Environmental 16S Amplicon Sequencing Protocol

Universal primers targeting the V4 hypervariable region of 16S ribosomal RNA (rRNA) genes were used to PCR amplify the gene fragments from DNA extracts from the environmental samples. We used a version of the 515F primer with a single-base change (in bold) to increase the coverage of certain taxonomic groups including the archaea (515F-Y, 5′-GTGYCAGCMGCCGCGGTAA; Parada et al., 2016 (link)). PCR reactions were carried out as described previously (Coskun et al., 2019 (link)). The 16S rRNA genes were subjected to dual-indexed barcoded sequencing of 16S rRNA gene amplicons on the Illumina MiniSeq as described previously (Pichler et al., 2018 (link)).
The MiniSeq reads (Pichler et al., 2018 (link)) were quality trimmed and assembled using USEARCH version 11.0.667 with the default parameters (Edgar, 2010 (link)). Reads were then de novo clustered at 97% identity using UPARSE; operational taxonomic units (OTUs) represented by a single sequence were discarded (Edgar, 2013 (link)). Taxonomic assignments were generated by QIIME 1.9.1 (Caporaso et al., 2010 (link)) using the implemented BLAST method against the SILVA rRNA gene database release 132 (Quast et al., 2013 (link)). The genera Pseudomonas, Ralstonia, Variovorax, or Streptococcus were also removed as these are common contaminants of molecular reagent kits (Salter et al., 2014 (link)) and we typically find these genera in DNA extraction blanks from our lab (Coskun et al., 2018 (link); Pichler et al., 2018 (link)). Statistical analyses and plots were performed using R. Studio version 3.3.0 (RStudio Team, 2015 ). The 16S rRNA gene sequence data is stored in the NCBI Short Read Archive under BioProject ID PRJNA888248.