DNA profiling in India: Addressing issues of sample preservation, databasing, marker selection, & statistical approaches.

DNA technology is the gold standard with respect to the identification of individuals from biological evidence. The technology offers the convenience of a universally similar approach and methodology for analysis across the globe. However, the technology has not realised its full potential in India due to the lack of a DNA database and lacunae in sample collection and preservation from the scene of crime and victims (especially those of sexual assault). Further, statistical interpretation of DNA results is non-existent in the majority of cases. Though the latest technologies and developments in the field of DNA analysis are being adopted and implemented,very little has been enacted practically to improve optimise sample collection and preservation. This article discusses current casework scenarios that highlight the pitfalls and ambiguous areas in the field of DNA analysis, especially with respect DNA databases, sampling, andstatistical approaches to genetic data analysis. Possible solutions and mitigation measures are suggested.

Population dynamics and genetic isolation in the Central Himalayan region: insights from Sikkim population, India.

India's rich diversity encompasses individuals from varied geographical, cultural, and ethnic backgrounds. In the field of population genetics, comprehending the genetic diversity across distinct populations plays a crucial role. This study presents significant findings from genetic data obtained from the Sikkimese population of India. Autosomal markers were crucial for evaluating forensic parameters, with a combined paternity index of 1 × 109. Notably, Penta E emerged as a distinguishing marker for individual identification in the Sikkim population. Fst genetic distance values revealed insights into genetic isolation among different groups, enhancing our understanding of population dynamics in the central Himalayan region. The NJ-based phylogenetic tree highlighted close genetic relationships, of the Sikkim population with the Nepalese population surrounding neighbouring Himalayan populations providing glimpses into common ancestry. In summary, this study contributes valuable data to population genetics and underscores the importance of genetic variation in comprehending population dynamics and forensic applications.

Genetic Diversity of Autosomal STR Markers in the Brahmin Population of Rajasthan and Haryana: Significance in Population and Forensic Genetics.

The aim of the study is to evaluate the suitability of STRs for molecular characterization and forensic applications in unrelated Brahmins of Rajasthan and Haryana states, India. Materials and Methods: A total of 203 male DNA samples from various districts of Haryana (n=104) and Rajasthan (n=99) were genotyped using the GlobalFiler® PCR Amplification Kit. Allelic frequencies and different forensic parameters like PD, PE, PIC, PM, Ho, He, UHe, and TPI were calculated with different software. Results: More than 200 alleles were present in both populations, ranging from 6.0 to 35.2 and SE33 was the most polymorphic marker. The combined power of discrimination was 1. To know the relatedness with other Indian Brahmin populations, the UPGMA dendrogram and principal component analysis plot were visualized to show that both populations are close to each other and in nearby Saraswat Brahmins of Himachal Pradesh. This study showed a genetic relationship and forensic examination in the Haryana and Rajasthan Brahmin populations and various ethno-linguistically diverse populations of India. Conclusion: The results imply that the highly polymorphic 21 autosomal STR loci might be applied for individuals' forensic identification and parentage testing. This study also suggests that the kit having both autosomal and Y-STR markers is appropriate for a better understanding of the genetic and forensic examination in the Brahmin population of Haryana and Rajasthan.

Evaluation of genetic polymorphisms at 21 autosomal STR loci in Ramgharia Sikh population of Punjab, India.

BACKGROUND: STR (Short Tandem Repeat) markers are highly polymorphic markers, which are widely used in forensics DNA analysis and aid to ascertain unique genotype profiles of individuals and determine the genetic diversity of the given population. AIM: In the present study, an attempt has been made to evaluate the population genetic diversity of the Ramgharia Sikh population of Punjab, India, using 21 autosomal STR loci (D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, SE33, D10S1248, D1S1656, D12S391, and D2S1338) to augment the emerging forensic database related to the indigenous population of India. SUBJECTS AND METHODS: For generation of the database, 200 (blood on FTA card) samples were obtained from genetically unrelated Ramgharia Sikhs residing in the state of Punjab. Twenty-one autosomal STR markers were analysed using the Globalfiler® PCR amplification kit. RESULTS: With the help of various statistical tools, a total of 232 alleles were observed and 11.048 ± 1.284 (mean ± standard deviation) alleles per locus were recorded. No locus deviated from Hardy Weinberg Equilibrium. SE33 locus was found to be the most polymorphic and exhibited the highest discrimination power, that is, 0.99. Moreover, results further indicated that Ramgharia Sikhs of Punjab showed a high affinity with Bhils of Madhya Pradesh (India). Thus, the studied population showed genetic proximity with the geographically close populations of India and showed significant genetic variations with distant populations, which was evident from the UPGMA tree and Principal Component Analysis plot. CONCLUSION: Overall, the 21 autosomal STRs were found to be polymorphic in the Ramgharia population and suitable for forensic casework and studies on population genetics.

Y-23 mediated genetic data analysis of endogamous Brahmin population of Rajasthan, India.

India's largest state Rajasthan is known for its variable population groups including castes, communities and tribes. In the present article, Y-STR polymorphisms of hundred unrelated healthy male volunteers from the Brahmin population of Rajasthan, India were investigated using the Powerplex® Y-23 PCR amplification kit. Total 94 distinct haplotypes were obtained out of them 93 were singletons. Haplotype Diversity (HD) and Discrimination Capacity (DC) for the population were 0.644 and 0.9894 respectively. The Intra-population relationship between the present population data and other reported Indian populations was examined through Multidimensional Scaling (MDS) Plot, which shows the Brahmin population of Rajasthan lies in a cluster with the Brahmin populations of Haryana and Maharashtra. Data generated with 23 Y-STR markers is submitted on Y chromosome haplotype reference database (YHRD) (yhrd.org) and it will robust the forensic database of the Rajasthan population of India.

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.

Key genetic and forensic attributes of Uttarakhand population of India.

The present study aimed to investigate the genetic and forensic attributes of Uttarakhand population based on 20 autosomal STR loci implemented in PowerPlex® 21 System. Results unveiled 219 alleles ranging from 6 (TH01, TPOX) to 20 (Penta E). Forensic analysis revealed locus Penta E as the most polymorphic and discriminative loci, whereas genetic analysis indicated presence of higher heterozygosity within population. Phylogenetic analysis indicated higher genetic affinity of Uttarakhand population with the neighbouring Himachal Pradesh population. Overall, the study indicates that the PowerPlex® 21 System is suitable for genetic and forensic analysis in Uttarakhand population.

Applicability of human-specific STR systems, GlobalFiler™ PCR Amplification Kit, Investigator 24plex QS Kit, and PowerPlex® Fusion 6C in chimpanzee (Pan troglodytes).

OBJECTIVES: Human identification systems based on STRs are widely used in human population genetics and forensic analysis. This study aimed to validate the cross-reactivity of three widely known human-specific STR identification systems i.e. GlobalFiler™ PCR Amplification Kit, Investigator 24plex QS Kit, and PowerPlex® Fusion 6C in chimpanzee. RESULTS: The present study revealed the successful amplification of 18 loci using GlobalFiler™ PCR Amplification Kit, 18 loci using Investigator 24plex QS Kit, and 20 loci using PowerPlex® Fusion 6C system. The marker Amelogenin (AMEL) showed differential allele size between male and female revealing the gender identity of chimpanzees and thus validates their application concerning forensic examination, population estimation, and genetic analysis.

Genetic evidence of shared ancestry among diverse ethno-linguistic human populations of Himachal Pradesh.

In Indian montane system, human populations often exhibit an unparallel social organization where inter-caste marriages are still not common. This attribute affects the demography and population genetic structure of the resident populations. Further, human populations residing in the mountains in India are poorly studied for their genetic make-up and allele distribution patterns. In the present study, we genotyped 594 unrelated individuals using PowerPlex® 21 System (Promega, USA) from eight different populations belonging to 12 districts of Himachal Pradesh which differed in ethnicity, language, geography and social organization. Altogether, we obtained 1415 alleles with a mean of 8.84 ± 0.26 alleles per locus and 0.80 mean observed heterozygosity. Locus Penta E showed the highest combined power of discrimination and was found most informative for forensic purposes. Interestingly, phylogenetic analysis grouped the populations of Rajputs, Scheduled castes and Brahmins into one cluster, which indicated a deep genetic admixture in the ancestral populations. This study documents the first-ever report on the population genetic assignment of various castes in Himachal Pradesh and unveils the facts of cryptic gene flow among the diverse castes in the northern hilly state of India. Our results showed a genetic relationship among the various ethno-linguistically diverse populations of India.

Genetic diversity of X-STR markers in Jat Sikh population of Punjab, India and its comparison with other 39 global populations.

BACKGROUND: The Jat Sikh population is the largest endogamous group of Punjab, a state in north-west India, and has not yet been explored for genetic polymorphism based on X-STR genetic markers. In India, which is the second most populous country in the world, only two population studies based on X-STR markers have been reported so far. AIM: To explore the genetic diversity of 12 X chromosomal STR genetic markers in the Jat Sikh population of Punjab and expand the X-STR polymorphism database. SUBJECTS AND METHODS: In this study, a total of 200 Jat Sikh individuals (100 males and 100 females) residing in Punjab were investigated for 12 X-STR markers using the Investigator Argus X-12 QS Kit. RESULTS: The highest power of discrimination (PD) in females (PDf) and males (PDm) was observed to be 0.965 (DXS10135) and 0.929 (DXS10135 and DXS10148), respectively. DXS10135 was found to be the most polymorphic and discriminating locus among all the studied loci in both males and females with highest values of power of discrimination (PD) and polymorphic information content (PIC) as well. CONCLUSION: Overall, the studied markers of the Argus 12 X-STR kit provide high polymorphic information which may prove to be an important tool in resolving issues such as missing person identification, incest, immigration disputes, kinship analysis and genealogical studies. The dataset obtained from this study will add to the present database of X-STRs.

Genetic polymorphism of 20 autosomal short tandem repeats (STRs) in Himachal Pradesh population, India.

Genetic diversity indices and forensic parameters at 20 autosomal short tandem repeat (STR) loci were evaluated in 233 unrelated individuals of Himachal Pradesh, India. In total, 255 alleles were observed with an average of 11.25 ± 0.88 alleles per locus. None of the loci deviated from Hardy Weinberg Equilibrium. The most polymorphic and discriminative locus was Penta E. The combined power of exclusion and the combined power of discrimination were found to be 0.99 and 1. Additionally, the genetic relationship of the Himachal Pradesh population with the populations of the neighboring state was also investigated. All the STR loci were found polymorphic, and the select panel of STRs was found suitable for population genetic studies and forensic analysis.

Assessment of genetic polymorphism at 20 autosomal STR loci in scheduled caste population of Himachal Pradesh.

In this study, we evaluated the genetic diversity indices and forensic parameters of scheduled caste population of Himachal Pradesh, India, at 20 autosomal STR loci. Altogether, 233 alleles were observed with an average of 11.65 ± 0.88 alleles per locus. In exception of locus D13S317, no loci deviated from Hardy Weinberg equilibrium. Locus Penta E was found to be the most polymorphic and discriminative loci. The combined power of discrimination and the combined power of exclusion were 1 and 0.999. Further, we established the phylogenetic relationship between the scheduled caste population of Himachal Pradesh and 14 other populations of India. We found 20 autosomal STR loci used in the present study were polymorphic and can be used in population genetic studies and forensic related case works.

Evaluation of genetic polymorphism at 20 autosomal STR loci in Rajput population of Himachal Pradesh, India.

In the present study, genetic diversity indices and forensic parameters of Rajput population of Himachal Pradesh, India, were obtained at 20 autosomal short tandem repeat (STR) loci included in the PowerPlex® 21 System (Promega, USA). With 227 alleles and 11.35 ± 0.89 alleles per locus, none of the locus deviated from the Hardy-Weinberg equilibrium (HWE). The most polymorphic and discriminative locus was Penta E. Phylogenetic tree showed high genetic affinity of Rajput population of Himachal Pradesh with Rajputs of Madhya Pradesh and Yadav of Bihar. Overall, the tested 20 autosomal STRs were polymorphic in Rajput population and found suitable in forensic casework and population genetic studies.

Cross-species validation of human specific STR system, SureID® 21G and SureID® 23comp (Health Gene Technologies) in Chimpanzee (Pan Troglodytes).

OBJECTIVES: The human specific commercially available STRs system are often not tested in non human primates for their cross applicability. The aim of this study is to test Cross-species validation of two commercially available human specific STR kits i.e. SureID® 21G and SureID® 23comp (Health Gene Technologies) for their positive application in Chimpanzee (Pan troglodytes). RESULT: In SureID® 21G, 19 loci amplified and while 20 loci amplified in SureID® 23comp. All the amplified loci in both STR kits were found polymorphic and the locus Amelogenin showed differential banding patterns between male and female revealing their known gender. The present study validates the applicability of these human specific STR kits in Chimpanzee that can be used in forensics analysis, paternity testing and population genetic studies.

Functional validation of human-specific PowerPlex® 21 System (Promega, USA) in chimpanzee (Pan troglodytes).

OBJECTIVE: This study was aimed to test the PowerPlex® 21 System (Promega, USA), used for human identification applications for its positive cross-species applicability in Chimpanzees (Pan troglodytes) in order to identify heterologous STRs which can be used for individual identification, paternity testing, relatedness establishment and reconstruction of pedigrees and studbook records for captive and wild chimpanzee breeding populations. RESULTS: Of 21 STRs in PowerPlex® 21 System (Promega, USA), 19 loci amplified and found to be polymorphic. Locus Aml showed differential banding patterns in males and females similar to those seen for humans and correctly assigned sexes of known identity. Altogether, 58 different alleles were found with an average 3.05 ± 0.28 alleles per locus. Mean observed (Ho), and expected heterozygosity (He) were 0.93 ± 0.03 and 0.52 ± 0.05, respectively.

Identification of a forensic case using microscopy and forensically informative nucleotide sequencing (FINS): a case study of small Indian civet (Viverricula indica).

The exhibits obtained in wildlife offence cases quite often present a challenging situation for the forensic expert. The selection of proper approach for analysis is vital for a successful analysis. A generalised forensic analysis approach should proceed from the use of non-destructive techniques (morphological and microscopic examination) to partially destructive and finally destructive techniques (DNA analysis). The findings of non-destructive techniques may sometime be inconclusive but they definitely help in steering further forensic analysis in a proper direction. We describe a recent case where a very small dried skin piece (<0.05 mg) with just one small trimmed guard hair (0.4 cm) on it was received for species identification. The single guard hair was examined microscopically to get an indication of the type of species. We also describe the extraction procedure with a lower amount of sample, using an automated extraction method (Qiagen Biorobot EZ1) and PCR amplification of three mitochondrial genes (16s rRNA, 12s rRNA and cytochrome b) for species identification. Microscopic examination of the single hair indicated a viverrid species but the initial DNA analysis with 16s rRNA (through NCBI BLAST) showed the highest homology (93%) with a hyaenid species (Hyaena hyaena). However, further DNA analysis based on 12s rRNA and cytochrome b gene proved that the species was indeed a viverrid i.e. Viverricula indica (small Indian civet). The highest homology shown with a Hyaenid species by the 16s rRNA sequence from the case sample was due to lack of a 16s rRNA sequence for Viverricula indica in the NCBI data base. The case highlights the importance of morphological and microscopic examinations in wildlife offence cases. With respect to DNA extraction technology we found that automatic extraction method of Biorobot EZ1 (Qiagen) is quite useful with less amount of sample (much below recommended amount).

Identification of mongoose (genus: Herpestes) species from hair through band pattern studies using discriminate functional analysis (DFA) and microscopic examination.

India is home to seven species of mongoose (Herpestes sp). Mongooses are being poached primarily for their hair, which is used in the production of painting and shaving brushes. Prior to September 2002, mongooses were listed under Schedule-IV of the Wildlife (Protection) Act 1972 (India). Indiscriminate poaching of the mongoose created an immediate threat to their survival and hence mongooses have now been placed under Schedule-II of the Wildlife (Protection) Act-1972 (India). In order to convict a person under this legislation, species identification of case related samples is necessary. Four species of mongoose i.e. H. edwardsii, H. smithii, H. palustris and H. urva were characterised by performing discriminate functional analysis (DFA) on measurements of their dorsal guard hair banding pattern and by microscopic hair characteristics (Cuticular, medullar and cross section). It was possible to distinguish between the four species studied, based on both these methods.

Microscopic hair characteristics of a few bovid species listed under Schedule-I of Wildlife (Protection) Act 1972 of India.

Dorsal guard hairs of 10 bovid species of India, listed under Schedule-I of Wildlife (Protection) Act 1972 of India and some of them quite frequently encountered in illegal trade, were studied using light microscopy. We discuss characteristics including colour, hair thickness, cuticular pattern, medulla pattern, medulla index, cross-section and scale count index for species characterisation/identification to deal wildlife offence cases. Although some species could be identified very easily based on one or few microscopic hair characteristics, however there were some overlaps of few hair characteristics among some species. Species like Pantholops hodgsonii could be characterised most easily, just based on cuticular pattern and similarly Capricornis sumatraensis could be characterised simply by medulla pattern. For species showing overlaps in some of the microscopic hair characteristics, a combination of all the characteristics was most useful. We suggest the use of maximum number of parameters for distinguishing sympatric and closely related species. In addition to wild species, hair characteristics of three domestic species have been examined and compared with the selected wild species.

Dealing wildlife offences in India: role of the hair as physical evidence.

India is known for its rich biodiversity, with a wide variety of wild floral and faunal species. This wildlife treasure of ours faces the threat of extinction due to rampant poaching and illegal trade. With most of the wildlife offence cases related to mammals having hair as physical evidence, it becomes imperative to use this evidence in the best possible way for wildlife crime investigation. We discuss the value of hair evidence with special reference to species characterization/identification using microscopic hair characteristics, keratin patterns, and mitochondrial DNA typing. The relevance of the techniques with respect to Indian scenario is specially taken care of and microscopic hair characteristics of one of the highly endangered species along with its keratin pattern are described. Finally, the use of mitochondrial DNA for species identification is also discussed.

Identification of shed or plucked origin of Indian peafowl (Pavo cristatus) tail feathers: preliminary findings.

Indian Peafowl (Pavo cristatus) tail covert feathers were studied to investigate the difference between shed and plucked feathers in the context of wildlife offence cases involving the killing of the Indian national bird for the purpose of plucking feathers. Plucked feathers were distinguished from shed feathers by examining their roots under low magnification of a stereoscopic microscope. A chemical test to show the presence of blood on the roots of plucked feathers was used to corroborate the plucked origin of feathers.