title: 16S rDNA human fecal sample M-ARCOL
design: The bacterial V3-V4 regions of 16S ribosomal DNA (rDNA) were amplified with primers V3_F357_N and V4_R805. Amplicons were generated using a Fluidigm Access Array followed by high-throughput sequencing using the Illumina MiSeq system (Illumina, San Diego, CA, USA) performed at the Carver Biotechnology Center of the University of Illinois Urbana-Champaign (Urbana, IL, USA). Bioinformatic analysis was performed using R software version 4.1.2 (1 November 2021) and the DADA2 pipeline [32]. Demultiplexed raw sequence data in fastq format were quality-filtered; reads with N bases were eliminated and reads under 50 bp length were removed. Decontamination steps were carried out to filter out sequences corresponding to PhiX DNA, used as a spikein control for MiSeq runs. Filtered sequences were dereplicated and cleaned for chimeras. Remaining sequences were dereplicated and amplicon variant sequences (ASVs) were inferred using the dada function from the dada2 package. A table with ASV counts for each sample and representative sequences of ASVs in fasta format were generated. Taxonomic affiliation of all ASVs were performed with the assignTaxonomy function using the SILVA database release 138.1 (80% bootstrap cut-off) as a reference. A phylogenetic tree was constructed based on ASV representative sequences using functions from the phangorn package and relative bacterial abundance was plotted at the phylum, family, and genus levels. For the - and -diversity analysis, unweighted and weighted Unifrac distance matrices were generated, and a redundancy analysis (RDA) ordination was used for visualization.