title: Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine.
abstract: Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylogenetic resolution. Most established protocols on the different massively parallel sequencing (MPS) platforms, however, invariably involve the PCR amplification of large fragments, typically several kilobases in size, which may fail due to mtDNA fragmentation in the available degraded materials. We introduce a MPS tiling approach for simultaneous whole human mt genome sequencing using 161 short overlapping amplicons (average 200 bp) with the Ion Torrent Personal Genome Machine. We illustrate the performance of this new method by sequencing 20 DNA samples belonging to different worldwide mtDNA haplogroups. Additional quality control, particularly regarding the potential detection of nuclear insertions of mtDNA (NUMTs), was performed by comparative MPS analysis using the conventional long-range amplification method. Preliminary sensitivity testing revealed that detailed haplogroup inference was feasible with 100 pg genomic input DNA. Complete mt genome coverage was achieved from DNA samples experimentally degraded down to genomic fragment sizes of about 220 bp, and up to 90% coverage from naturally degraded samples. Overall, we introduce a new approach for whole mt genome MPS analysis from degraded and nondegraded materials relevant to resolve and infer maternal genetic ancestry at complete resolution in anthropological, evolutionary, medical, and forensic applications.
authors.affiliation: Department of Genetic Identification, Erasmus MC University Medical Center, Rotterdam, The Netherlands.
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Chaitanya
authors.affiliation: Department of Genetic Identification, Erasmus MC University Medical Center, Rotterdam, The Netherlands.
authors.forename: Arwin
authors.initials: A
authors.lastname: Ralf
authors.affiliation: Department of Genetic Identification, Erasmus MC University Medical Center, Rotterdam, The Netherlands.
authors.forename: Mannis
authors.initials: M
authors.lastname: van Oven
authors.affiliation: Institute of Forensic Research, Section of Forensic Genetics, Kraków, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Kupiec
authors.affiliation: Thermo Fisher Scientific, South San Francisco, California, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Chang
authors.affiliation: Thermo Fisher Scientific, South San Francisco, California, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Lagacé
authors.affiliation: Department of Genetic Identification, Erasmus MC University Medical Center, Rotterdam, The Netherlands.
authors.forename: Manfred
authors.initials: M
authors.lastname: Kayser
mesh_terms: D058893:DNA Barcoding, Taxonomic / Q000295:instrumentation / Q000379:methods / Q000592:standards; D004272:DNA, Mitochondrial; D054629:Genome, Mitochondrial; D023281:Genomics / Q000295:instrumentation / Q000379:methods / Q000592:standards; D006239:Haplotypes; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation* / Q000379:methods* / Q000592:standards; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004272:DNA, Mitochondrial
keywords: MPS; NGS; massively parallel sequencing; mitochondria; mtDNA; next-generation sequencing