engleski

Complete mitochondrial genomes from targeted, deep sequencing perspective

Mitochondrial DNA (mtDNA) analysis experienced revival when massively parallel sequencing (MPS) became widely accessible in science. In contrast to Sanger sequencing, MPS enables fast and cost-effective insight into complete individual mitochondrial genomes, typically covered by huge number of sequencing reads. The question is whether we can utilize discoveries propelled by the new technology, in order to enhance forensic investigations. We aim at providing some answers by exploring applicative potential of mtDNA analysis by deep sequencing. Our journey of building knowledge on both the technology and mitochondrial genetics started with sequencing of the whole mtDNA at ∼6500x average coverage, extracted from more than 300 reference buccal swab samples of Croatian citizens. We performed sequencing and data analysis using Illumina® solutions: NexteraXT Kit for library preparation, MiSeq v2 chemistry for sequencing on MiSeq FGx instrument and BaseSpace mtDNA applications for variant calling and visualization. We determined haplogroups by web application HaploGrep2 and by manual inspection according to human mtDNA phylogenetic tree (Phylotree, Build 17). Macrohaplogroup distribution of the Croatian population (Figure 1.) fits into general European phylogenetic context. Total of 199 haplogroups were assigned for 304 full mtDNA sequences. In several multi-member haplogroups, there is an indication for further branching of Phylotree. Since the full potential of deep sequencing comes to the fore in mtDNA heteroplasmy detection, we analyzed point heteroplasmy (PHP) distribution within our sample pool at minor allele frequency (MAF) above 1%. In total, we detected 407 PHPs, 79% of which are below approximate PHP threshold of Sanger sequencing1. Moreover, we identified 72% of PHP-positive samples, which is a huge increase compared to 6% typically found by Sanger-based approach in mtDNA control region of buccal cells2. The amount of information on mtDNA sequence produced by MPS is clearly overwhelming and creates the need to test the potential use in existing and novel forensic applications. Apart from primarily being used as a forensic database of whole mtDNA sequences in future forensic casework, the results of present work will also contribute to other scientific fields such as molecular medicine and anthropology. Figure 1. Distribution of mitochondrial haplogroups in Croatian population (middle pie chart) with substructure of the two most prevalent macrohaplogroups H (right) and U (left)

human mitochonrial genome ; deep sequencing ; mitochondrial heteroplasmy ; novel forensic markers

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