title: Massively parallel pyrosequencing of the mitochondrial genome with the 454 methodology in forensic genetics.
abstract: of sequencing of whole mitochondrial genome, HV1 and HV2 DNA with the second generation system (SGS) Roche 454 GS Junior were compared with results of Sanger sequencing and SNP typing with SNaPshot single base extension detected with MALDI-TOF and capillary electrophoresis. We investigated the performance of the software analysis of the data, reproducibility, ability to sequence homopolymeric regions, detection of mixtures and heteroplasmy as well as the implications of the depth of coverage. We found full reproducibility between samples sequenced twice with SGS. We found close to full concordance between the mtDNA sequences of 26 samples obtained with (1) the 454 SGS method using a depth of coverage above 100 and (2) Sanger sequencing and SNP typing. The discrepancies were primarily observed in homopolymeric regions. The 454 SGS method was able to sequence 95% of the reads correctly in homopolymers up to 4 bases, and up to 6 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%. We detected previously unreported heteroplasmy in the GM9947A component of the NIST human mitochondrial DNA SRM-2392 standard reference material.
authors.affiliation: Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Frederik V's Vej 11, DK-2100 Copenhagen Ø, Denmark. Electronic address: martin.mikkelsen@sund.ku.dk.
authors.forename: Martin
authors.initials: M
authors.lastname: Mikkelsen
authors.affiliation: Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Frederik V's Vej 11, DK-2100 Copenhagen Ø, Denmark.
authors.forename: Rune
authors.initials: R
authors.lastname: Frank-Hansen
authors.affiliation: Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Frederik V's Vej 11, DK-2100 Copenhagen Ø, Denmark.
authors.forename: Anders J
authors.initials: AJ
authors.lastname: Hansen
authors.affiliation: Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Frederik V's Vej 11, DK-2100 Copenhagen Ø, Denmark. Electronic address: niels.morling@sund.ku.dk.
authors.forename: Niels
authors.initials: N
authors.lastname: Morling
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000235:genetics*; D019075:Electrophoresis, Capillary; D053803:Forensic Genetics; D054629:Genome, Mitochondrial; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial
keywords: 454 sequencing; NGS; SGS; SRM-2392; heteroplasmy; mtDNA