Though investigations into the use of massively parallel sequencing technologies for the generation of complete mitochondrial genome (mtGenome) profiles from difficult forensic specimens are well underway in multiple laboratories, the high quality population reference data necessary to support full mtGenome typing in the forensic context are lacking. To address this deficiency, we have developed 588 complete mtGenome haplotypes, spanning three U.S. population groups (African American, Caucasian and Hispanic) from anonymized, randomly-sampled specimens. Data production utilized an 8-amplicon, 135 sequencing reaction Sanger-based protocol, performed in semi-automated fashion on robotic instrumentation. Data review followed an intensive multi-step strategy that included a minimum of three independent reviews of the raw data at two laboratories; repeat screenings of all insertions, deletions, heteroplasmies, transversions and any additional private mutations; and a check for phylogenetic feasibility. For all three populations, nearly complete resolution of the haplotypes was achieved with full mtGenome sequences: 90.3-98.8% of haplotypes were unique per population, an improvement of 7.7-29.2% over control region sequencing alone, and zero haplotypes overlapped between populations. Inferred maternal biogeographic ancestry frequencies for each population and heteroplasmy rates in the control region were generally consistent with published datasets. In the coding region, nearly 90% of individuals exhibited length heteroplasmy in the 12418-12425 adenine homopolymer; and despite a relatively high rate of point heteroplasmy (23.8% of individuals across the entire molecule), coding region point heteroplasmies shared by more than one individual were notably absent, and transversion-type heteroplasmies were extremely rare. The ratio of nonsynonymous to synonymous changes among point heteroplasmies in the protein-coding genes (1:1.3) and average pathogenicity scores in comparison to data reported for complete substitutions in previous studies seem to provide some additional support for the role of purifying selection in the evolution of the human mtGenome. Overall, these thoroughly vetted full mtGenome population reference data can serve as a standard against which the quality and features of future mtGenome datasets (especially those developed via massively parallel sequencing) may be evaluated, and will provide a solid foundation for the generation of complete mtGenome haplotype frequency estimates for forensic applications.

In a previous study we presented an assay for targeted mRNA sequencing for the identification of human body fluids, optimised for the Illumina MiSeq/FGx MPS platform. This assay, together with an additional in-house designed assay for the Ion Torrent PGM/S5 platform, was the basis for a collaborative exercise within 17 EUROFORGEN and EDNAP laboratories, in order to test the efficacy of targeted mRNA sequencing to identify body fluids. The task was to analyse the supplied dried body fluid stains and, optionally, participants' own bona fide or mock casework samples of human origin, according to specified protocols. The provided primer pools for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms included 33 and 29 body fluid specific targets, respectively, to identify blood, saliva, semen, vaginal secretion, menstrual blood and skin. The results demonstrated moderate to high count values in the body fluid or tissue of interest with little to no counts in non-target body fluids. There was some inter-laboratory variability in read counts, but overall the results of the laboratories were comparable in that highly expressed markers showed high read counts and less expressed markers showed lower counts. We performed a partial least squares (PLS) analysis on the data, where blood, menstrual blood, saliva and semen markers and samples clustered well. The results of this collaborative mRNA massively parallel sequencing (MPS) exercise support targeted mRNA sequencing as a reliable body fluid identification method that could be added to the repertoire of forensic MPS panels.

A large number of new microhaplotype loci were identified in the human genome by applying a directed search with selection criteria emphasizing short haplotype length (<120 nucleotides) and maximum levels of polymorphism in the composite SNPs. From these searches, 107 autosomal microhaplotypes and 11 X chromosome microhaplotypes were selected, with well-spaced autosomal positions to ensure their independence in relationship tests. The 118 microhaplotypes were assembled into a single multiplex assay for the analysis of forensic DNA with massively parallel sequencing (MPS). A single AmpliSeq-adapted primer set was made for Illumina MiSeq and Thermo Fisher Ion S5 MPS platforms and the performance of the assay was comprehensively evaluated in both systems. Five microhaplotypes showed critical sequencing failures in both MPS platforms and were removed, while a further 13 required manual checks and the application of sequence quality thresholds in one or both systems to ensure the successful analysis of low-level DNA in these loci. The targeting of short microhaplotype spans during marker selection, with an average length of 51 nucleotides in the 118 loci, led to a high level of sensitivity for the panel when sequencing the very degraded DNA typically encountered in forensic casework and the identification of missing persons.