title: 16S rRNA gene amplicon of biofilms: pH treatments
design: The 16S rRNA gene amplicon sequencing was performed using primers 515F/806R (V4 region of the 16S gene) and the Earth Microbiome Project conditions (Version 4_13)1. All samples were sequenced on a single Illumina MiSeq run. Sequences were first processed in Qiime (version 1.9.1) using default parameters2 including minimum read length of 75 bp, min number of consecutive high quality base calls to include a read as a fraction of the input read length of 0.75, Phred quality score of 3, no ambiguous bases allowed, and no mismatches allowed in primer sequence3. All sequences kept for analysis were 151 bp. Sequence clustering (97% sequence similarity) into Operational Taxonomic Units (OTUs) was done using the SILVA (version 119) reference library4 and UCLUST5 following the open-reference Operational Taxonomic Unit (OTU) picking protocol. Taxonomy assignments were done using BLAST against the SILVA database (max-e value = 0.001)6. Subsampling and rarefactions (10 times) were performed to equal read depths of 8,000 per sample, and samples below that threshold were removed. After rarefaction, all 10 OTU tables were merged and exported for further processing in R7. The rarified biom file was processed using the phyloseq package8.