Ion 316 chip v2

Archaeal and bacterial hypervariable regions V4–V5 of the 16S rRNA gene were amplified and then sequenced according to the protocol described in Poirier et al. (2016 (link)) and Madigou et al. (2019 ), with some modifications. The targeted region was amplified by PCR with fusion primers 515F (5′‐Ion A adapter–Barcode–GTGYCAGCMGCCGCGGTA‐3′) (Wang et al., 2007 (link)) and 928R (5′‐Ion trP1 adapter–CCCCGYCAATTCMTTTRAGT‐3′) (Wang & Qian, 2009 (link)). The detailed protocol for library preparation is provided in the Supplementary Information (Section 1.2). Sequencing was performed on an Ion Torrent Personal Genome Machine using Ion 316 Chip V2 (Life Technologies) and Ion PGM Hi‐Q View Sequencing Kit (Life Technologies) according to the manufacturer's instructions. Sequencing data were processed with the Torrent Suite Software. All diversity analyses were performed with the R phyloseq package.

For each single genome amplicon, 100 ng of second round PCR product was enzymatically sheared to 400 bp followed by barcoding using the Ion Xpress Plus Fragment Library & Ion Xpress Barcode Adapters kits (LifeTechnologies, ThermoFisher Scientific). Quantification was performed using a 2100 Bioanalyzer (DNA 1000 kit, Agilent Technologies, Sunnyvale, CA). DNA size-selection was performed using Blue Pippin 2% dye free cassette with internal standard marker V1 (Sage Science). The size-selected product was equalized using the Ion Equalizer kit (LifeTechnologies, ThermoFisher Scientific) following manufacturer’s instructions. All purifications used Agencourt AMPure XP Reagent (Beckman Coulter).
Emulsion PCR (ePCR) and enrichment for 400 bp sheared product used the Ion OneTouch 400 bp Template kit (LifeTechnologies, Carlsbad, CA) on the OneTouch and ES instruments. Sequencing was carried out using the Ion PGM 400 kit and Ion 316 chip v2 on the IonTorrent PGM platform (LifeTechnologies, Carlsbad, CA), following manufacturer’s instructions. For scalability experiments, the ePCR/enrichment of libraries derived from 43 different amplicons were carried out on the Ion Chef instrument with Ion PGM Hi-Q Chef kit (LifeTechnologies, Carlsbad, CA) and were sequenced on Ion 318 chips v2 BC.