DNA Typing Strategies for Identification of Human Remains via Real-Time Nanopore Sequencing

Human remains are frequently encountered in forensic laboratories, coming from crime scenes, mass graves, historical samples, mass disasters, and military conflicts. The problem faced by forensic laboratories when analyzing challenged samples of human remains is they must choose between depleting sample volumes repeating individualizing STR analysis or performing costly, time-consuming, and less discriminatory mtDNA analysis. New DNA sequencing methodologies may provide a more effective platform for identification of human remains that overcome the most common challenges associated with processing of bone fragments, aged tissue, and hair samples. While the gold standard for forensic DNA identification is analysis of autosomal STR loci, skeletal remains have often been exposed to environments that trigger degradation of the nuclear DNA and introducing PCR inhibitors. Likewise, rootless hairs yield little to no usable nuclear DNA fragment. For this reason, remains processing often relies upon sequencing of the mtDNA control region, as mtDNA can often be extracted and amplified in samples where nuclear DNA cannot. However, mtDNA is not individualizing because each person shares their mtDNA with all their maternal relatives. Single nucleotide polymorphisms (SNPs) occur throughout the genome and account for 85% of genetic variability observed between individuals. SNP interrogation involves assessing the variation of individual nucleotides, rather than strings of nucleotides seen in STRs, and provide the discriminatory power necessary for positive forensic identification, making them ideal for low template or degraded DNA samples. In instances of low level and fragmented DNA, probe capture has shown effective for enrichment. We will develop and validate alternative sequencing approach via nanopore single molecule sequencing for an all-encompassing process that will simultaneously interrogate STRs, mtDNA, and SNPs. For high-volume samples, such as whole bones, we will establish a rapid, whole genome sequencing method. For instances of low-level samples, we will utilize RNA baiting to target our regions of interest. Through this process, we will develop reliable and comprehensive new methods for human remains identification that can also lend itself to predictive phenotyping for the development of investigative leads and the analysis of historical samples. We have a team of researchers with extensive forensic knowledge and over ten years of collective experience with ONT sequencing. We will establish working protocols, including DNA input suggestions based on the quality and quantity of the starting sample, as well as interpretation guidelines for human identification laboratories consistent with current standards laid out by the Scientific Working Group on DNA Analysis Methods.

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