Microbiome Diversity Analysis Pipeline

Sequences were demultiplexed at the sequencing facility with the bcl2fastq v2.17.1.14 Conversion Software (Illumina, San Diego, CA), allowed 0 mismatches in the barcode sequences. De-multiplexed forward (read 1) and reverse reads (read 2) were further processed using the QIIME software package (27 (link)) The paired-end reads were merged, filtered and split into libraries as previously described (26 (link)). The representative operational taxonomic units (OTU) picking, chimera removing and construction of phylogenetic tree were performed as described by Monaco et al. (26 (link)). The representative sequence of each OTU was assigned to different taxonomic levels using Ribosomal Database Project naïve Bayesian rRNA Classifier (28 (link)) at 80% confidence level on the Greengenes reference database v.13.8. An OTU table was created and further filtered to remove non-aligned and chimeric OTUs and singletons. Alpha diversity (observed OTUs, Chao1 and Shannon and Simpson reciprocal indices) and beta diversity analysis was performed from the filtered OTU table after rarefying to 26,350 reads for each sample.