Alternative DNA technologies for obtaining DNA profiles from cartridge cases

Abstract

Obtaining a DNA profile from unfired cartridges and fired cartridge cases is desirable in a forensic investigation as it can link an individual to an offence. Conventional nuclear DNA (nDNA) profiling technologies have proven largely unsuccessful for this evidence type, as any DNA recovered is often low in quality and quantity. The advent of massively parallel sequencing (MPS) has allowed for the simultaneous analysis of many short DNA amplicons with a greater sensitivity for highly degraded samples. This research aimed to determine whether two different MPS technologies, the Precision ID Whole mtDNA Genome Panel and the ForenSeqTM DNA Signature Prep Kit, could successfully generate DNA profiles from unfired cartridges and fired cartridge cases. Previously developed custom MPS workflows were trialled on touch DNA samples recovered from unfired .223 Remington cartridges. Mitochondrial DNA (mtDNA) variants concordant with the corresponding reference haplotype were generated from 82.1% of the unfired samples, with 25% generating fully concordant mtDNA haplotypes. STR and iiSNP alleles could be recovered from unfired cartridges, with typed iiSNPs more likely to be typed concordant to a reference profile. The addition of a second purification, post-library preparation, minimised the impact of adapter-dimers on MiSeq FGxTM sequencing performance and was included into the final mtDNA workflow. Less DNA was recovered from a fired cartridge case, resulting in the smaller concentrations of mitochondrial and ForenSeqTM libraries. mtDNA haplotypes were generated in 78.6% of the fired samples, with 17.9% generating fully concordant mtDNA haplotypes. The presence of non-concordant minor mtDNA variants suggests that mtDNA haplotypes should be called using only major variants, ignoring any low-level heteroplasmy. STR and iiSNP alleles were also recovered from fired cartridge cases; however, allele dropouts were common, indicating that only partial loci profiles can be used in a forensic context. These methods could successfully obtain DNA profiles from cartridges and cartridge cases. Ultimately, this research demonstrates that MPS technologies should be strongly considered for further optimisation, validation, and implementation into the analysis of firearm evidence recovered from crime scenes.