Ion Torrent PGM sequencing
NA
Libraries were prepared for genomic DNA sequencing using the Ion Xpress Plus gDNA Fragment Library kit, with Ion Shear Plus reagent fragmentation for 300-base read libraries, according to the manufacturer�۪s instructions. In brief, 1 �g genomic DNA was fragmented for approximately 10 minutes using the Ion Shear Plus protocol, an enzymatic fragmentation generating blunt-ended fragments ready for adapter ligation with no additional end repair step required. Fragment size was assessed using the Agilent Bioanalyzer with Agilent High Sensitivity DNA kit, and median fragment sizes were found to be approximately 390 bp.  Barcoded adapters were ligated and nick repair performed, and the resulting barcoded libraries were run on an E-gel SizeSelect 2 % Agarose gel (Invitrogen), and DNA of approximately 450 bp was extracted. This was then subject to 5 cycles of PCR amplification before being assessed using the Agilent High Sensitivity DNA kit with the Agilent 2100 Bioanalyzer. Libraries were then pooled at equimolar concentrations, and diluted to 20 pM in preparation for sequencing. In accordance with the recommended protocols provided, sequencing template preparation was performed using the Ion OneTouch system in conjunction with the Ion PGM Template OT2 400 kit, where template positive Ion Sphere Particles (ISPs) were prepared and subsequently enriched.  Sequencing was then performed on an Ion Personal Genome Machine System, using an Ion 318 Chip v2 with the Ion PGM Sequencing 400 Kit.