Hypersuppressive mitochondrial DNAs are thought to be linear tandem repeats of the base unit of specific ORI regions on the mitochondrial genome. Here we confirm the linear tandem repeats using SMRT sequencing technology on four hypersuppressive clones. Mitochondrial DNA from four Saccharomyces cervisiae hypersuppressive mutants and a wild-type control was enriched and sequenced using SMRT sequencing technology.

The mtDNA isolation for SMRT sequencing was done using two phenol chloroform extractions on pellets enriched for mitochondria using differential ultracentrifugation. Isolated mtDNA was sequenced using SMRT sequencing technology on Pacific Biosciences Sequel II. 10kb insert size was selected for on 0.75% agarose Blue Pippin cassettes obtaining 12-13kb mean length inserts and mean read length of 6-7kb.