Ion 316tm chip v2 bc

Obtained PCR products were used to prepare libraries for diversity analyses by next generation sequencing (NGS) approach on Personal Genome Machine (Life Technologies, Carlsbad, CA, USA) according to Milani et al. [71 (link)]. 200 ng of DNA from each sample was used to prepare sequencing libraries by NEBNext® Fast DNA Library Prep Set kit (New England Biolabs, Ipswich, MA, USA) according to manufacturer’s protocol. The Ion Xpress Barcode adapters (Thermo Fisher Scientific, Waltham, MA, USA) were used to label each sample. The adaptor ligated libraries were purified and simultaneously size-selected using AMPure XP bead sizing (Beckman Coulter, Brea, CA, USA). The barcoded libraries were pooled in equimolar amount (about 26 pM). The pool of libraries was used to prepare sequencing template by emulsion PCR on Ion Sphere Particles (ISPs) using Ion PGMTM Hi-QTM View OT2 400 Kit (Thermo Fisher Scientific) in Ion OneTouchTM 2 instrument. The enrichment of the template positive ISPs were performed on Ion OneTouchTM ES instrument. The enriched template positive ISPs were then loaded in Ion 316TM Chip v2 BC (Thermo Fisher Scientific). The sequencing was then performed on an Ion Torrent PGM sequencer (Thermo Fisher Scientific, Waltham, MA, USA) using Ion PGMTM Hi-QTM View Sequencing solutions kit (Thermo Fisher Scientific).