16S rRNA sequencing and microbiome analysis

PCR amplification was performed using the Phusion High-Fidelity DNA polymerase with primers designed to flank the V4/V5 region of the 16S rRNA gene. Samples were submitted to the Genomics and Sequencing Center at the University of Rhode Island for PrepX NGS library preparation. Amplicons were sequenced using the Illumina MiSeq platform, yielding paired-end, 250-base-pair reads.

DADA2 pipeline [66] was used in R (version 3.3.4) and truncated reads where average Phred scores <30. The RDP classifier algorithm with the RDP training set 14 was used to perform taxonomic assignment [67]. ASV table was imported into R using phyloseq package [68]. Bar plots were made by converting sample counts into percentages to account for variations in sampling depth and exported into Prism software (GraphPad). Principal Coordinates of Analysis (PCoA) plots were generated using phyloseq and normalized by converting counts into relative abundance. Distance matrices were generated using both weighted and unweighted UniFrac distance metrics [69].