2.7. 16S rRNA-based polymerase chain reaction, Illumina library preparation, and data analysis

The 16S gene was amplified by PCR as previously described [2]. Next, PCR primers targeted the 338–806 bp region of the 16S rRNA encoding gene (Table 1) that also contained Illumina 3′ adapter sequences as and 12-bp barcodes. Sequencing was carried out on an Illumina MiSeq DNA sequencer at Argonne National Laboratory. Sequences were trimmed and classified with the QIIME toolkit (version 1.8.0). Using the QIIME wrappers, OTUs were picked at 97% sequence identity for each OTU. Representative sequences were aligned using PyNAST and taxonomy assigned using uclust. The PyNAST-aligned sequences were utilized for phylogenetic analysis and weighted UniFrac distances.