Discordance of Penta D x.4 microvariant alleles results between three capillary electrophoresis and one massively parallel sequencing short tandem repeat kits

Abstract

Forensic Biology is contingent upon matching DNA profiles between a crime sample and a reference sample. There are several capillary electrophoresis kits available to generate a short tandem repeat (STR) profile from DNA samples, while newer methods using massively parallel sequencing are slowly being implemented in forensic laboratories worldwide. During evaluation of a newer capillary electrophoresis kit, Applied Biosystems™ VeriFiler™ Plus, a discordance was observed in the Penta D locus. The previous kit, Promega PowerPlex 21® System produced a 13.4,14 genotype, whilst VeriFiler™ Plus produced a 14,14 genotype. An expanded investigation into Penta D microvariant alleles revealed that multiple discordances were observed for DNA profiles containing larger x.4 variants. There was full concordance between PowerPlex® 21 and QIAGEN Investigator® 26plex, however discordances were observed between VeriFiler™ Plus and the other three kits tested, including the massively parallel sequencing kit, Verogen ForenSeq® MainstAY. Notably, four of these discordances resulted in null alleles with the VeriFiler™ Plus kit. A review of the Penta D DNA sequences in MainstAY revealed fully concordant microvariant alleles involved deletions within the repeat region, whilst variability in the discordances observed were dependent on the location of the variation outside the repeat region and the analysis method used. Variations observed within the 5’ flanking region produced the same allele designation across all capillary electrophoresis kits. However, deletions within the 3’ region either produced a null allele for VeriFiler™ Plus where the deletion is thought to overlap the primer binding site, or microvariant alleles for the PowerPlex® 21 and Investigator 26plex kits, which produced longer Penta D amplicons. The discovery of these variations in the Penta D flanking sequences is informative as it increases the awareness of Penta D discordances between different kit chemistries in nominated reference DNA profile comparisons and DNA database searching and matching alike, and provides support for this phenomenon when providing evidence as to the admissibility of such results in trial proceedings.