Detection of minor DNA components in biological mixtures has increased as molecular techniques have become more sensitive. Accordingly, mixture deconvolution has become a major concern and topic of debate in the forensic DNA community. Short tandem repeat (STR) profile data generated with capillary electrophoresis and massively parallel sequencing (MPS) are subject to inherent issues that complicate mixture deconvolution, such as PCR stutter artifacts and allele length biases. Deconvolution may be improved by sequencing microhaplotypes as they are not subject to the amplification noise artifacts and stochastic effects that impact STRs. Before microhaplotypes can be implemented in casework, the following considerations should be addressed: definition of a consistent panel of microhaplotype loci; increased population studies to determine relevant haplotype allele frequencies; incorporation of advanced sequencing technologies into forensic laboratories; development of user-friendly bioinformatic analysis and mixture deconvolution methods; and assessment of the infrastructure requirements necessary to build a searchable microhaplotype criminal database. Ultimately, implementation requires a thorough understanding of the benefits and limitations of the new marker set as well as the practical investment of time and effort put forth for its adoption by the laboratory.
By coupling a highly discriminatory microhaplotype MPS assay with probabilistic genotyping methods such NexGenID, a novel software platform optimized for mixture deconvolution and probabilistic genotyping of sequence data, or EuroForMix, a widely used open-source probabilistic genotyping software modifiable for use with microhaplotype sequence data, microhaplotype analysis may be efficiently implemented by practitioners. The proposed microhaplotype panel demonstrated high discriminatory power with combined match probabilities ranging from 9.53E-52 to 4.79E-63 and the ability to infer biogeographical ancestry. The assay proved to be sensitive down to 50 pg inputs and applicable to inhibited or degraded trace samples. Application to complex DNA mixture samples demonstrates the assay’s potential to exceed minor-contributor detection when compared to STR deconvolution, help solve complex cases, increase the number of samples considered suitable for comparison, and enable retesting of cold cases where a minor contributor was assumed present but was not suitable for comparison. This effort also identified novel research areas that were technically explored to inform future solutions for the forensic community.
(Author abstract provided.)