16S rRNA sequencing
For 16S rRNA profiling, primers were designed to amplify variable regions 2, 4, and 8 in a single tube with the resulting amplicon fragments of ~250 base pairs (bp), ~288 bp, and ~295 bp, respectively. In a second single tube, a multiplex PCR reaction targets variable regions 3, 6-7, and 9 with resulting amplicon fragments of ~215 bp, ~260 bp, and ~209 bp, respectively.  The PCR amplification products were used to create a library via the Ion Plus Fragment Library Kit with sample indexing using the Ion Xpress Barcode Adapters 1-96 Kit. Template preparation was performed using the Ion OneTouch 2 System. Sequencing was conducted on the Ion Torrent S5 sysytem.
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