16S Ribosomal Gene Amplification and Sequencing

Ribosomal 16S rRNA gene sequences were amplified using the 16S Metagenomics Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA), consisting of primer pools to amplify multiple variable regions (V2-4–8 and V3–6) [7 (link),8 (link),9 (link)] of the 16S rRNA. The libraries were created using the Ion Plus Fragment Library Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Barcodes were added to each sample using the Ion Xpress Barcode Adapters kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Emulsion PCR and sequencing of the amplicon libraries were performed on an Ion 520 chip (Ion 520^TM Chip Kit) via the Ion Chef System and Torrent S5^TM system, respectively, using the Ion 520^TM/530^TM Kit-Chef (Thermo Fisher Scientific Inc., Waltham, MA, USA). Base calling and run demultiplexing were performed using Torrent Suite^TM Server software (Thermo Fisher Inc., Waltham, MA, USA), version 5.4.0, with default parameters for 16S Target Sequencing (bead loading ≤ 30, key signal ≤ 30, and usable sequences ≤ 30).