Long-read sequencing of ATP-binding cassette sub-family A member 2 (ABCA2) cDNA from Pectinophora gossypiella
The symmetric barcode-tailed PCR primers were designed based on the PacBio¨ multiplex PCR primer guidelines. Prior to SMRTbellTM library preparation, ~5 kb PCR products were gel purified with QIAquick¨ Gel extraction kit (Qiagen, Germantown, MD) and quantified using QubitTM dsDNA High Sensitivity Assay Kit with the QubitTM 2.0 Fluorometer (Thermo Fisher Scientific). Barcoded samples (n = 22) were pooled in equimolar concentrations and SMRTbellTM libraries were prepared with 0.5 µg of the pooled amplicons following the standard procedures for blunt ligation of hairpin adapters using SMRTbellTM Template Prep Kit 1.0 (Pacific Biosciences). DNA sequencing was performed on the PacBio¨ RSII sequencer using P6-P4 enzyme chemistry at the University of Arizona, Arizona Genomics Institute (Tucson, Arizona). Data was captured using 3-hour movies.