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Massively parallel sequencing of whole mitochondrial genomes: Croatian population study

Mitochondrial genomes are particularly suitable for forensic analysis, due to their stable circular structure, small size (16569 bp), and large copy number per cell. During evolution, mtDNA accumulated variations throughout the sequence, thereby differentiating thousands of mitochondrial haplogroups known today. From these variations it is possible to decipher population’s matrilineal origins and historical migrations. In forensics, in order to assign a certain weight of evidence to mtDNA profile, a population study is the necessary prerequisite for establishing haplogroup frequencies. Therefore, we aimed at sequencing whole mt genomes in a representative sample of Croatian population, comprising 299 volunteers. DNA was extracted from buccal swabs, purified and quantified. Long-range PCR approach was adopted for amplification of whole mtDNA in two fragments (9, 1 kb and 11, 2 kb). Libraries were prepared using Nextera XT Library Prep Kit (Illumina). Normalized and pooled libraries were sequenced on MiSeq instrument (Illumina). Sequencing and quality metrics strongly correlated with manufecturer’s specifications, sometimes even exceeding the specified values. Our data were of sufficient quality in order to produce confident haplogroup determination. The composition of Croatian population sample displayed the prevalence of mt haplogroups from branch H (36%). Second most common haplogroups came from U and K branches (29%, combined), and J and T branches (16%, combined). Furthermore, almost 28% of samples contained point heteroplasmies with minor allele frequencies ≥10%, which is comparable with the level of heteroplasmy detection by Sanger sequencing. The most common heteroplasmy was 16093Y (in 10 out of 83 heteroplasmic samples). Detection of heteroplasmies is also a subject of interest in forensics, which could strengthen the power of discrimination in mtDNA analysis. Overall, frequencies of mt haplogroups detected in our samples are concordant with the distribution of haplogroups in Europe. It is our intention that this population study forms the basis of a database to be used for purposes of forensic identification, when such need arises in casework. We also propose that mt haplotypes detected in our population sample contribute to further development and branching of the global phylogenetic mtDNA tree.

Whole mtDNA sequencing, MiSeq, Population study, Mitochondrial haplogroups, Croatia, Heteroplasmy

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