Validation of the Idseek® Omnistr™ Global Autosomal Str Profiling Kit, Reverse Complement Pcr as an Improved Tool/Method for Routine Massively Parallel Sequencing of Short Tandem Repeats

Abstract

Massively Parallel Sequencing (MPS) has gained interest in the forensic community over the past decade. Most of the published MPS methods focus on specialty applications intended for use in a limited number of samples with protocols that are relatively laborious. Recent developments using Reverse-Complement PCR enable an efficient MPS protocol suited for routine analysis of high numbers of samples. This method is implemented in the IDseek® OmniSTR™ Global Autosomal STR Profiling kit (Nimagen) for sequencing 28 of the most commonly used forensic autosomal STRs, one Y-chromosomal STR and Amelogenin.This study describes the validation of this kit and focuses on sensitivity, inhibitor tolerance, sequence variation detection and performance with mixtures up to 5 contributors. Results are compared to a Capillary Electrophoresis method (the PowerPlex® Fusion 6C system, Promega) and the first commercial forensic MPS kit (ForenSeq™ DNA Signature prep, Qiagen). Analysis settings in FDSTools are deduced and discussed, and an almost completely automated analysis is achieved. Using FDSTools noise correction, contributions in a mixture down to a level of 1.5% of the major allele of a marker can be detected.

Keywords: MPS, STR sequencing, IDseek OmniSTR, SE33, FDSTools, Mixture analysis, PCR inhibition, Reverse complement PCR