Multiplex DNA amplification and barcoding in a single reaction for 454 Roche sequencing: A comprehensive study on the control region of the mitochondrial genome |
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| Affiliation: | 1. Laboratory of Forensic Genetics and Molecular Archaeology, UZ Leuven, Belgium;2. Forensic Medicine, UZ Leuven, Belgium;3. Faculty of Medicine, University of Perugia, Italy;4. Faculty of Health and Medical Sciences, University of Surrey, UK;5. Department of Imaging and Pathology, KU Leuven, Belgium;1. Immunological Research, Universy of Cartagena, Cartagena, Colombia;2. Laboratorio GENES Ltda, Medellín, Colombia;3. Instituto de Biología, Universidad de Antioquia, Medellín, Colombia;4. Laboratorio de Genética Molecular, Cruz Roja Ecuatoriana, Quito, Ecuador;5. Universidad Pontificia Bolivariana, Medellín, Colombia;6. IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Portugal;1. Laboratorio de Genética Molecular de Cruz Vital – Cruz Roja Ecuatoriana Quito, Ecuador;2. Laboratorio GENES Ltda, Medellin, Colombia;1. Flinders Centre for Nanoscale Science and Technology, Flinders University, Sturt Road, Bedford Park, Adelaide, South Australia, Australia;2. School of Biological Sciences, Flinders University, Sturt Road, Bedford Park, Adelaide, South Australia, Australia;1. Network of Forensic Science Institutes, Institute of Forensic Medicine, DNA Laboratory, Budapest, Hungary;2. Research Centre for Natural Sciences of the HAS, Department of Complex Systems, Budapest, Hungary;1. Forensics Genetic Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal;2. Cencifor, Forensic Science Centre, Portugal;3. National Institute of Legal Medicine and Forensic Sciences, I.P., Portugal;4. Department of Biology, University of Aveiro, Portugal |
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| Abstract: | The analysis of the non-coding region of the mitochondrial genome using Sanger sequencing remains a laborious and time-consuming assay with too low resolution for the identification of low-frequency heteroplasmy or for mixture interpretation. In this study, an experimental design was tested in which the complete hypervariable region of the mitochondrial genome was sequenced using a novel barcoding strategy. The strategy involves a single-step multiplex nested PCR and we demonstrate its effectiveness by sequencing two multiplex reactions of two amplicons each covering the complete hypervariable region of the mitochondrial genome for 58 reference samples, 30 of which were analysed in triplicate, and 10 casework samples, each analysed in triplicate, on a 454 Roche DNA pyrosequencer with GS FLX chemistry using Multiplex Identifier (MID) primers to discriminate between samples. The generated reads for forensic (±3600 reads/MID) and reference samples (±466 reads/MID) allowed us to evaluate the accuracy in SNP calling and the variation in heteroplasmy and sequencing error rates in homopolymeric stretches between replicates. |
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| Keywords: | 454 Pyrosequencing Single-step nested multiplex PCR |
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