Development of a Probe Capture Next-Generation Sequencing Assay for Analysis of the Mitochondrial Genome and Nuclear STR and SNP Markers

Forensic biological samples from cases, such as missing persons, can often be highly compromised (degraded, limited, mixed). To analyze such samples, we developed a probe capture Next-Generation Sequencing (NGS) system targeting the entire mitochondrial genome and greater than 450 nuclear SNPs. This approach uses DNA probes to enrich targeted regions from randomly fragmented DNA libraries for clonal, massively parallel sequencing, thereby maximizing recovery of short DNA fragments characteristic of forensic samples. NGS analyzes the components of a mixture separately and the composition of a mixture can be determined by counting the number of sequence reads assigned to each individual contributor of the mixture. We applied this system to sequence the mitochondrial genome and nSNPs of limited and highly degraded DNA from hair and bones as well as mixtures. We have also demonstrated the feasibility of two different software programs for reconstructing the mitochondrial genome sequence of the contributors in a mixture. Although sequencing the entire mitochondrial genome and nSNP markers provide greater discriminatory capability, currently nSNP databases are limited. Our goal is to develop a probe capture NGS system that can be used to analyze both mtDNA and nuclear markers (STRs and SNPs) from a single shotgun DNA library for improved resolution of mixtures and increased discriminatory power of challenging forensic samples with backwards compatibility with current CODIS databases by expanding our nuclear probe capture panel to include autosomal and X/Y STRs. The proposed nuclear probe panel will target greater than 500 markers including STRs, SNPs, and microhaplotypes. The addition of the STR category is critical for use with existing databases. This combined system has the potential to overcome major technical challenges associated with highly degraded, limited, and mixed samples while still providing a high discrimination power. In cases where the DNA sample is limited in amount, such as shed hairs, practitioners currently have to choose between analyzing nuclear or mtDNA markers. Our strategy can allow for the analysis of both mitochondrial and nuclear markers from DNA samples limited in amount, since only a single DNA library is required for both analyses. Additionally, this combined system has the potential to greatly improve the capability of analyzing mixed DNA samples through the analysis of both haploid and multi-allelic markers. If successful, this grant proposal promises to make a significant contribution to the criminal justice system by providing a new method for analyzing challenging forensic specimens that is backwards compatible with existing databases.

This project contains a research and/or development component, as defined in applicable law, and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14).

CA/NCF