Internal validation study of the next generation sequencing of Globalfiler™ PCR amplification kit for the Ion Torrent S5 sequencer

Abstract

In the last few years, massively parallel sequencing (MPS) techniques have improved greatly in the field of forensic genetics, providing several advantages over the traditionally capillary electrophoresis (CE) system in terms of resolution, scalability, and throughput [1,2]. Nevertheless, before implementing MPS into routine forensic applications, both workflow and output results must be rigorously validated with respect to robustness, performance and compatibility to CE-based data [3].We present the validation study of MPS technology for short tandem repeat (STR) genotyping using Precision ID GlobalFiler™ NGS STR Panel v2 on the Ion S5™ platform (both Thermo Fisher Scientific), that include experiments to assess concordance, sensitivity, reliability and mixture analysis.MPS allowed to generate a concordant result with those by CE system, demonstrating the reliability of both methods. Sensitivity study revealed that single source full-profile could be obtained using a significantly less amount of DNA compared to the quantity recommended in the protocol. In addition, STR genotypes obtained were reproducible and consistent among multiple typing replicates. Partial STR genotypes of minor contribute could be detected up to a 1:80 mixture. However, analysis of MPS-based STR revealed more noise artefacts (e.g. stutter artefacts) respect to CE, that were not independently filtered by Converge™ software NGS Analysis Module supported by Life Technologies platform.