Validation of the Verogen ForenSeq™ DNA Signature Prep kit/Primer Mix B for phenotypic and biogeographical ancestry predictions using the Micro MiSeq® Flow Cells

Abstract

In anticipation of offering phenotypic and biogeographical ancestry predictions to help resolve cases, the Verogen ForenSeq™ DNA Signature Prep kit/Primer Mix B was evaluated in the context of Micro MiSeq® Flow Cells. These flow cells were determined as the best format for a quick turnaround time response and cost effective approach compared to standard flow cells. The phenotype informative SNPs (piSNPs) and ancestry informative SNPs (aiSNPs) were thoroughly examined through sensitivity, reproducibility and repeatability, concordance, robustness (mock casework) and low level DNA mixture studies purposely selecting individuals with different phenotypes (hair and eye color) when possible and different biogeographical ancestry. SNP locus-specific interpretation thresholds were established for the Universal Analysis Software (UAS) based on surviving alleles and SNP predictor rank to minimize false homozygous genotypes and maximize the information that can be derived from an unknown sample. Dropin alleles' intensity determined an appropriate threshold to minimize false heterozygous SNP genotypes. The selection of inappropriate interpretation thresholds was shown to have major consequences on phenotypic predictions. A 3.2% and 4.8% minor DNA component contribution to a DNA mixture had no impact on ancestry predictions whereas a 9.1% contribution did. The multi-locus SNP genotypes generated using the ForenSeq™ DNA Signature Prep kit/Primer Mix B were shown to be reliable, reproducible, concordant and resulted in predictions that were also reliable, reproducible and concordant based on the limited number of donors (N = 19) used in this study.

Keywords: Biogeographical ancestry predictions; ForenSeq™; Forensic DNA phenotyping; Micro MiSeq® Flow Cells; SNP-specific interpretation thresholds; Validation.