Retrofitting massively parallel sequencing (MPS) for HLA-DQA1 and polymarker (PM) in forensic casework

Abstract

Genotype profiling has played a major role in forensics for decades. The technology for detection and discrimination has advanced substantially, from serology to DNA sequence analysis. Currently, there may be situations where there is a need for re-analysis of forensic DNA data that was produced using methodology that is no longer available. An example of this is the allele-specific oligonucleotide hybridization assays used in the 1990s. In the study presented herein, we have developed a multiplex system combining PCR and massively parallel sequencing (MPS) technologies to identify DNA polymorphisms. Our results are consistent with those found in the widely utilized AmpliType PM + DQA1 Amplification and Typing Kit originally marketed by Perkin Elmer. During the course of our studies, it became apparent that paralogous genes for two of the loci, GYPA and HBG2 (formerly HBGG), could have confounded the interpretation of the original assays, and we describe the technical solutions we developed to overcome ambiguity in genotype assignment. This study results in a novel resource enabling the re-analysis of DNA profiling results produced decades past using current day technology.

Keywords: Allele-specific oligonucleotide hybridization; AmpliType forensic DNA amplification and typing kit; Forensic genotyping; Massively parallel sequencing; Paralogous genes.