Detection of minor DNA components in biological mixtures has increased as molecular techniques have become more sensitive

Thus, DNA mixture deconvolution and adequate interpretation methods are under scrutiny within the forensic DNA community and require reassessment of standard practice, as detailed by the 2021 NIST scientific foundation review on mixture interpretation. Short tandem repeat (STR) profile data generated with capillary electrophoresis and massively parallel sequencing (MPS) are subject to inherent issues that complicate mixture deconvolution. Ultimately, a new DNA typing system that 1) has equivalent discriminatory power and sensitivity; 2) is not subject to STR's inherent amplification artifacts and 3) can run on modern equipment, is needed. 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. In two phases, this study will optimize and assess an MPS workflow and analysis package for improved mixture deconvolution using microhaplotypes. Analysis will be performed with nexgenid, a novel software platform optimized for mixture deconvolution and probabilistic genotyping of sequence data. Phase i objectives will include evaluation and down-selection of microhaplotype loci optimal for individualization and mixture deconvolution; construction of wet-bench target assay; haplotyping of donor samples to obtain expanded population allele frequency data; and assessment of the projected performance of the microhaplotype allele calling analysis workflow. Phase II objectives will include evaluating the benefits and limitations of mixture deconvolution and probabilistic genotyping using the microhaplotype wet-bench assay with illumina sequencing and nexgenid analysis by applying the workflow to in vitro mixtures and constructed mock evidence and also comparing outcomes from nexgenid to analysis of microhaplotype mixtures using a retrofitted version of euroformix. By coupling a highly discriminatory microhaplotype MPS assay with nexgenid, microhaplotype analysis can be efficiently implemented by practitioners. The proposed microhaplotype workflow has the 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. CA/NCF