16S rDNA adult human feces
Bacterial V3-V4 regions of 16S ribosomal DNA (rDNA) and the archaeal 16S rDNA were amplified using primers described in Table 2. Amplicons were generated using a Fluidigm Access Array followed by high throughput sequencing on an Illumina MiSeq system (Illumina, IL, USA) performed at the Carver Biotechnology Center of the University of Illinois (Urbana, USA). Bioinformatic analysis was performed using R studio software and using rANOMALY package (Theil and Rifa, 2021). Prior to analysis, raw data were demultiplexed and quality filtered using DADA2 R-package (Callahan et al., 2016). Reads with N bases or low phred quality score (less than or equal 2) were eliminated and reads under 100 pb length were removed. Decontamination steps were carried out to filter out sequences corresponding to PhiX DNA used as a spike-in control for MiSeq runs. Filtered sequences were dereplicated and cleaned for chimeras (DADA2) and amplicon variant sequences (ASVs) were inferred using the dada function from DADA2 R-package. Taxonomic affiliation was performed with idtaxa function from DECIPHER package (Murali et al., 2018) using SILVA database release 138(Quast et al., 2013). A phylogenetic tree was constructed based on ASVs representative sequences using functions from phangorn package (Schliep, 2011).