Developmental validation of a whole genome sequencing workflow for use in a forensic laboratory

Abstract

The generation of large single nucleotide variant (SNV) profiles are at the forefront of the minds of many forensic practitioners, largely in response to the growing demand for forensic genetic genealogy (FGG) analyses on both missing persons and forensic cases. Current options available to laboratories today include PCR-based targeted amplification, SNV microarrays, and whole genome sequencing, each with multiple commercially available kits from which to choose. In addition to molecular considerations, bioinformatic workflows (and accompanying tools) must be constructed, validated, and implemented. To aid this process, a developmental validation study of a whole genome sequencing workflow was completed. This workflow consisted of library preparation with the KAPA HyperPrep Kit and sequencing on the NovaSeq 6000. Validation of this workflow included studies on PCR optimization, library stability, and library plexity. Sensitivity studies (ranging from 10 nanograms to 50 picograms of DNA) demonstrated the dynamic range and limit of detection. Libraries were generated by multiple individuals to allow for assessment of reproducibility. The potential for contamination was assessed through evaluation of negative controls for evidence of exogenous DNA. Finally, mock casework samples and mixtures at ratios ranging from 1:1–1:49 were sequenced to determine performance with potentially challenging samples. A portable end-to-end bioinformatic workflow (Tapir) was developed, allowing for a seamless transition from raw data from Illumina instruments (BCL) to sample genotypes in a format compatible with GEDmatch. Tapir includes various summaries of sample composition, including information on coverage (breadth and depth), duplication rates and call rates, and it supports both modern genotype imputation (i.e., genotype refinement) as well as classical genotyping techniques. The detailed breakdown of this workflow and the associated validation data will serve as an important tool in enabling the implementation of whole genome sequencing in forensic laboratories.