Over the last 10 years, next generation sequencing (NGS) became the gold standard for both diagnosis and discovery of new disease genes responsible for heterogeneous disorders, such as mitochondrial encephalomyopathies. The application of this technology to mtDNA mutations poses extra challenges compared to other genetic conditions because of the peculiarities of mitochondrial genetics and the requirement for proper NGS data management and analysis. Here, we describe a detailed, clinically relevant protocol to sequence the whole mtDNA and quantify heteroplasmy levels of mtDNA variants, starting from total DNA through the generation of a single PCR amplicon.
Mitochondrial DNA Sequencing and Heteroplasmy Quantification by Next Generation Sequencing / A. Legati, D. Ghezzi, C. Viscomi (METHODS IN MOLECULAR BIOLOGY). - In: Mitochondrial DNA / [a cura di] T.J. Nicholls, J.P. Uhler, M. Falkenberg. - [s.l] : Humana, 2023. - ISBN 978-1-0716-2921-5. - pp. 381-395 [10.1007/978-1-0716-2922-2_26]
Mitochondrial DNA Sequencing and Heteroplasmy Quantification by Next Generation Sequencing
D. GhezziSecondo
;C. ViscomiUltimo
2023
Abstract
Over the last 10 years, next generation sequencing (NGS) became the gold standard for both diagnosis and discovery of new disease genes responsible for heterogeneous disorders, such as mitochondrial encephalomyopathies. The application of this technology to mtDNA mutations poses extra challenges compared to other genetic conditions because of the peculiarities of mitochondrial genetics and the requirement for proper NGS data management and analysis. Here, we describe a detailed, clinically relevant protocol to sequence the whole mtDNA and quantify heteroplasmy levels of mtDNA variants, starting from total DNA through the generation of a single PCR amplicon.File | Dimensione | Formato | |
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