engleski

Mitochondrial DNA heteroplasmy pattern in swab samples of oral epithelium

Massively parallel sequencing (MPS) technology shed new light on the phenomenon of mitochondrial DNA (mtDNA) heteroplasmy, potentially leading to novel forensic applications. Therefore, we analyzed distribution of whole mitogenomic point heteroplasmies (PHPs) within sample pool of 301 buccal swabs obtained from Croatian citizens. After sequencing of libraries prepared by Nextera® XT Library Preparation Kit (Illumina®) on Illumina® MiSeq® FGx instrument, mitotypes were determined by Illumina® BaseSpace® Sequence Hub Applications (mtDNA Variant Processor and mtDNA Variant Analyzer). Here we report PHPs identified with at least ten reads for the minor allele that is represented above 1% detection threshold at specific mtDNA position. Positions exceeding Q40 combined score, accounting for base call quality scores, alignment score and the presence of low abundance reads, were considered. PHPs identified as sequencing artefacts appearing in majority of samples were discarded. Of analyzed samples, 28% do not display, while 33% harbors one PHP. Percentage of samples with PHP is inversely related with the number of PHPs per sample. The maximal number of PHPs per sample in our sample pool is nine (found twice). In total, 407 PHPs are discovered, 320 with minor allele frequency (MAF) ranging from 1% to 10%, and 87 above 10%. Considering mtDNA nucleotide positions, 214 of them exhibit MAF of 1-10% and 72 exhibit MAF>10%. Regardless of MAF, position 16093 is by far the most variable, with PHPs detected in 24 samples. We also identified seven additional control region PHP hotspots (≥ 7 PHPs per position). Coding region PHPs predominantly appear once across all samples. Five positions are affected twice and one position three times. PHP substitutions are mostly transitions (393) with merely 14 transversions. We will consider presented results as a starting point for optimization of PHP detection by both introducing experimental modifications and developing bioinformatics pipeline.

massively parallel sequencing, mitochondrial DNA, point heteroplasmy

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