mtDNA was amplified using 1 ng total DNA and long-range proofreading PCR (TaKaRa LR DNA Polymerase) with primers specific for the mitochondrial genome (Supplemental Table 1 ). 500 pg of mtDNA amplicon per sample were used for sequencing library preparation with Nextera XT reagents (Illumina) similar to previously described (Masser et al. 2013 (link)). Libraries were diluted to 4 nM and pooled for benchtop next generation sequencing (MiSeq, Illumina) using 600 cycle (2x250bp) reagents (Illumina) at a final library concentration of 12 pM. Detailed bioinformatics analysis methods are provided in the Supplemental Methods .
Protocol detail
Mitochondrial Genome Sequencing Pipeline
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Dna polymerase
Library
Mitochondrial genome
Mtdna
Primers
Corresponding Organization : Oklahoma Medical Research Foundation
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Variable analysis
independent variables
- Amount of total DNA used for amplification (1 ng)
- Mitochondrial DNA (mtDNA) sequencing data
- Primers specific for the mitochondrial genome
- Use of long-range proofreading PCR (TaKaRa LR DNA Polymerase)
- Amount of mtDNA amplicon used for sequencing library preparation (500 pg)
- Sequencing library preparation method (Nextera XT reagents)
- Sequencing platform (MiSeq, Illumina)
- Sequencing read length (2x250bp)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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