Advancements in differentiation between sperm cells and epithelial cells for efficient forensic DNA analysis in sexual assault cases.

Most of the sexual assault casework samples are of mixed sources. Forensic DNA laboratories are always in the requirement of a precise technique for the efficient separation of sperm and non-sperm DNA from mixed samples. Since the introduction of the differential extraction technique in 1985, it has seen significant advancements in the form of either chemicals used or modification of incubation times. Several automated and semi-automated techniques have also adopted the fundamentals of conventional differential extraction techniques. However, lengthy incubation, several manual steps, and carryover over non-sperm material in sperm fraction are some of the major limitations of this technique. Advanced cell separation techniques have shown huge promise in separating sperm cells from a mixture based on their size, shape, composition, and membrane structure and antigens present on sperm membranes. Such advanced techniques such as DEParray, ADE, FACS, LCM, HOT and their respective pros and cons have been discussed in this article. As current-day forensic techniques should be as per the line of Olympic slogan i.e., faster, higher, stronger, the advanced cell separation techniques show a huge potential to be implemented in the casework samples.

Evaluation of face masks as a valuable forensic DNA evidence in the post-COVID era.

After the onset of COVID-19 pandemic, a sharp surge in the usage of the face-masks throughout the globe has been observed. Pre-experiment survey of 252 individuals indicated a higher use of cotton-make masks (41%), followed by N-95 make (31%), and surgical disposable masks (26%). It was also further revealed that a higher fraction of individuals wear a face-mask more than 3 times (37%) before its disposal. In order to assess the potential usability of different mask types as forensic DNA evidence, a study was conducted on 50 healthy individuals. DNA content of different fractions such as the portion of mask covering the mouth region and the ear-piece showed a good source of host DNA. Though no statistically significant difference (P < 0.05) was found in the DNA quantity obtained from different face mask types, an increasing trend was obtained in the order: cloth make type (7.031 ± 0.31 ng), N-95 make (4.711 ± 0.15 ng), and surgical disposable type (2.17 ± 0.13 ng). The time of wearing of a face-mask showed a positive correlation with the yield of DNA irrespective of the face-mask type used. Samples retrieved from both the portions covering the mouth area and the ear-piece showed a good source of genomic DNA yielding an average of 4.82 ± 0.11 ng and 4.44 ± 0.10 ng of DNA, respectively. Irrespective of the face-mask types, number of reuse, and the portion of the mask, 66.66-96.11% of samples showed a complete autosomal STR DNA profile. This suggests that if a face-mask is found at the crime scene, it should be collected and preserved as a potential source of DNA evidence for routine forensic DNA analysis.

Geolocation prediction from STR genotyping: a pilot study in five geographically distinct global populations.

BACKGROUND: Traditional CE-based STR profiles are highly useful for the purpose of individualisation. However, they do not give any additional information without the presence of the reference sample for comparison. AIM: To assess the usability of STR-based genotypes for the prediction of an individual's geolocation. SUBJECTS AND METHODS: Genotype data from five geographically distinct populations, i.e. Caucasian, Hispanic, Asian, Estonian, and Bahrainian, were collected from the published literature. RESULTS: A significant difference (p < 0.05) in the observed genotypes was found between these populations. D1S1656 and SE33 showed substantial differences in their genotype frequencies across the tested populations. SE33, D12S391, D21S11, D19S433, D18S51, and D1S1656 were found to have the highest occurrence of "unique genotype's" in different populations. In addition, D12S391 and D13S317 exhibited distinct population-specific "most frequent genotypes." CONCLUSIONS: Three different prediction models have been proposed for genotype to geolocation prediction, i.e. (i) use of unique genotypes of a population, (ii) use of the most frequent genotype, and (iii) a combinatorial approach of unique and most frequent genotypes. These models could aid the investigating agencies in cases where no reference sample is available for comparison of the profile.

Microbial community signatures for estimation of postmortem time intervals.

The human body provides a complex ecosystem for symbiotic habitation of a huge number of microorganisms. These commensal microorganisms provide a huge benefit to the living host by acting against many deadly infections. Once the host dies, many changes in the complex ecosystem of the human body take place. The personalized microbes of a human body undergo successional change as many exogenous microbes attack the nutrient-rich cadaver after death. The succession pattern change of microbes in human cadaver allows postulating different models for estimation of Postmortem time interval (PMI). Estimation of PMI has a broad prospect from the criminal investigation point of view. Though many techniques are being used nowadays to estimate PMI, all of them have their pros and cons. With the advent of advanced molecular biological techniques, studies on the thanatomicrobiome of a human cadaver have gained pace and provide a superior alternative for conventional methods of PMI estimation. This chapter summarizes the recent advancements in the changes in signature microflora postmortem with change in human microenvironment to postulate a consensus model for estimation of PMI.

Forensic characterization of 124 SNPs in the central Indian population using precision ID Identity Panel through next-generation sequencing.

With the advent of next-generation sequencing technology, SNP markers are being explored as a useful alternative to conventional capillary electrophoresis-based STR typing. Low mutation rate and short-sized amplicons are added advantages of SNP markers over the STRs. However, to achieve a sufficient level of discrimination among individuals, a higher number of SNPs need to be characterized simultaneously. Hence, the NGS technique is highly useful to analyze a sufficiently higher number of SNPs simultaneously. Though the technique is in its nascent stage, an attempt has been made to assess its usability in the central Indian population by analyzing 124 SNPs (90 autosomal and 34 Y-chromosome) in 95 individuals. Various quality parameters such as locus balance, locus strand balance, heterozygosity balance, and noise level showed a good quality sequence obtained from the Ion GeneStudio S5 instrument. Obtained frequency of SNP alleles ranged from 0.001 to 0.377 in autosomal SNPs. rs9951171 was found to be the most informative SNP in the studied population with the highest PD and lowest MP value. The cumulative MP of 90 SNPs was found to be 4.76698 × 10-37. Analysis of 34 Y-chromosome SNPs reveals 11 unique haplogroups in 54 male samples with R1a1 as the most frequent haplogroup found in 22.22% of samples. Interpopulation comparison by FST analysis, PCA plot, and STRUCTURE analysis showed genetic stratification of the studied population suggesting the utility of SNP markers present in the Precision ID Identity Panel for forensic demands of the Indian population.

CRISPR-CasB technology in forensic DNA analysis: challenges and solutions.

CRISPR-Cas technology has revolutionized the field of biotechnology with its precise therapeutic use from genetic as well as infectious diseases point of view. This technology is rapidly evolving to single tool enabling site-directed cut in the genome and highly specific activation or inhibition of gene expression or the exchange of single bases. Besides clinical applications, CRISPR-Cas technology has also shown promising use in the field of forensic DNA analysis. Enrichment of targeted genetic marker for identification followed by sequencing and non-PCR-dependent technique ensures the use of CRISPR-Cas technology in challenging forensic biological samples. The use of this advanced technology is also deemed helpful in mixed profile attribution, mostly in LCN contributors and the generation of a useful DNA profile in degraded samples. Besides its useful applications in forensic DNA analysis, CRISPR-Cas technology poses a huge threat from the generation of ghost DNA profiles by modification/alteration of target genetic markers. Forensic DNA analysts should carry out analysis of additional markers such as non-CODIS markers, Y-, X-chromosome markers, and mitochondrial DNA sequencing in a suspected ghost DNA profile case. KEY POINTS: • CRISPR-Cas9 technique is useful in analyzing LCN, mixed and degraded samples • Alteration of DNA using this technique can lead to generation of ghost DNA profiles • Alternative genetic markers and methylation pattern may detect a ghost DNA profile.

Forensic characterization and genetic evaluation in the Central Indian population using 27 Y-STRs.

INTRODUCTION: Forensic characterization and genetic evaluation study in the 539 randomly selected unrelated adult healthy individuals belonging to the Central Indian population was undertaken. METHODS: The study was performed using a multiplex of 27 Y-STRs incoporated in Yfiler™ Plus multiplex kit. RESULTS: Out of 539 samples, 6 samples were observed for large deletion and tri-allelic patterns, which were removed from the analysis, and out of 533 samples, a total of 507 haplotypes were found, and out of these haplotypes, 482 unique haplotypes were found in this piece of work. The forensically important parameters, i.e., gene diversity (GD) and discrimination capacity (DC), were found to be 0.669 and 0.951, respectively, for the tested Y STR loci. The genetic data of this study will enrich the Y STR data bank and being used as a potential tool for forensic DNA and various genetic studies.

Genetic diversity and forensic characterization of 15 autosomal microsatellite markers in the North-east Indian Tripuri population.

This study was conducted to explore the genomic diversity and forensic characterization of 15 autosomal microsatellite markers in the East Indian Tripuri population. In the studied population, we observed 158 different alleles with the average 10.53 alleles per locus. The locus D2S1338 (PIC= 0.862) was found to be the most polymorphic wheres locus TPOX (PIC= 0.647) as the least polymorphic, among all the studied loci. The locus FGA was found with the highest number of effective alleles (Nall=19) whereas locus TH01 showed least number of effective alleles (Nall=6). The cumulative values for matching probability (CPm), power of discrimination (CPD), power of exclusion CPE), and paternity index (CPI) were found as 1.94×10-18, 1, 0.999998, and 4.8×105 respectively. The studied population showed genetic closeness with the Gorkha population. In neighbor-joining tree, Tripura population pooled with the population of Nepal and Tibet. The genetic data obtained from the present study will not only enrich the existing autosomal STR database but will also be useful for forensic DNA application and genealogical studies.

Useful autosomal STR marker sets for forensic and paternity applications in the Central Indian population.

BACKGROUND: Many countries have developed their core set of STR loci for forensic application and database generation, which India lacks. AIM: To assess the usefulness of various combinations of autosomal STR marker sets for their superior use in the central Indian population for forensic and paternity applications. SUBJECTS AND METHODS: 19 STR marker sets were analysed on 200 central Indian populations and 20 paternity cases to assess their usefulness. RESULTS: Two marker sets each comprising 19 STR markers are found to be superior to 20 expanded CODIS loci in the studied population. These marker sets also showed their effectiveness in 20 paternity cases having CPI values of 7.62 × 1011 and 7.16 × 1011. Three non-CODIS STR markers Penta E, Penta D, and SE33 showed amplification in 50 challenging samples with >0.80 heterozygosity. CONCLUSION: Population-specific STR marker sets are useful in forensic and paternity applications, as well as database generation, and it is envisioned that Penta E, Penta D, and SE33 markers will be included in the list of core STR loci in the central Indian population.

Prevalence and characterisation of size and sequence-based microvariant alleles at nine autosomal STR markers in the Central Indian population.

BACKGROUND: Though microvariant alleles are widely reported in global populations, they are not well characterised to date. AIM: To study the prevalence and characterisation of size and sequence-based microvariant alleles. SUBJECTS AND METHODS: Next Generation Sequencing (NGS) was used to sequence microvariant alleles at nine autosomal STR markers in 138 samples. RESULTS: After sequencing 31 STR markers using Precision ID GlobalFilerTM NGS STR panel v2, only nine markers, i.e. D12S391, D19S433, D1S1656, D21S11, D2S441, D7S820, FGA, Penta D, and TH01 showed the prevalence of microvariant alleles. Occurrence of microvariant alleles was positively correlated with Total Possible Alleles (p < 0.005), Power of Discrimination (p < 0.01), Polymorphic Information Content (p < 0.01), and Power of Exclusion (p < 0.05) and negatively correlated with the Matching Probability (p < 0.01). The average allele frequency of the microvariant alleles was found to be significantly less than the allele frequency value of the complete alleles (p = 0.88). Further, sequencing of these microvariant alleles reveals the deletion of nucleotides from the start, end, or middle of the repeat unit is responsible for the generation of a microvariant allele. CONCLUSIONS: Prevalence of microvariant alleles is rare in nature and is limited to 9 STR loci out of 31 STR loci tested in the central Indian population. The occurrence of microvariant alleles in a locus increases its forensic and paternity application.

Evaluation of diallelic STR markers with inter-population allelic database for their usefulness in paternity trios in the Central Indian population.

BACKGROUND: Most of the forensic DNA laboratories have migrated to new generation STR kits of 6 dye chemistry with more than 20 autosomal STRs. The population-specific databases of such STR markers are lacking in many regions. AIM: To evaluate the effect of the inter-population database in 100 paternity trios with no inconsistencies at 23 STRs. SUBJECTS AND METHODS: 100 paternity trios were evaluated considering inter-population allelic frequency values for calculation of Combined Paternity Index (CPI) and Probability of Paternity (POP). RESULTS: No significant variation (p < 0.05) among the allele frequencies at the interpopulation level was observed. The number of obligate alleles and the likelihood of transferring obligate alleles from the putative father showed a positive correlation (p < 0.005) with Power of Discrimination (PD), Polymorphic Information Content (PIC), Power of Exclusion (PE), Paternity Index (PI), Observed and Expected Heterozygosity (Ho and He), and a statistically significant negative correlation (p < 0.005) with Matching Probability (Pm). The average Combined Paternity Index (CPI) and Probability of Paternity (POP) did not show any statistically significant difference (p < 0.05) at the interpopulation level. CONCLUSION: The allelic database showed no effect on the CPI and POP in 100 paternity trios. This suggests no urgent need for using population-specific databases for statistical evaluation of paternity trios without inconsistencies.

Genomic portrait of Odisha, India drawn by using 21 autosomal STR markers.

In order to find out the genetic diversity in the eastern Indian population of Odisha consisting of various linguistic and ethnic groups, we undertook a study on 508 unrelated healthy individuals belonging to Odisha, India. We assessed genetic variation and compared the data with published literature of Indian population consisting of different ethnic groups from different geographical areas using 21 autosomal STR markers. The most polymorphic and discriminatory STR locus in the studied population was found to be SE33 with the calculated values of 0.94 and 0.991 respectively for both the parameters. The combined power of discrimination (CPD) and combined power of exclusion (CPE) were found to be 1 and 0.999999999704865 respectively. The combined probability of match (CPm) and combined paternity index (CPI) for all 21 autosomal STR loci were found to be 8.01 × 10-26 and 3.45 × 109 respectively. Though, the studied eastern Indian population of Odisha shared its closest genetic affinity with nearest Indian geographical regions, i.e., the population of Jharkhand which is geographically located in eastern India as well as the Central Indian population.

Forensic effectiveness and genetic distribution of 23 autosomal STRs included in Verifiler PlusTM multiplex in a population sample from Madhya Pradesh, India.

We report here the first ever global study on genetic polymorphism using a Verifiler PlusTM autosomal STR multiplex system. The study evaluated genetic characteristics of 23 autosomal STRs in 200 unrelated residents of Guna district of Madhya Pradesh, India. Allele frequencies and forensic parameters are reported. Population comparison analysis was also performed using NJ tree and PCA plot. Penta E marker showed highest power of discrimination (0.938) among all 23 studied markers. The study also presents the first ever global forensic assessment in Indian population on D6S1043 marker (PD 0.937). The results demonstrated that all the 23 markers were highly polymorphic and the Verifiler PlusTM kit is suitable for forensic purposes in Indian population.

Thanatomicrobiome and epinecrotic community signatures for estimation of post-mortem time interval in human cadaver.

Estimation of post-mortem time interval (PMI) is a key parameter in the forensic investigation which poses a huge challenge to the medico-legal experts. The succession of microbes within different parts of the human body after death has shown huge potential in the determination of PMI. Human body harbors trillions of microorganisms as commensals. With the death of an individual when biological functions are stopped, these microorganisms behave contrarily along with the invasion of degrading microbes from the environment. Human cadaver becomes a rich source of nutrients due to autolysis of cells, which attracts various invading microorganisms as well as macroorganisms. At different stages of degradation, the succession of microorganisms differs significantly which can be explored for accurate PMI estimation. With the advent of microbial genomics technique and reduction in the cost of DNA sequencing, thanatomicrobiome and epinecrotic community analysis have gained huge attention in PMI estimation. The article summarizes different sources of microorganisms in a human cadaver, their succession pattern, and analytical techniques for application in the field of microbial forensics. KEY POINTS: • Thanatomicrobiome and epinecrotic microbiome develop in postmortem human body. • Lack of metabolic, immune, neuroendocrine systems facilitate microbial succession. • Analysis of postmortem microbial communities predicts accurate PMI.

Forensic genetic analysis of population of Madhya Pradesh with PowerPlex Fusion 6C™ Multiplex System.

Performance of PowerPlex Fusion 6C kit (PP F6C) was assessed in 374 unrelated individuals belonging to Madhya Pradesh, an Indian state. The study evaluated the forensic parameters for the loci included in PP F6C Multiplex System. The combined discrimination power (CPD) and combined exclusion power (CPE) were 1 and 0.999999995, respectively, for all 23 autosomal STR loci. SE33 showed the greatest power of discrimination (0.990) in the studied population, whereas TPOX showed the lowest (0.843). The availability of three Y-STR loci in the Multiplex System is suitable for assessing male contribution and amelogenin deletion in a single Multiplex PCR simultaneously. The study also presents the first global report on polymorphism in the Indian population on SE 33 autosomal STR loci and PP Fusion 6C Multiplex System. The results revealed that the studied STR Multiplex System is highly polymorphic and suitable for forensic purposes.

PCR inhibitors and facilitators - Their role in forensic DNA analysis.

Since its inception, DNA typing technology has been practiced as a robust tool in criminal investigations. Experts usually utilize STR profiles to identify and individualize the suspect. However, mtDNA and Y STR analyses are also considered in some sample-limiting conditions. Based on DNA profiles thus generated, forensic scientists often opine the results as Inclusion, exclusion, and inconclusive. Inclusion and exclusion were defined as concordant results; the inconclusive opinions create problems in conferring justice in a trial- since nothing concrete can be interpreted from the profile generated. The presence of inhibitor molecules in the sample is the primary factor behind these indefinite results. Recently, researchers have been emphasizing studying the sources of PCR inhibitors and their mechanism of inhibition. Furthermore, several mitigation strategies- to facilitate the DNA amplification reaction -have now found their place in the routine DNA typing assays with compromised biological samples. The present review paper attempts to provide a comprehensive review of PCR inhibitors, their source, mechanism of inhibition, and ways to mitigate their effect using PCR facilitators.

Sequence variations, flanking region mutations, and allele frequency at 31 autosomal STRs in the central Indian population by next generation sequencing (NGS).

Capillary electrophoresis-based analysis does not reflect the exact allele number variation at the STR loci due to the non-availability of the data on sequence variation in the repeat region and the SNPs in flanking regions. Herein, this study reports the length-based and sequence-based allelic data of 138 central Indian individuals at 31 autosomal STR loci by NGS. The sequence data at each allele was compared to the reference hg19 sequence. The length-based allelic results were found in concordance with the CE-based results. 20 out of 31 autosomal STR loci showed an increase in the number of alleles by the presence of sequence variation and/or SNPs in the flanking regions. The highest gain in the heterozygosity and allele numbers was observed in D5S2800, D1S1656, D16S539, D5S818, and vWA. rs25768 (A/G) at D5S818 was found to be the most frequent SNP in the studied population. Allele no. 15 of D3S1358, allele no. 19 of D2S1338, and allele no. 22 of D12S391 showed 5 isoalleles each with the same size and with different intervening sequences. Length-based determination of the alleles showed Penta E to be the most useful marker in the central Indian population among 31 STRs studied; however, sequence-based analysis advocated D2S1338 to be the most useful marker in terms of various forensic parameters. Population genetics analysis showed a shared genetic ancestry of the studied population with other Indian populations. This first-ever study to the best of our knowledge on sequence-based STR analysis in the central Indian population is expected to prove the use of NGS in forensic case-work and in forensic DNA laboratories.

Microbial Degradation of Forensic Samples of Biological Origin: Potential Threat to Human DNA Typing.

Forensic biology is a sub-discipline of biological science with an amalgam of other branches of science used in the criminal justice system. Any nucleated cell/tissue harbouring DNA, either live or dead, can be used as forensic exhibits, a source of investigation through DNA typing. These biological materials of human origin are rich source of proteins, carbohydrates, lipids, trace elements as well as water and, thus, provide a virtuous milieu for the growth of microbes. The obstinate microbial growth augments the degradation process and is amplified with the passage of time and improper storage of the biological materials. Degradation of these biological materials carriages a huge challenge in the downstream processes of forensic DNA typing technique, such as short tandem repeats (STR) DNA typing. Microbial degradation yields improper or no PCR amplification, heterozygous peak imbalance, DNA contamination from non-human sources, degradation of DNA by microbial by-products, etc. Consequently, the most precise STR DNA typing technique is nullified and definite opinion can be hardly given with degraded forensic exhibits. Thus, suitable precautionary measures should be taken for proper storage and processing of the biological exhibits to minimize their decaying process by micro-organisms.

Bacillus and biopolymer: Prospects and challenges.

The microbially derived polyhydroxyalkanoates biopolymers could impact the global climate scenario by replacing the conventional non-degradable, petrochemical-based polymer. The biogenesis, characterization and properties of PHAs by Bacillus species using renewable substrates have been elaborated by many for their wide applications. On the other hand Bacillus species are advantageous over other bacteria due to their abundance even in extreme ecological conditions, higher growth rates even on cheap substrates, higher PHAs production ability, and the ease of extracting the PHAs. Bacillus species possess hydrolytic enzymes that can be exploited for economical PHAs production. This review summarizes the recent trends in both non-growth and growth associated PHAs production by Bacillus species which may provide direction leading to future research towards this growing quest for biodegradable plastics, one more critical step ahead towards sustainable development.