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Mitochondrial DNA (mtDNA) is a small but significant part of the human genome, whose applicability potential has gradually increased with the advent of massively parallel sequencing (MPS) technology. Knowledge of the particular workflow, equipment, and reagents used, along with extensive usage of negative controls to monitor all preparation steps constitute the prerequisites for confident reporting of results. In this study, we performed an assessment of Illumina® Human mtDNA Genome assay on MiSeq FGx™ instrument. Through analysis of several types of negative controls, as well as mtDNA positive controls, we established thresholds for data analysis and interpretation, consisting of several components: minimum read depth (220 reads), minimum quality score (41), percentage of minor allele sufficient for analysis (3.0%), percentage of minor allele sufficient for interpretation (6.0%), and percentage of major allele sufficient for homoplasmic variant call (97.0%). Based on these criteria, we defined internal guidelines for analysis and interpretation of mtDNA results obtained by MPS. Our study shows that the whole mtDNA assay on MiSeq FGx™ produces repeatable and reproducible results, independent of the analyst, which are also concordant with Sanger-type sequencing results for mtDNA control region, as well as with MPS results produced by NextSeq®. Overall, established thresholds and interpretation guidelines were successfully applied for the sequencing of complete mitochondrial genomes from high-quality samples. The underlying principles and proposed methodology on the definition of internal laboratory guidelines for analysis and interpretation of MPS results may be applicable to similar MPS workflows, e.g. targeting good-quality samples in forensic genetics and molecular diagnostics.

Background: Optimized and efficient library preparation workflow is one of the most important prerequisites for obtaining high quality and quantity of results in massively parallel sequencing (MPS). Our aim was to assess and optimize different steps of Illumina® Nextera® XT assay for analysis of whole mitochondrial genomes.Methods and Results: Among the three long-range high-fidelity DNA polymerases tested here, PrimeSTAR® GXL performed best in aspects of specificity and yield for mitochondrial DNA (mtDNA) enrichment. Furthermore, library quantification combined with individual library-by-library dilution outperformed bead-based normalization in terms of more equal distribution of reads per library, reduced hands-on time and simplified workflow. Increasing the number of amplification cycles in the index-adapters-adding PCR step had no adverse effect on the level of sequencing noise, which remained low both in negative controls and in samples.Conclusions: Optimizations described herein provide beneficial insights for laboratories aiming at implementation and/or advancement of similar MPS workflows (e.g. small genomes, PCR amplicons and plasmids).

In order to implement X-chromosome short tandem repeat (X-STR) typing into routine forensic practice, reference database of a given population should be established. Therefore we extended already published data with additional 397 blood samples from unrelated Croatian citizens, and analyzed the total of 995 samples (549 male and 446 female) typed by Investigator® Argus X-12 Kit. To test genetic homogeneity of consecutively processed five historic-cultural regions covering the entire national territory, we calculated pairwise Fst genetic distances between regions based on allele and full haplotype frequencies. Since the comparison did not yield any statistically significant difference, we integrated STR profile information from all regions and used the whole data set to calculate forensic parameters. The most informative marker is DXS10135 (polymorphism information content (PIC = 0.929) and the most informative linkage group (LG) is LG1 (PIC = 0.996). We confirmed linkage disequilibrium (LD) for seven marker pairs belonging to LG2, LG3 and LG4. By including LD information, we calculated cumulative power of discrimination that amounted to 0.999999999997 in females and 0.999999005 in males. We also compared Croatia with 13 European populations based on haplotype frequencies and detected no statistically significant Fst values after Bonferroni correction in any LG. Multi- dimensional scaling plot revealed tight grouping of four Croatian regions amongst populations of southern, central and northern Europe, with the exception of northern Croatia. In this study we gave the first extensive overview of aberrant profiles encountered during Investigator® Argus X-12 typing. We found ten profiles consistent with single locus duplication followed by tetranucleotide tract length polymorphism. Locus DXS10079 is by far the most frequently affected one, presumably mutated in eight samples. We also found four profiles consistent with X-chromosome aneuploidy (three profiles with XXX pattern and one profile with XXY pattern). In conclusion, we established integral forensic Croatian X- chromosome database, proved forensic pertinence of Investigator® Argus X-12 Kit for the entire Croatian population and identified locus DXS10079 as a potential duplication hotspot.

The purpose of this study was to establish allele frequencies and forensic parameters for 20 STR loci (15 established STR loci and five additional ESS STR loci), as a maximum number of loci used for the production of the DNA profiles.