Mitochondrial Genome Sequencing and Annotation

Whole genomic DNA was extracted using Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich) according to the manufacturer’s protocols. Sanger sequencing of the entire mtDNA was performed following a quality-check protocol as previously described [49 (link)]. Mitochondrial DNA mutations were confirmed using a second PCR reaction. In silico prediction of the pathogenic potential of missense mutations was performed with PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) as previously described [50 (link)]. FASTA files were used as input for MToolBox [51 (link)] in order to annotate mitochondrial variants and related features, which read mapping, post-mapping processing, genome assembly, haplogroup prediction and variant annotation. Nucleotide site-specific variability was estimated on the multi-alignment of the updated healthy genomes reported in HmtDB [52 (link)] and in HmtVar [53 (link)].