title: Forensic strategy to ensure the quality of sequencing data of mitochondrial DNA in highly degraded samples.
abstract: Mitochondrial DNA (mtDNA) is widely used for DNA analysis of highly degraded samples because of its polymorphic nature and high number of copies in a cell. However, as endogenous mtDNA in deteriorated samples is scarce and highly fragmented, it is not easy to obtain reliable data. In the current study, we report the risks of direct sequencing mtDNA in highly degraded material, and suggest a strategy to ensure the quality of sequencing data. It was observed that direct sequencing data of the hypervariable segment (HVS) 1 by using primer sets that generate an amplicon of 407 bp (long-primer sets) was different from results obtained by using newly designed primer sets that produce an amplicon of 120-139 bp (mini-primer sets). The data aligned with the results of mini-primer sets analysis in an amplicon length-dependent manner; the shorter the amplicon, the more evident the endogenous sequence became. Coding region analysis using multiplex amplified product-length polymorphisms revealed the incongruence of single nucleotide polymorphisms between the coding region and HVS 1 caused by contamination with exogenous mtDNA. Although the sequencing data obtained using long-primer sets turned out to be erroneous, it was unambiguous and reproducible. These findings suggest that PCR primers that produce amplicons shorter than those currently recognized should be used for mtDNA analysis in highly degraded samples. Haplogroup motif analysis of the coding region and HVS should also be performed to improve the reliability of forensic mtDNA data.
authors.affiliation: Department of Legal Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Yamanashi 409-3898, Japan. Electronic address: nadachi@yamanashi.ac.jp.
authors.forename: Noboru
authors.initials: N
authors.lastname: Adachi
authors.affiliation: Department of Forensic Medicine, Faculty of Medicine, Yamagata University, 2-2-2 Iida-nishi, Yamagata 990-2331, Japan. Electronic address: kumetsu@med.id.yamagata-u.ac.jp.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Umetsu
authors.affiliation: Department of Legal Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Yamanashi 409-3898, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Shojo
mesh_terms: D004272:DNA, Mitochondrial / Q000032:analysis*; D053803:Forensic Genetics / Q000379:methods*; D006801:Humans; D017422:Sequence Analysis, DNA
chemical_list: D004272:DNA, Mitochondrial
keywords: Contamination; Degraded DNA; Forensic identification; Mitochondrial DNA; PCR