title: individual 'COII-G177S mtDNA'  fly #10, re-isolated from original heteroplasmic w1118 stock, subjected to duplex sequencing of fly mitochondrial genome for sensitive mutation detection
design: Duplex Sequencing of fly mtDNA was performed as previously described with several modifications (Kennedy et al, Nature Protocols, 2014, PMID 25299156). Total DNA was purified from individual fly heads using a QIAamp DNA micro kit. The DNA was sonicated using a Covaris AFA S2 ultrasonicator with the following settings: Duty cycle: 10%; Intensity: 5; Cycles/burst: 100; Time: 15s x 3. The sheared DNA was then end-repaired and ligated using the NEBNext Ultra 2 end-repair and dA-tailing kit (New England Biolabs) according to the manufacturer’s instructions. Duplex Sequencing adapters, described previously (Kennedy et al 2014), were ligated to the DNA library using the NEBNext Ultra 2 ligation kit (New England Biolabs) according to the manufacturer’s instructions. 1.5 ng of total DNA was then PCR amplified using KAPA HiFi DNA polymerase (Roche) according to the manufacturer’s recommendations. After amplification, the mtDNA was enriched by targeted capture using xGen target capture probes (Integrated DNA Technologies) specific to the fly mitochondrial genome. The samples were then sequenced on an Illumina NextSeq500 with 150bp paired-end reads. The data were processed as previously described in Kennedy et al.