THE APPLICATION OF CELL-FREE FETAL DNA (cff-DNA) AND SIBLINGS DNA METHODS IN THE PROCESS OF PATERNITY TEST THROUGH CODIS STR LOCI (CSF1PO, THO1, TPOX, AND vWA)

Background: The non-invasive cff-DNA and siblings DNA methods are the latest breakthroughs in the forensic identification process. The use of cff-DNA and siblings DNA as non-invasive techniques in the forensic identification process has, hitherto, not been widely proven. Methods and Materials: This was an analytic observational study. The sample of this study consisted of peripheral blood of women in the second trimester of pregnancy and their two biological children. The kinship analysis was carried out through siblings' DNA and cff-DNA from the mothers through CODIS STR loci (CSF1PO, THO1, TPOX, and vWA). Results: The means of allele sharing between full siblings in loci CSF1PO, THO1, TPOX, and vWA were 0 (13.75%), 1 (44.75%), and 2 (41.50%). The allele sharing found in the study is in line with the one in previous research conducted by Wenk (1998) and the theory proposed by O'Connor (2011), indicating that one allele sharing dominates, contrasting with the finding of previous research conducted by Sosiawan (2020) revealing that 2-allele sharing was more superior. The variation is caused by the ethnicity having a different genetic contribution among the population. The variation can be attributed to historical and demographical processes leading to genetic drift. Conclusion: The mean of SI in 1 allele sharing in CODIS STR loci (CSF1PO, THO1, TPOX, and vWA) has the highest value of 44.5%. The use of cff-DNA of pregnant women as one of the non-invasive techniques can serve as an alternative material in a paternity test.

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.

Secondary DNA transfer on denim using a human blood analogue

DNA quantification technology has increased in accuracy and sensitivity, now allowing for detection and profiling of trace DNA. Secondary DNA transfer occurs when DNA is deposited via an intermediary source (e.g. clothing, tools, utensils). Multiple courtrooms have now seen secondary transfer introduced as an explanation for DNA being present at a crime scene, but sparse experimental studies mean expert opinions are often limited. Here, we used bovine blood and indigo denim substrates to quantify the amount of secondary DNA transfer and quality of STRs under three different physical contact scenarios: passive, pressure, and friction. We showed that the DNA transfer was highest under a friction scenario, followed by pressure and passive treatments. The STR profiles showed a similar, albeit less pronounced trend, with correctly scored alleles and genotype completeness being highest under a friction scenario, followed by pressure and passive. DNA on the primary substrate showed a decrease in concentration and genotype completeness both immediately and at 24 hours, suggestive of a loss of DNA during the primary transfer. The majority of secondary transfer samples amplified less than 50% of STR loci regardless of contact type. This study showed that while DNA transfer is common between denim, this is not manifested in full STR profiles. We discuss the possible technical solutions to partial profiles from trace DNA, and more broadly the ubiquity of secondary DNA transfer.

Forensic Application Evaluation of a Novel Canine STR System in Pembroke Welsh Corgi and Shiba Inu Groups

Aim. To evaluate the forensic application values of 19 autosomal short tandem repeat (STR) loci in canines. Methods. The 19 STR loci in two canine groups (Pembroke Welsh Corgis, n = 200 ; Shiba Inus, n = 175 ) were analysed by the capillary electrophoresis platform. The allele frequencies and forensic parameters were calculated, and the genetic relationships between these two canine groups and a previously reported Labrador group were analysed. Results. These two canine groups conformed to the Hardy-Weinberg equilibrium at all STRs except for locus VGL3438 in the Pembroke Welsh Corgi group, and there was no linkage disequilibrium among pairwise loci at the 19 STRs. All STRs were polymorphic in the Pembroke Welsh Corgi and Shiba Inu groups, of which the locus C38 had the highest polymorphism. And it was found that the genetic relationship between the Pembroke Welsh Corgi and Labrador groups were closer in the three canine groups (Pembroke Welsh Corgi, Shiba Inu and Labrador). Conclusion. The 19 STR loci had high genetic polymorphisms and forensic application values in Pembroke Welsh Corgi and Shiba Inu groups.

The Mutational Dynamics of Short Tandem Repeats in Large, Multigenerational Families

Short tandem repeats (STRs) are tandemly repeated sequences of 1-6 bp motifs. STRs compose approximately 3% of the genome, and mutations at STR loci have been linked to dozens of human diseases including amyotrophic lateral sclerosis, Friedreich ataxia, Huntington disease, and fragile X syndrome. Improving our understanding of these mutations would increase our knowledge of the mutational dynamics of the genome and may uncover additional loci that contribute to disease. Here, to estimate the genome-wide pattern of mutations at STR loci, we analyzed blood-derived whole-genome sequencing data for 544 individuals from 29 three-generation CEPH pedigrees. These pedigrees contain both sets of grandparents, the parents, and an average of 9 grandchildren per family. Using HipSTR we identified de novo STR mutations in the 2nd generation of these pedigrees. Analyzing ~1.6 million STR loci, we estimate the empircal de novo STR mutation rate to be 5.24*10-5 mutations per locus per generation. We find that perfect repeats mutate ~2x more often than imperfect repeats. De novo STRs are significantly enriched in Alu elements (p < 2.2e-16). Approximately 30% of STR mutations occur within Alu elements, which compose only ~11% of the genome, and ~10% are found in LINE-1 insertions, which compose ~17% of the genome. Phasing these de novo mutations to the parent of origin shows that parental transmission biases vary among families. We estimate the average number of de novo genome-wide STR mutations per individual to be ~85, which is similar to the average number of observed de novo single nucleotide variants.

STRling: a k-mer counting approach that detects short tandem repeat expansions at known and novel loci

Expansions of short tandem repeats (STRs) cause dozens of rare Mendelian diseases. However, STR expansions, especially those arising from repeats not present in the reference genome, are challenging to detect from short-read sequencing data. Such "novel" STRs include new repeat units occurring at known STR loci, or entirely new STR loci where the sequence is absent from the reference genome. A primary cause of difficulty detecting STR expansions is that reads arising from STR expansions are frequently mismapped or unmapped. To address this challenge, we have developed STRling, a new STR detection algorithm that counts k-mers (short DNA sequences of length k) in DNA sequencing reads, to efficiently recover reads that inform the presence and size of STR expansions. As a result, STRling can call expansions at both known and novel STR loci. STRling has a sensitivity of 83% for 14 known STR disease loci, including the novel STRs that cause CANVAS and DBQD2. It is the first method to resolve the position of novel STR expansions to base pair accuracy. Such accuracy is essential to interpreting the consequence of each expansion. STRling has an estimated 0.078 false discovery rate for known pathogenic loci in unaffected individuals and a 0.20 false discovery rate for genome-wide loci in unaffected individuals when using variants called from long-read data as truth. STRling is fast, scalable on cloud computing, open-source, and freely available at https://github.com/quinlan-lab/STRling.