DNA extracted from biological forensic samples can be extremely fragmented and in low quantity. In such cases, chances of allelic dropouts are high in STR analysis because large DNA fragments with intact primer binding sites are required. One approach to analyzing highly degraded and limited DNA is SNP typing as single base polymorphisms are analyzed as opposed to the large repeat polymorphisms for STRs. However, primer extension assays used in conventional SNP typing methods can be applied to only a limited number of SNPs and require intact primer binding sites. On the other hand, new technologies such as massively parallel clonal sequencing allow for analysis of many SNPs across multiple samples (single source and mixed) simultaneously. Furthermore, a novel method of library preparation called probe capture has shown success in capturing and enriching targeted mitochondrial DNA fragments for massively parallel sequencing of whole mitochondrial genome. The goal of this project was to design and test a probe capture assay targeting forensically relevant nuclear SNPs for massively parallel sequencing of degraded and limited DNA samples, and mixed DNA samples.
To develop the custom probe panel, 451 SNPs were selected, including Identity Informative SNPs, Ancestry Informative SNPs, Phenotypically Informative SNPs, Lineage SNPs, Tri-allelic SNPs, Tetra-allelic SNPs, and Micro-haplotype SNPs. The custom assay was then tested for average depth of coverage per SNP, sensitivity, performance with size selected and mock degraded samples, and performance with DNA mixtures. 3 SNPs failed reads in several samples during the average depth of coverage test. The remaining 448 SNPs were then analyzed for sensitivity, performance with size selected, mock degraded, and mixed DNA samples. The sensitivity test showed that sample amounts of 5ng and higher exhibited complete allele calls for all 448 SNPs (100% full SNP call or 100% “SNP profile”). Allelic and complete dropout increased with decreasing sample amount, however, the 100pg sample amount exhibited a 23% full SNP call (103 SNPs) which is more than the minimum number of SNPs (50-60) required for useful investigative purposes.
The size selection experiment carried out for ≤75bp, ≤100bp, ≤150bp, ≤200bp, and ≤250bp yielded at least 96% SNPs with coverage ≥10X, and 95% full SNP allele calls. The mock degradation test samples (at an average length of 150bp) yielded full SNP call for over 90% of SNPs for samples amounts ≥1ng. Finally, two-person male-male mixtures were tested in contributor ratios ranging from 60:40 to 97.5:2.5. The mixtures were detected at all ratios and at least 80% of the unique minor contributor alleles were detected for ratios between 60:40 and 90:10. Furthermore, the haploid and multi-allelic markers aided in detection of mixtures in all samples without the need of reference samples. Based on these results, the custom probe panel was highly successful in capturing SNP regions in degraded and mixed DNA samples, and has the potential to be applied to forensic samples.
(Publisher abstract provided.)