Systematic optimisation of crude buccal swab lysate protocols for use with the ForenSeq™ DNA Signature Prep Kit.

Abstract

The ForenSeq™ DNA Signature Prep kit has not been thoroughly tested with crude buccal swab lysates in large-scale population studies using massively parallel sequencing (MPS). Commonly used lysis buffers for swabs intending to undergo direct polymerase chain reaction (PCR) are SwabSolution™ and STR GO! Lysis Buffers, and these have been successfully used to generate population data using capillary electrophoresis (CE) systems. In this study, we investigated the performance and optimisation of SwabSolution™ and STR GO! lysates with the ForenSeq™ DNA Signature Prep workflow and addressed the challenge of failed MPS profiles in initial trials. To mitigate PCR inhibition in SwabSolution™ lysates, three optimisation methods were evaluated: dilution of lysates, addition of 5X AmpSolution^® reagent, and purification with magnetic beads. For STR GO! lysates, we explored spin-column purification using the QIAamp^® DNA Investigator kit, magnetic bead purification, and a pH adjustment with 1 M hydrochloric acid. Our findings indicated that the addition of 5X AmpSolution^® was effective for overcoming PCR inhibition in SwabSolution™ lysates, thereby maintaining a direct PCR approach. Spin-column purification, however, is recommended for STR GO! lysates to minimise MPS profile failure rates. These improvements enhance first-time success rates of crude swab lysates, and reduce the need for repeat sampling and re-sequencing, making the workflow more suitable for large-scale population studies in forensic laboratories.