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 assessment of Illumina® Human mtDNA Genome assay on MiSeq FGx TM 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 TM produces repeatable and reproducible results, independent of 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 sequencing of complete mitochondrial genomes from high-quality samples. The underlying principles and proposed methodology on definition of internal laboratory guidelines for analysis and interpretation of MPS results may be applicable to similar MPS workflows, primarily in forensic genetics and molecular diagnostics.

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.

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The aim of the study was to assess forensic pertinence of 12 short tandem repeats (STRs) on X-chromosome in south Croatia population. Investigator Argus X-12 kit was used to co-amplify 12 STR loci belonging to four linkage groups (LGs) on X-chromosome in 99 male and 98 female DNA samples of unrelated donors. PCR products were analyzed by capillary electrophoresis. Population genetic and forensic parameters were calculated by the Arlequin and POPTREE2 software, and an on-line tool available at ChrX-STR.org. Hardy-Weinberg equilibrium was confirmed for all X-STR markers in female samples. Biallelic patterns at DXS10079 locus were detected in four male samples. Polymorphism information content for the most (DXS10135) and the least (DXS8378) informative markers was 0.9212 and 0.6347, respectively. In both male and female samples, combined power of discrimination exceeded 0.999999999. As confirmed by linkage disequilibrium test, significant association of marker pair DXS10074-DXS10079 (P = 0.0004) within LG2 and marker pair DXS10101-DXS10103 (P = 0.0003) within LG3 was found only in male samples. Number of observed haplotypes in our sample pool amounted 3.01, 7.53, 5 and 3.25% of the number of possible haplotypes for LG1, LG2, LG3 and LG4, respectively. According to haplotype diversity value of 0.9981, LG1 was the most informative. In comparison of south Croatia with 26 world populations, pair-wise [Formula: see text] values increase in parallel with geographical distance. Overall statistical assessment confirmed suitability of Investigator Argus X-12 kit for forensic casework in both identification and familial testing in the population of south Croatia.