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

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 6 C system, Promega) and the first commercial forensic MPS kit (ForenSeq™ DNA Signature prep, Qiagen) and for a concordance study with data from the Powerseq® MPS kit as well. 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. [Display omitted] •OmniSTR performance for minimal DNA amounts exceeds current MPS and CE methods.•SE33 sequence variation results in many more variants compared to length variation.•OmniSTR tolerance for inhibitors Humic and Tannic acid is comparable to PPF6C but improved for EtOH.•Automated analysis of references achieved 99.94 % accuracy for 96.7 % called markers.•Minor alleles down to a level of 1.5 % of the major can be called in mixtures for OmniSTR.