core CODIS loci. This additional polymorphism increases discrimination power significantly, whereby the seven most polymorphic STR loci have a discrimination power equivalent to the 10 most discriminating of the CODIS core loci. An analysis of substructure among the three population groups revealed a higher θ than would be observed compared with using alleles designated by nominal length, i.e., repeats solely. Two loci, D3S1358 and vWA produced θ estimates of 0.0477 and 0.0234, respectively, when the expanded allele complement (i.e., nominal allele and SNPs) was considered compared to 0.0145 and 0.01266, respectively when only nominal repeat number was considered. These differences may indicate underlying population specific allele distributions exist within these populations. A system of nomenclature has been developed that facilitates the databasing, searching and analyses of these combined data forms.

Nine polymorphic STR loci in the HLA region in the Shaanxi Han population of China

A large number of microsatellite genetic markers have been identified in the human leukocyte antigen (HLA) region. We investigated genetic polymorphism of the nine short tandem repeat (STR) loci (D6S276, MOGCA, D6S265, MIB, D6S273, G51152, TAP1CA, RING3CA, and D6S291) in the HLA region in the Shaanxi Han population. Using a fluorescence-labeled multiplex-PCR STR typing method, 6-13 alleles were detected in these nine STR loci in 150 unrelated Han Chinese from the region of Shaanxi, China. The distributions of the genotypes at these nine loci were in Hardy-Weinberg equilibrium. We conclude that these nine STR loci have a high level of genetic polymorphism; they would be useful for population genetic studies, pre-transplantation HLA typing, forensic and paternity testing, etc.

Population studies of 17 equine STR for forensic and phylogenetic analysis

As a consequence of the close integration of horses into human society, equine DNA analysis has become relevant for forensic purposes. However, the information content of the equine Short Tandem Repeat (STR) loci commonly used for the identification or paternity testing has so far not been fully characterized. Population studies were performed for 17 polymorphic STR loci (AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG10, LEX3 and VHL20) including 8641 horses representing 35 populations. The power of parental exclusion, polymorphic information content, expected and observed heterozygosity and probability of identity were calculated, showing that the set of 17 STRs has sufficient discriminating power for forensic analysis in almost all breeds. We also explored the reliability of individual assignment tests in identifying the correct breeds of origin for unknown samples. The overall proportion of individuals correctly assigned to a population was 97.2%. Finally, we demonstrate the phylogenetic signal of the 17 STR. We found three clusters of related breeds: (i) the cold-blooded draught breeds Haflinger, Dutch draft and Friesian; (ii) the pony breeds Shetland and Miniature horse with the Falabella, Appaloosa and Icelandic; and (iii) The Warmblood riding breeds, together with the hot-blooded Standard-bred, Thoroughbred and Arabian.

Analysis of TPOX short tandem repeat locus with matrix-associated laser desorption/ionization time-of-flight-based restriction fragment mass polymorphism assay

Short tandem repeat (STR) loci are routinely analyzed by capillary electrophoresis. However, this method has several disadvantages, including long operational time, low throughput, and inaccuracy. As a result of the introduction of matrix-associated laser desorption/ionization time-of-flight (MALDI–TOF) and electrospray ionization (ESI), mass spectrometry has become an alternative method for genotyping polymorphic STR loci. Here we established a restriction fragment mass polymorphism (RFMP) assay for genotyping STR locus, TPOX, by typeIIS restriction endonuclease cleavage of polymerase chain reaction (PCR) amplicon followed by MALDI–TOF mass spectrometry. The resulting TPOX genotypes from this assay were in good agreement with the results from direct DNA sequencing and GeneScan assays. Our results showed that the RFMP assay is an accurate and high-throughput method for analyzing long DNA fragments such as STR markers. Further research with multiple STR loci may allow this assay to be used for diverse applications such as forensics, paternity tests, and detection of genetic disorders.