Comparison of Next Generation Sequencing Methodology on the Ion PGM™ System Performance versus that on the Sanger Sequencing Method for HV1 and HV2 Regions of mtDNA

Analysis of mitochondrial DNA in forensic applications has enabled the identification of a missing person through comparison with additional maternal relatives. Most forensic applications are based on sequencing of both hypervariable regions of the mtDNA. Sequencing of these regions has been commonly done using Sanger-type sequencing (STS) methodology, which is expensive, time-consuming and laborious. Next Generation Sequencing (NGS) technology, such as the Ion Torrent PGM™ System platform, overcomes most of these issues. In this study, samples from the Guatemalan population (n=40) were sequenced with both Ion Torrent PGM™ technology and STS methods. A high level of consistency (98%) was observed among data derived from both methods. Most of the discrepancies were point heteroplasmy, which were more easily detected by PGM™ technology. In terms of performance, the NGS method was shown to be quick, with high-throughput and more efficient compared to the traditional STS method. More accurate and reliable sequencing data were obtained from the Ion Torrent PGM™ method due to its high level of coverage. Sequencing data for all individuals, representing 19 different family groups, were obtained using the NGS technology. Sequence polymorphisms were detected in 55 positions, from which 26 were observed only in relatives belonging to the same family and were not observed for any other family group. In a forensic context, haplotype specific polymorphisms are the basis for identification and comparison between evidence and reference samples purposes. Haplotypes between maternally related individuals were consistent in 18 family groups.