As submitted by the proposer:
The quality of human remains recovered for identification vary substantially, from pristine to highly degraded and/or substantially contaminated (i.e., co-mingled with other victims or containing materials that may inhibit downstream analysis). There will be situations in which environmental insults are so great that standard STR typing protocols will not yield sufficient results to obtain a reliable identification. Alternate DNA analysis techniques will be needed that are robust and highly informative. In order to improve our ability to analyze challenged samples and thus resolve more missing persons cases, a more discriminatory approach than simply using STR and mtDNA markers is needed. Forensic massively parallel sequencing (MPS) panels may provide that solution for missing persons and DVI cases when samples are too degraded for conventional analysis and/or family reference samples are inadequate, by increasing the number of markers examined for greater success in typing, and reducing the reliance on more family members for identification.
To utilize the full potential of MPS for identifying the challenging samples commonly encountered in missing persons and DVI cases, sample preparation methods must be optimized for maximum compatibility with the various MPS chemistries used for forensic analysis. This 24-month project will use human cadavers (N=3) subjected to various environmental conditions to develop and test the best possible sample preparation methods for skeletal and decomposing human remains for identification using emerging MPS HID platforms.
Sample preparation strategies are needed so that the DNA recovered from biological evidence is sufficiently pure for downstream analyses; sample preparation workflows integrate well with MPS; and robust interpretation guidelines are developed that address data quality, error rates, results analysis, data storage issues, and time and cost of analysis. This study will address the front end of the DNA/MPS analysis system and its downstream effect from common inhibitors, and various DNA extraction methods for environmentally challenged tissues and skeletal samples. We will focus on identifying the most efficient sample preparation methods that maximize DNA quality and quantity for sequencing, and also develop the most effective data interpretation strategies for the vast amounts of data obtained from the MPS forensic identify panels in order to improve our ability to resolve more missing persons and DVI cases. Quality metrics are generated during the analytical process and include quality scores for base calling, read-level quality control (to include trimming of low quality bases on fragment ends), alignment, GC content, depth of coverage, strand bias, and variant calling.
This project contains a research and/or development component, as defined in applicable law.