The effect of library preparation protocol on the efficiency of heteroplasmy detection in mitochondrial DNA using two massively parallel sequencing Illumina systems

Massively parallel sequencing (MPS) technology has become the gold standard in mitochondrial DNA research due to its high
sensitivity in detecting mtDNA heteroplasmy, a prognostic marker in various medical applications. Various MPS technologies
and platforms used for mtDNA analysis exist. Obtaining reliable and sensitive results requires deep and uniform coverage
of the entire mtDNA sequence, which is heavily infuenced by the choice of library preparation method and sequencing
platform. Here, we present a comparison of the sequencing coverage and the ability to heteroplasmy detection using two
library preparation protocols (Nextera XT DNA Library Preparation Kit and Nextera DNA Flex Library Preparation Kit) and
two diferent (MiSeq FGx and ISeq 100) Illumina MPS platforms. Our study indicates that the Nextera DNA Flex Library
protocol provides a more balanced coverage along the mitogenome and a reliable heteroplasmy detection with both MiSeq
and iSeq Illumina MPS systems