title: Amplicon Massively Parallel Sequencing of homo sapiens mitochondrial DNA: patients diagnosed with Tuberous Sclerosis Complex
design: High-quality DNA was isolated from 102 buccal swaps (BuccalFix Plus DNA Isolation Kit, Isohelix, UK) as well as other 46 tissue samples using standard methods following the manufacturers instructions (QIAamp Qiagen Mini Kit, cat#51306, Germany). The DNA concentration was assessed using a Qubit 4.0 fluorometer assay (Invitrogen by Thermo Fisher Scientific, Oregon, USA). High-depth, targeted sequencing analysis by amplicon based MPS for the entire mitochondrial DNA was performed for 148 samples (LC Sciences, LLC; VariantProTM -Mitochondrial Mutations Screening Panel, Houston, TX, USA), achieving median coverage of 7,349 reads. The assay uses 110 primer pairs (VariantPro primer design VariantPro primer pools) generating ~200bp amplicons and providing 100% amplicon coverage of the 16,569 bp of the human mitochondrial genome. Briefly, a total of 100ng DNA was used for library construction following the manufacturers instructions. The sequencing libraries were generated by VariantPro protocol (LC Sciences, USA) and sequenced on NovaSeq6000 (Illumina, San Diego, USA) with PE150 (paired-end 150bp). To minimize the risk of allelic dropout during PCR due to primers that are not able to bind, SNPs with a frequency >10% were not allowed in the primer priming region. The VariantProTM utilizes a nested PCR thermal cycling protocol: including 30 cycles to anneal specific primers and initial annealing of library primers. A total of 15 cycles were used for amplicon generation.  mtDNA variants with heteroplasmy levels >5% were included in the analysis, whereas variants in positions where common sequencing errors occur due to a homopolymer stretch (m.302315 and m.31053109) were excluded.