8518
For library preparation, RT-PCR products from 8 mitochondrial genes, including partial sequences of atp1, 4, 8 and 9, and cox1 and 3, and full sequences of atp6 and cox2 containing 5’ and 3’ UTR regions were pooled in an equimolar ratio to produce the pooled concentration of 30 nM per genotype.     Non-barcoded cDNA library was generated using the Ion XpressTM Plus Fragment Library Kit (Life Technologies, Foster City, CA) as described by the manufacturer’s protocol. One-hundred nanogram of equimolar pooled RT-PCR amplicons per genotype were prepared to be enzymatically fragmented by the incubation for 25 min at 37°C using Ion ShearTM Plus Reagents, and subsequently purified using the Agencourt® AMPure® XP Kit (Beckman Coulter, Brea, California, USA). The fragmented amplicon sizes were assessed using Agilent High sensitivity DNA Kit (Agilent Technologies, Santa Clara, California, USA) with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA). Adapter ligation and nick repair to the fragmented amplicons was performed and followed by the purification of adapter ligated amplicons employing the Agencourt® AMPure® XP Kit. The final cDNA library was eluted in 20 µL of low TE buffer and stored at –20°C. DNA concentration was determined utilizing the Qubit® dsDNA HS Assay Kit (Life technologies, Foster City, California, U.S) with the Qubit® 2.0 Fluorometer (Life technologies, Foster City, California, U.S). The final fragment size of the adapter ligated libraries per genotype was evaluated using Agilent High Sensitivity