Tetranucleotide STRs Research Articles

Development of the first DNA database and identification portal for identification of Unidentified bodies in India – UMID

Identifying missing persons and unidentified dead bodies is a well-documented global problem in recent years. To curb this issue, countries such as the USA, UK, and Australia already have well-established DNA databases. Considering the alarming number of unidentified/unclaimed dead bodies reported in India every year, it is evident that the current practices are not sufficient to establish their identities. Forensic medicine professionals are ethically, morally, and dutybound to collect information about missing and unidentified persons and work with the government agencies to determine their identity. Concerning the social and public interest, we have developed the first-ever identification portal and DNA database of unidentified dead bodies autopsied at the Department of Forensic Medicine and Toxicology, AIIMS, New Delhi, India. After the investigation officer's informed consent, biological samples from unidentified dead bodies and a detailed phenotypic description, anthropological data and other visual characteristics of the deceased are recorded at the time of autopsy. This information is uploaded on our database which is available for public access, and the genotypic information generated through STR analysis is only available for internal usage.Claimants (biological relatives) may browse through the URL (https://umid-aiims.icmr.org.in/), and if they wish to claim an unidentified dead body, they may approach as per the given guidelines. The DNA profiles generated include a total of 16 STRs (15 autosomal tetranucleotide microsatellite STRs and 1 Sex Chromosome Specific STR). The claimant's STR profile is run through the questioned database to look for a potential match. If positive, the investigating officer of that particular case is informed for further necessary action. Until December 2020, our database consisted the information of 255 individuals and two unidentified cadavers were identified. This project's success can also lead to a pioneering National DNA database of unidentified and missing persons in India.

Large-scale genetic admixture suggests high dispersal in an insect pest, the apple fruit moth.

Knowledge about population genetic structure and dispersal capabilities is important for the development of targeted management strategies for agricultural pest species. The apple fruit moth, Argyresthia conjugella (Lepidoptera, Yponomeutidae), is a pre-dispersal seed predator. Larvae feed on rowanberries (Sorbus aucuparia), and when rowanberry seed production is low (i.e., inter-masting), the moth switches from laying eggs in rowanberries to apples (Malus domestica), resulting in devastating losses in apple crops. Using genetic methods, we investigated if this small moth expresses any local genetic structure, or alternatively if gene flow may be high within the Scandinavian Peninsula (~850.000 km2, 55o - 69o N). Genetic diversity was found to be high (n = 669, mean He = 0.71). For three out of ten tetranucleotide STRs, we detected heterozygote deficiency caused by null alleles, but tests showed little impact on the overall results. Genetic differentiation between the 28 sampling locations was very low (average FST = 0.016, P < 0.000). Surprisingly, we found that all individuals could be assigned to one of two non-geographic genetic clusters, and that a third, geographic cluster was found to be associated with 30% of the sampling locations, with weak but significant signals of isolation-by-distance. Conclusively, our findings suggest wind-aided dispersal and spatial synchrony of both sexes of the apple fruit moth over large areas and across very different climatic zones. We speculate that the species may recently have had two separate genetic origins caused by a genetic bottleneck after inter-masting, followed by rapid dispersal and homogenization of the gene pool across the landscape. We suggest further investigations of spatial genetic similarities and differences of the apple fruit moth at larger geographical scales, through life-stages, across inter-masting, and during attacks by the parasitoid wasp (Microgaster politus).

A 13-plex of tetra- and penta-STRs to identify zebrafish

The zebrafish species Danio rerio has become one of the major vertebrate model organisms used in biomedical research. However, there are aspects of the model that need to be improved. One of these is the ability to identify individual fish and fish lines by DNA profiling. Although many dinucleotide short tandem repeat (diSTR) markers are available for this and similar purposes, they have certain disadvantages such as an excessive polymerase slippage (“stutter”) that causes difficulties in automated genotyping and cross-laboratory comparisons. Here we report on the development of a 13-plex of tetranucleotide and pentanucleotide STRs (tetraSTRs and pentaSTRs, respectively) that have low stutter. The system uses an inexpensive universal primer labelling system, which can easily be converted to a direct labeling system if desired. This 13-plex was examined in three zebrafish lines (NHGRI-1, kca33Tg, and kca66Tg, originally obtained from ZIRC). The average observed heterozygosity (Ho) and expected heterozygosity (He) in these highly inbred lines were 0.291 and 0.359, respectively, which is very similar to what has been found with diSTRs. The probability of identity (PI) for all fish tested was 2.1 × 10−5 and the PI for siblings (PIsib) was 6.4 × 10−3, as calculated by the Genalex package. Ninety percent of the fish tested were correctly identified with their respective strains. It is also demonstrated that this panel can be used to confirm doubled-haploid cell lines. This multiplex should find multiple uses for improving the accuracy and reproducibility of studies using the zebrafish model.

UrsaPlex: An STR multiplex for forensic identification of North American black bear (Ursus americanus)

UrsaPlex is a forensic quality 5-dye multiplexed tetranucleotide STR and sex identification panel for individual genetic identification of North American black bears (Ursus americanus). The panel is validated for the identification of black bears involved in human-wildlife conflict events and poaching investigations. This is the first single multiplex panel composed solely of tetranucleotide STRs derived from black bear and bear-specific sex markers. UrsaPlex produces complete genetic profiles from as little as 78 pg of DNA template and has a probability of identity of 2.63 × 10−13. The panel has also been tested for utility in other ursids, and our results indicate with minor modifications, UrsaPlex should prove valuable in identification investigations involving these species as well.

Development of a multiplex, PCR-based genotyping assay for African and Asian elephants for forensic purposes.

Wildlife crimes and the threats they present to elephant populations raise the need to develop and implement DNA-based methodology as an aid for wildlife forensic investigations and conservation efforts. This study describes the development of a tetra-nucleotide repeat STR multiplex, genotyping assay that will identify Asian elephant (Elephas maximus) and African elephant (Loxodonta africana) DNA. The assay targets six tetra-nucleotide STRs and two sex-typing markers simultaneously in both genera of elephants, a first for elephant genotyping assays. The developed assay has potential application in wildlife investigations to associate a biological sample to a particular individual elephant and additionally in conservation science for population management.

Analysis of four novel X-chromosomal short tandem repeats within 71kb of the Xp22.3 region.

DNA testing using X-chromosomal short tandem repeat (X-STR) polymorphisms has been used in maternity/paternity and complex kinship cases. Analyses of repeat sequences, surveys on racial statistics, and development of practical applications for DNA testing continue to be reported. In this study, we identified four novel tetranucleotide STR loci located in the X chromosome, which is the basis of X-STR research. These four tetranucleotide STRs were located within 71kb of the chromosome Xp22.3 region. Using sequence analysis of the structure of repeat sequences, we identified simple repeat sequences of TAAA, CTTT, TATC, and GATA with rare insertions. We then calculated forensic statistical parameters using base length analysis. In the Japanese population, the polymorphism information content was 0.597-0.687, power of discrimination in females was 0.829-0.884, and power of discrimination in males was 0.635-0.729. As these tetranucleotide STRs are closely linked, we conducted haplotype analysis and detected that three loci (LC149476, LC149479, and LC149480) were in linkage disequilibrium. We demonstrated that the simultaneous analysis of these loci may be useful in complex kinship cases. Because these four loci can be detected by multiplex PCR, the detection of alleles at these loci can be rapidly and easily achieved. We conclude that the X-STR loci detected in this study may be useful tools in complex kinship cases and may increase the reliability of genetic testing.

Establishing a DNA identification system for pigs (Sus scrofa) using a multiplex STR amplification

In this study we establish a novel STR multiplex using 13 tetra-nucleotide STRs and the amelogenin marker for the forensic identification of pigs. The genotypes and allele frequency were generated based on 341 samples from 11 pig breeds in Taiwan. Genetic variation was tested including Na, Ne, Ho, He, F-statistics, PIC, Pm and PE for each STR locus and for each breed. Based upon the 341 samples in this study, the CPm and CPEtrio of the 13 STR loci were 1.31 E-11 and 0.9996 respectively. The CPItrio based on ten family sets ranged from 4.012 E+4 to 4.332 E+6 for paternity test. Validation of the multiplex included: determining the sensitivity of the test, where reproducible full DNA profiles were obtained using an initial template of between 0.25 and 1 ng; a comprehensive range of tissue types generated the same genotype; and the specificity was confirmed as no DNA full profile was generated for any species other than Sus scrofa. Based on the phylogenetic analysis, the European domestic breeds clustered separately from the Asian breeds, as expected, and their hybrids formed unique clades respectively between the clades of Asian and European breeds. Eleven test samples, acting as unknown samples, matched all expected breeds. We demonstrate that this novel 14-plex PCR system is valuable in pig individualization, parentage testing, breed assessment, phylogenetic study and forensic applications.

Characterization of STR/microsatellite primers for the Gila monster, Heloderma suspectum screened from paired-end Illumina shotgun sequencing

We used Illumina paired-end shotgun sequencing to characterize microsatellite loci in Heloderma suspectum. We identified over 124,000 potentially amplifiable loci and describe PCR primers for 18 variable tri- and tetra-nucleotide STRs. The observed number of alleles per locus ranged from 5 to 16 and heterozygosity varied from 0.64 to 0.92. In addition 13 of these loci cross-amplified in the beaded lizard, Heloderma horridum. This method of microsatellite identification proved extremely efficient and cost effective. This novel marker set can be used by researchers to better understand these elusive species.

A new 15 autosomal STRs multiplex for domestic horse genotyping

Horse genotyping has a wide range of applications such as identification, pedigree verification, parentage test, forensic investigation, population genetics, analysis of diversity, legitimate registration, among others. Following the recommendations of the International Society for Forensic Genetics (ISFG) regarding the use of non-human (animal) DNA in forensic genetic investigations we have developed a multiplex PCR system of 15 autosomal tetra-nucleotide STRs loci to Equus caballus. The system includes the newly described ECAC2, ECAC4, ECAC5, ECAC9, ECAC10, ECAC12, ECAC14, ECAC15, ECAC18, ECAC21, ECAC23, ECAC26, ECAC28, ECAC29 and ECAC30 loci (on chromosomes 2, 4, 5, 9, 10, 12, 14, 15, 18, 21, 23, 26, 28, 29 and 30, respectively). The polymorphism is in average 8 alleles per marker with a maximum of eleven and a minimum of five for the population studied. All markers were in Hardy–Weinberg equilibrium, except ECAC5 (p=0.0007). The probabilities of paternity (W), exclusion (PE) and cumulative discrimination (PD) for all loci were greater than 0.9999. This work will contribute to the implementation of standardized horse genotyping systems in the forensic community and the horse industry.

Structure and polymorphism of 16 novel Y-STRs in Chinese Han Population

Y-chromosome short tandem repeats (Y-STRs) are useful tools for identifying paternity origin and male-female mixed samples because of their male-specificity, haploid inheritance and relatively simplicity. We focused on novel Y-STRs deposited in the human Genome database from DYS708 to DYS726. We typed 16 male-specific Y-STRs from males of a Chinese Han population residing in Shanxi Province (north China), including DYS708-719, DYS721-723, and DYS726, but failed in typing DYS720, DYS724 and DYS725. The 16 Y-STRs, with mean gene diversity (GD) of 0.79, included three trinucleotide Y-STRs (711, 718, 719), nine tetranucleotide STRs (708, 709, 710, 712, 713, 715, 722, 723, 726) and four pentanucleotide repeat STRs (714, 716, 717, 721). DYS712, consisting of eight alleles, was the most informative STR in our population, with a GD of 0.843. The STRs were classified as simple STRs and complex STRs, according to their structures based on sequencing. Genetic indexes, including allele frequencies, haplotype distribution and male-specificity were determined. The Y-STRs, especially those male-specific, tetra- and penta-nucleotide, with only one copy on Y-chromosome, and relative simple structures, such as DYS709, DYS714, DYS715, DYS716, DYS718, DYS719, and DYS726, were suggested for the future forensic DNA analysis, while DYS724 and DYS725 were not recommended for their multi-copy distribution. The population data provided putative Y-STRs for future genetic and forensic applications.

Forensic evaluation of three closely linked STR markers in a 13 kb region at Xp11.23

Searching for markers located in the region p11 of the X-chromosome the clone AF196972 was checked for its content of microsatellites. The two tetranucleotide STRs DXS10076 and DXS10078 and the trinucleotide STR DXS10077 were characterised and evaluated for their forensic efficiency in a population sample of 352 unrelated individuals from North-West Germany. Whereas at locus DXS10076 10 alleles with PIC and HET values of 0.767 and 0.747 and at locus DXS10078 13 alleles with PIC and HET values of 0.811 and 0.861 could be observed locus DXS10077 only consists of 5 alleles leading to much lower HET and PIC values (0.492 and 0.507) and therefore to a decreased individualization capacity.

Population genetic comparisons of three X-chromosomal STRs (DXS7132, DXS7133 and GATA172D05) in North and South Italy

Population genetic data of three tetranucleotide X-chromosomal STRs, DXS7132, DXS7133 and GATA172D05, were obtained by analyzing 295 unrelated healthy individuals living in North and South Italy (160 females and 135 males), and 40 family trios with female children. PCR primers for the loci DXS7132 and DXS7133 were redesigned in order to reduce the length of the amplification products compared with conventional design so that improved typing success rate when highly degraded DNA is used as a template. The comparison of the allele frequencies of these three ChrX markers gave similar distributions for North and South Italy although minor variations were found for some alleles. Additionally, some differences were found when comparing the allele frequencies of the male and female samples independently. Based on the investigated meiotic events, mutations were not found.

A Novel Multiplex PCR System Consisting of Y-STRs DYS441, DYS442, DYS443, DYS444, and DYS445

We have developed a new sensitive multiplex PCR system consisting of five male-specific and polymorphic tetranucleotide STRs--DYS441 (GDB: 10013873), DYS442 (GDB: 10030304), DYS443 (GDB: 10807127), DYS444 (GDB: 10807128), and DYS445 (GDB: 10807129) on the Y chromosome. Fifty pg DNA per 10 microL reaction volume was required for the correct typing of five STRs. Using this system, the five Y-STRs were correctly typed from blood and semen stains that had been stored for several years at room temperature.

Characterization and haplotype analysis of the polymorphic Y-STRs DYS443, DYS444 and DYS445 in a Japanese population.

From sequence database information we have newly identified three male-specific and polymorphic tetranucleotide STRs, DYS443 (GDB: 10807127), DYS444 (GDB: 10807128) and DYS445 (GDB: 10807129) on the Y chromosome. Analysis of 190 Japanese males revealed 6, 5 and 4 alleles in the DYS443, DYS444 and DYS445 systems, with calculated STR diversities of 0.68, 0.57 and 0.53, respectively. The cumulative haplotype diversity of the five Y-STRs DYS441, DYS442, DYS443, DYS444 and DYS445 was calculated to be 0.95 and therefore application of these STRs may yield very useful information for forensic individualization.

Identification and characterization of two novel human polymorphic STRs on the Y chromosome.

From sequence database information, we have identified two male-specific and polymorphic tetranucleotide STRs, DYS 441 (GDB:10013873) and DYS 442 (GDB: 10030304), on the Y chromosome. Analysis of 184 males allowed 7 and 5 alleles to be distinguished in the DYS 441 and DYS 442 systems, respectively, yielding 21 haplotypes. The gene diversities were 0.72 and 0.51, respectively and the haplotype diversity was 0.85.