Validation and beyond: Next generation sequencing of forensic casework samples including challenging tissue samples...

The proceeding developments in next generation sequencing (NGS) technologies enable increasing discrimination power for short tandem repeat (STR) analyses and provide new possibilities for human identification. Therefore, the growing relevance and demand in forensic casework display the need for reliable validation studies and experiences with challenging DNA samples. The presented validation of the MiSeq FGx system and the ForenSeq™ DNA Signature Prep Kit (1) investigated sensitivity, repeatability, reproducibility, concordance, pooling variations, DNA extraction method variances, DNA mixtures, degraded, and casework samples and (2) optimized the sequencing workflow for challenging samples from human corpses by testing additional PCR purification, pooling adjustments, and adapter volume reductions. Overall results indicate the system's reliability in concordance to traditional capillary electrophoresis (CE)-based genotyping and reproducibility of sequencing data. Genotyping success rates of 100% were obtained down to 62.5 pg DNA input concentrations. Autosomal STR (aSTR) profiles of artificially degraded samples revealed significantly lower numbers of locus and allelic dropouts than CE. However, it was observed that the system still exposed drawbacks when sequencing highly degraded and inhibited samples from human remains. Due to the lack of studies evaluating the sequencing success of samples from decomposed or skeletonised corpses, the presented optimisation studies provide valuable recommendations such as an additional PCR purification, an increase in library pooling volumes, and a reduction of adapter volumes for samples with concentrations ≥31.2 pg. Thus, this research highlights the importance of all-encompassing validation studies for implementing novel technologies in forensic casework and presents recommendations for challenging samples.