Miseq dna platform

To assess bacterial community structure, primers specific for 16S rRNA V4–V5 region (Forward: 338F: 5′-GTGCCAGCMGCCGCGGTAA-3′ and Reverse: 806R: 5′-GGACTACHVGGGTWTCTAAT-3′) that contained Illumina 3′ adapter sequences as well as a 12-bp barcode were used. Sequences were generated by an Illumina MiSeq DNA platform at Argonne National Laboratory and analyzed by the program Quantitative Insights Into Microbial Ecology (QIIME) [47 (link)]. Operational Taxonomic Units (OTUs) were picked at 97% sequence identity using open reference OTU picking against the Greengenes database (05/13 release) [48 (link)]. OTUs were quality filtered based on default parameters set in the open-reference OTU command in QIIME and sequences were rarified to an equal sampling depth of 5000 reads per sample. Representative sequences were aligned via PyNAST [47 (link)], taxonomy assigned using the RDP Classifier [49 (link)], and a phylogenetic tree was built using FastTree [50 (link)]. Beta Diversity is represented by measuring UniFrac distances calculated using both weighted and unweighted algorithms and visualized with PCoA plots generated in Emperor.

To assess bacterial community structure, primers specific for 16S rRNA V3-V4 region (forward: 341F: 5′- CCTACGGGNGGCWGCAG −3′ and reverse: 805R: 5′- GACTACHVGGGTATCTAATCC −3′) that contained Illumina 5′ adapter sequences, as well as a 12-bp barcode, were used. Sequences were generated by an Illumina MiSeq DNA platform at University of Wisconsin-Madison Biotech Center, followed by AxyPrep Mag PCR clean-up (Axygen biosciences), quality and quantity assessment of finished libraries using Agilent 4200 TapeStation DNA 1000 kit Qubit dsDNA HS Assay Kit. Paired-end reads were processed using QIIME2 version 2022.8. Sequences were imported, trimmed, dereplicated and denoised through DADA2 to generate ASVs. Taxonomy was assigned using a naive-bayes classifier, trained with the 341F/805R primers above against Greengenes 13_5. Beta-diversity was calculated using Bray-Curtis group differences assessed using PERMNOVA and ANOSIM. Pattern Search was performed using Pearson correlations coefficient on relative abundances.