Evaluation of the <scp>ForenSeq</scp> ™ <scp>MainstAY</scp> workflow for forensic <scp>DNA</scp> samples

This study evaluated the performance and limitations of the ForenSeq™ MainstAY chemistry on the MiSeq FGx® Sequencing System, which is approved for National DNA Index System (NDIS) use. This study included baseline performance, sensitivity and reproducibility studies, challenging casework samples, assessments of library plexities, comparisons with capillary electrophoresis (CE), standard versus enhanced PCR1 buffers, manual versus automated library preparation, and different flow cell types. To assess reproducibility across users, 33 samples were prepared by both an experienced and a novice user. Overall, the ForenSeq™ MainstAY Kit demonstrated high reliability with pristine samples and consistent performance across users, with minimal locus dropout until 62 pg. The kit outperformed CE for low-input and degraded samples, but limitations were observed at higher plexities. Increased sample numbers on a single flow cell disproportionately reduced read counts and locus recovery in degraded casework samples, while pristine samples were less affected. Strategic adjustments, such as batching degraded samples together or lowering plexity, may help mitigate these effects. Casework samples, including blood, touched items, bone, hair, and tissue, were tested with both standard and enhanced PCR1 buffers. In a 64-sample run (32 per buffer type), increased multiplexing reduced locus recovery in standard buffer samples, while the enhanced buffer improved recovery in 16 samples. Automated library preparation on the Opentrons OT-2 produced comparable or improved recovery relative to manual preparation, with no evidence of cross-contamination. These findings provide practical guidance for forensic laboratories adopting the MainstAY Kit, particularly in optimizing workflows for challenging samples and high-throughput sequencing environments.