Massively Parallel Pyrosequencing of the Mitochondrial Genome with the 454 Methodology in Forensic Genetics

• NCJ Number: 248139
• Journal: Forensic Science International Genetics Volume: 12 Dated: September 2014 Pages: 30-37
• Author(s): Martin Mikkelsen; Rune Frank-Hensen; Anders J. Hensen; Niels Morling
• Date Published: September 2014
• Length: 8 pages
• Publication Format: Article
• Publication Type: Research (Applied/Empirical); Report (Technical); Report (Technical Assistance); Report (Summary); Report (Study/Research)
• Language: English
• Country: Ireland

Annotation

This article presents the results of mitochondrial DNA (mtDNA) sequencing of two long overlapping PCR amplified DNA fragments (9033 and 8970 bp) obtained from high-quality biological samples with the Roche 454 GS junior in a forensic genetic laboratory, and the results were compared with those obtained with Sanger sequencing and SNP typing.

Abstract

This project found close to full concordance between the mtDNA sequences of the samples obtained with the 454 second generation system (SGS) method using depth of coverage above 100 and Sanger sequencing and SNP typing. The discrepancies were primarily observed in homopolymeric regions. The 454 technology was able to detect 95 percent of the reads correctly in homopolymers up to four bases, and up to six bases could be sequenced with similar success if the results were carefully visually inspected. The 454 technology was able to detect mixtures or heteroplasmy of approximately 10 percent. Researchers detected previously unreported heteroplasmy in the CM9947A component of the NIST human mtDNA SRM-2392 standard reference material. The researchers advise that because of the long initial PCRs, the approach presented will most likely not work on degraded DNA that is often found in forensic genetic crime casework. The presented method is best suited for samples with high-quality DNA, such as reference samples. 5 tables, 2 figures, and 29 references