Developmental Validation of a KAPA EvoPrep Whole Genome Sequencing Workflow

The forensic community’s options for producing large single nucleotide variant profiles span an array of PCR-based targeted amplification, microarray, and whole genome sequencing (WGS) workflows. Each option has commercially available kits from which to choose, and considerations such as platform, throughput, sensitivity, accuracy, and repeatability all impact laboratories’ decisions. To aid this process, a developmental validation study of a WGS workflow was completed, taking the SWGDAM and FBI QAS recommendations into account. The workflow consisted of library preparation with the KAPA EvoPrep Kit and sequencing on the NovaSeq 6000. The amplification, size selection, and pooling (i.e., plexity) steps of the workflow were assessed. Sensitivity studies, with inputs as low as 50 picograms of DNA, demonstrated the dynamic range and limit of detection. Generation of replicate libraries within and across individuals allowed reproducibility and repeatability to be evaluated. The potential for contamination was assessed through inclusion of traditional and bioinformatic negative controls for evidence of exogenous DNA or indices. Finally, mock casework samples and mixtures at ratios ranging from 1:1 to 1:49 were sequenced to evaluate performance with potentially challenging types of samples often encountered in forensic laboratories. Tapir, a portable end-to-end bioinformatic workflow, was used for analysis, providing summary statistics of sample composition, breadth and depth of coverage for the sequencing data, and call rates (with and without modern genotype imputation) for genotyping results. Data from these validation studies will assist laboratories considering WGS workflows and further implementation of WGS, as a whole, in forensic laboratories.