Develop non-PCR-based target enrichment reagents and methods for MPS analysis of forensic DNA

Limited quantity, low quality, degradation, and contamination are the major challenges in forensic DNA analyses. Current methods of analysis of short-tandem-repeats (STRs) markers and single nucleotide polymorphic sites (SNPs) require PCR amplification using target-specific primers. However, trace DNA often fails to amplify in PCR-based methods due to inhibition or degradation of priming sites, resulting in allelic dropout. Though various PCR optimization protocols have been developed, analysis of low quality and sparse forensic DNA samples is still challenging. Universal adapter ligation based massively parallel sequencing (MPS) methods have overcome the PCR amplification problems. Enrichment of target DNA regions is necessary for cost-effective MPS analysis. Hybridization capture methods have been developed to enrich whole mitochondrial DNA and SNPs for forensic analyses, yet the cost of baits prohibits wider adoption. Hence, I propose to develop methods to generate inexpensive non-PCR-based target enrichment baits for MPS analysis of DNA from difficult forensic samples. This proposal aims to generate baits for forensic markers and optimize hybridization capture methods by three objectives. First, demonstrate feasibility of an in-house method to generate non-PCR-based target enrichment baits for a small forensic panel consisting of ~2000 forensically relevant SNPs and STRs (2k panel). Second, develop hybridization capture target enrichment methods for forensic samples using the 2k panel. This will involve targeted sequencing of hair specimens from five volunteers, testing various input DNA and bait quantities and hybridization capture conditions, and comparing the resultant genotypes with microarray-generated genotypes. Finally, develop a 100,000 SNP/STRs panel to improve the discriminatory power for sample mixture and kinship analysis, for missing person identification and for gene-genealogy searches. The 100k panel will include SNPs used in forensic DNA phenotyping, SNPs informative of ancestry, externally visible characteristics, and individual identification. This panel will be tested for mixture deconvolution using mock mixtures of pre-genotyped control samples. The economic benefits of the in-house generation of baits for hybridization capture will be evaluated. Achievement of the proposed objectives will result in the development of methods and reagents valuable for the forensic community. Methods developed by and results of this project will be published in peer-reviewed journals and a detailed report will be submitted to NIJ for a broader audience. Protocols, design files, and bait panels will be made available for interested persons. Note: 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