Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
6553279 | Forensic Science International: Genetics | 2018 | 11 Pages |
Abstract
DNA from biological forensic samples can be highly fragmented and present in limited quantity. When DNA is highly fragmented, conventional PCR based Short Tandem Repeat (STR) analysis may fail as primer binding sites may not be present on a single template molecule. Single Nucleotide Polymorphisms (SNPs) can serve as an alternative type of genetic marker for analysis of degraded samples because the targeted variation is a single base. However, conventional PCR based SNP analysis methods still require intact primer binding sites for target amplification. Recently, probe capture methods for targeted enrichment have shown success in recovering degraded DNA as well as DNA from ancient bone samples using next-generation sequencing (NGS) technologies. The goal of this study was to design and test a probe capture assay targeting forensically relevant nuclear SNP markers for clonal and massively parallel sequencing (MPS) of degraded and limited DNA samples as well as mixtures. A set of 411 polymorphic markers totaling 451 nuclear SNPs (375 SNPs and 36 microhaplotype markers) was selected for the custom probe capture panel. The SNP markers were selected for a broad range of forensic applications including human individual identification, kinship, and lineage analysis as well as for mixture analysis. Performance of the custom SNP probe capture NGS assay was characterized by analyzing read depth and heterozygote allele balance across 15 samples at 25â¯ng input DNA. Performance thresholds were established based on read depth â¥500X and heterozygote allele balance within ±10% deviation from 50:50, which was observed for 426 out of 451 SNPs. These 426 SNPs were analyzed in size selected samples (at â¤75â¯bp, â¤100â¯bp, â¤150â¯bp, â¤200â¯bp, and â¤250â¯bp) as well as mock degraded samples fragmented to an average of 150â¯bp. Samples selected for â¤75â¯bp exhibited 99-100% reportable SNPs across varied DNA amounts and as low as 0.5â¯ng. Mock degraded samples at 1â¯ng and 10â¯ng exhibited >90% reportable SNPs. Finally, two-person male-male mixtures were tested at 10â¯ng in contributor varying ratios. Overall, 85-100% of alleles unique to the minor contributor were observed at all mixture ratios. Results from these studies using the SNP probe capture NGS system demonstrates proof of concept for application to forensically relevant degraded and mixed DNA samples.
Keywords
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Authors
Nikhil Bose, Katie Carlberg, George Sensabaugh, Henry Erlich, Cassandra Calloway,