Comparison of commercial targeted amplicon sequencing assays for human remains identification casework

Abstract

Targeted amplicon sequencing (TAS) facilitates the genotyping of forensically informative single nucleotide polymorphisms (SNPs) using massively parallel sequencing (MPS). For human remains identification, where any extracted DNA is likely to be degraded, TAS may succeed when short tandem repeat (STR) profiling using capillary electrophoresis fails. Further, as well as yielding identity information, SNPs can provide information about ancestry, phenotype, kinship and paternal lineage (Y chromosome haplotypes). Two TAS platforms were compared in this study: Ion AmpliSeq™ panels coupled with Ion Torrent sequencing on an Ion GeneStudio™ S5 Plus System, manufactured by Thermo Fisher Scientific, and the ForenSeq® Kintelligence Kit coupled with Illumina sequencing on the MiSeq FGx® Sequencing System, manufactured by QIAGEN. Four Ion AmpliSeq™ panels (Precision ID Identity, Precision ID Ancestry, DNA Phenotyping and HID Y-SNP) share 177 SNPs with the ForenSeq® Kintelligence Kit and all five were used to profile the DNA extracted from the petrous part of the temporal bone from six skeletonised cadavers. Of the 6 × 177 = 1,062 SNP genotype comparisons, 1,055 (99%) were concordant between the Ion AmpliSeq™ panels and Kintelligence Kit. Of the seven (< 1%) non-concordant SNPs, only three of them (0.3%) would have resulted in erroneous genotypes being reported as a result of allele dropout by either assay, using our optimised relative variant frequency windows for allele calling. We conclude that both the Ion AmpliSeq™ panels and the ForenSeq® Kintelligence Kit were suitable for TAS applied to the human remains in this study.