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

• Published in: Journal of applied genetics Vol. 65; no. 3; pp. 559 - 563
• Main Authors: Daca-Roszak, Patrycja, Fiedorowicz, Joanna, Jankowski, Maciej, Ciesielka, Marzanna, Teresiński, Grzegorz, Lipska-Zietkiewicz, Beata, Zietkiewicz, Ewa, Grzybowski, Tomasz, Skonieczna, Katarzyna
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.09.2024; Springer Nature B.V
• Subjects: Animal Genetics and Genomics; Biomedical and Life Sciences; DNA sequencing; DNA, Mitochondrial - genetics; Gene Library; Gene sequencing; Genome, Mitochondrial - genetics; Heteroplasmy; Heteroplasmy - genetics; High-Throughput Nucleotide Sequencing - methods; Human Genetics; Human Genetics • Short Communication; Humans; Life Sciences; Medical research; Microbial Genetics and Genomics; Mitochondrial DNA; Nucleotide sequence; Plant Genetics and Genomics; Platforms; Sensitivity analysis; Sequence Analysis, DNA - methods; Heteroplasmy detection; Nextera Flex; mtDNA; MPS library preparation; Nextera XT
• ISSN: 1234-1983; 2190-3883
• DOI: 10.1007/s13353-023-00821-4

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 influenced 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 different (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.