Evaluation of Library Preparation Workflows and Applications to Different Sample Types Using the PowerSeq 46GY System with Massively Parallel Sequencing.

This project evaluated the prototype PowerSeq 46GY System using donor DNA and casework-type samples. The goal of this study was to determine whether modifications to the manufacturer's protocol could increase read coverage and improve sample results. Buccal and casework-type libraries were prepared using the TruSeq DNA PCR-Free HT kit or the KAPA HyperPrep kit. Both kits were evaluated unmodified, and by substituting AMPure XP beads for the beads of the most optimal kit. Two qPCR kits, the PowerSeq Quant MS System and KAPA Library Quantification Kit, were also evaluated along with a KAPA size-adjustment workbook, which was compared as a third quantification method. Libraries were sequenced using the MiSeq FGx and data were analyzed with STRait Razor. Results suggested that all three quantification methods overestimated library concentration, but the PowerSeq kit was most accurate. Samples prepared with the TruSeq library kit provided the highest coverage and the fewest instances of dropout and below-threshold alleles compared with the KAPA kit. Additionally, all bone and hair samples demonstrated full profile completeness, with bone samples yielding a higher average coverage than hair samples. Overall, our study demonstrated that the 46GY manufacturer's protocol produced the best quality results compared to alternative library preparation options.