Next-generation mtDNA sequencing

DNA extracted from cells and tissues was assessed for quality and purity using an Agilent TapeStation analyzer. mtDNA sequencing was performed upon amplification of mitochondrial genomes from 1 to 10 μl of whole genomic DNA extracted from blood, cells, tumor tissues, and surrounding normal kidney tissues using the QIAseq Targeted DNA Panel (DHS-105Z Human Mitochondria Panel, QIAGEN) according to the manufacturer’s instructions. The resulting libraries were pair-end sequenced on a MiSeq instrument (Illumina). Read processing, alignment, and variant calling were carried out on the QIAseq Targeted Sequencing Data Analysis Portal with the Biomedical Genomics Analysis plugin (QIAGEN).

Variants below 0.03 variant allele fraction were filtered out, and variant lists were compared between tumor and normal DNA samples (blood or normal kidney) to identify somatic changes. Sorting Intolerant From Tolerant (SIFT) (80), Protein Variation Effect Analyzer (PROVEAN) (81), PolyPhen-2 (82), and MutationAssessor (83) algorithms were used together to predict the consensus pathogenicity of missense mutations detected in FH-deficient cell lines and tumors.