Optimization of microhaplotypes for advanced DNA mixture deconvolution

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.
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.