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Resolution of DNA Mixtures and Analysis of Degraded DNA Using the 454 DNA Sequencing Technology

NCJ Number
Cassandra D. Calloway; Hanna Kim; Henry Erlich
Date Published
August 2014
175 pages
This report presents the methodology and results of a project whose goal was to develop methods for analyzing mixed, limited, and degraded DNA samples using the 454 next generation sequencing (NGS) technology for deep sequencing mtDNA and STR markers.
NGS is capable of analyzing sequence polymorphisms, such as mtDNA and SNPs, as well as length polymorphisms (e.g., STRs) on the same platform. Overall, the project succeeded in using NGS for the analysis of challenging forensic samples. A duplex PCR assay that targeted the mtDNA NVI/HVII regions was developed by using eight sets of 454 MID tagged fusion primers in a combinatorial approach for deep sequencing 64 samples in parallel. The project designed and showed proof-of-concept for a solution phase sequence capture and NGS assay for targeted enrichment and deep sequencing of the entire mitochondria genome for increased discrimination power. Moreover, a DNA fragmentation method that uses mechanical shearing (Covaris) was optimized and shown to be DNA quantity and quality independent. This is essential for preparing highly degraded or limited samples. Proof-of-concept for using the optimized fragmentation method for analysis of degraded samples was established with artificially degraded samples. In addition, NGS assays that targeted the CODIS STR loci were developed using 454 mini STR fusion primers in a multiplex PCR. Amplification and sequencing of 50 pg of DNA was accomplished. Also, proof-of-concept for sequencing mtDNA and STR markers in a single 454 NGS run was achieved. Software was modified for mtDNA and STR NGS alignment and analyses. 72 figures, 91 references, and listing of occasions and products for the dissemination of project findings

Date Published: August 1, 2014