IonTorrent S5 sequence reads of S griseoincarnatus R-35
Library prepartion and sequencing was performed by the Central Analytical Facilities at the Stellenbosch University as per follows: library preparation was performed from 50ng purified PCR product for each sample using the Ion Plus Fragment Library Kit (ThermoFisher Scientific) according to the protocol; (Ion Xpress Plus gDNA Fragment Library Preparation User Guide). Briefly, DNA was fragmented using the Covaris S2 focused ultrasonicator (Covaris, Inc.; Woburn, MA, USA) with a 10% duty cycle, 5% intensity and 200 cycles/burst in two cycles; consisting of 60sec treatment, each. Sheared DNA was purified using 1.8x volume AMPure XP reagent (Beckman Coulter, Brea, CA, USA). The fragmented DNA was end-repaired at room temperature for 20 min and purified using 1.8x volume AMPure XP reagent (Beckman Coulter). End-repaired DNA was blunt-end ligated to IonCode Barcode Adapters (ThermoFisher Scientific) at 22C for 15min and purified, using 1.4x volume AMPure XP reagent (Beckman Coulter). The adapter-ligated libraries were amplified across nine polymerase chain reaction (PCR) cycles. PCRs were performed on the SimpliAmp Thermal Cycler (ThermoFisher Scientific). The amplified libraries were size-selected on the Pippin Prep (Sage Science, Beverly, MA, USA) using 2% dye-free gel cassettes with marker L, to retain DNA fragments of ~275bp. The size selected libraries were purified, using 1.8x volume AMPure XP reagent (Beckman Coulter) and assessed for fragment size-distribution on the LabChip GX Touch 24 Nucleic Acid Analyzer (PerkinElmer, Waltham, MA, USA), using the X-Mark DNA LabChip and HT DNA NGS 3K Reagent Kit according to the manufacturers protocol; NGS 3K Assay Quick Guide. The final, purified-libraies were quantified using the Ion the Ion Library TaqMan Quantitation Kit; according to the manufacturers protocol (Ion Library TaqMan Quantitation Kit User Guide). Quantitative PCR amplification was performed using the StepOnePlus Real-Time PCR System (ThermoFisher Scientific). The library was diluted to a target concentration of 60pM for template preparation using the Ion 540 Chef Kit (ThermoFisher Scientific). Twenty five microliters of diluted, pooled barcoded library was loaded onto the Ion Chef liquid handler for template preparation and enrichment using Ion 540 Chef Reagents, Solutions and Supplies according to the protocol, MAN0010851 F.0. Enriched ion sphere particles were loaded onto an Ion 540 Chip. The library was diluted to a target concentration of 60pM for template preparation using the Ion 540 Chef Kit (ThermoFisher Scientific). Twenty five microliters of diluted, pooled barcoded library was loaded onto the Ion Chef liquid handler for template preparation and enrichment using Ion 540 Chef Reagents, Solutions and Supplies according to the protocol, MAN0010851 F.0. Enriched ion sphere particles were loaded onto an Ion 540 Chip. Massively parallel sequencing was performed on the Ion GeneStudio S5 Prime System (ThermoFisher Scientific, Waltham, MA, USA) using Sequencing Solutions, Reagents according to the protocol, MAN0010851 F.0.