DNA was isolated as previously described (Kumar et al., 2011b (link)) and multiplexed bacterial tag-encoded FLX 16S pyrosequencing was performed using the Titanium platform (Roche Applied Science, Indianapolis, IN, USA). Two regions of the 16S rRNA genes were sequenced: V1–V3 (spanning E.coli 16S gene regions 8–27 and 519–536) and V7–V9 (spanning E.coli 16S gene regions 1099–1114 and 1528–1541). The primers used for sequencing have been previously described (Kumar et al., 2011b (link)). Each primer is capable of detecting a range of genera that the other fails to recover. Together they allow the recovery of a wider range of the microbiome than is possible with a single primer alone. However, some genera are picked up by both primers. Thus, to prevent over counting, the number of sequences assigned to an OTU by both primers was reduced by half. Primer averaging was carried out as previously described (Kumar et al., 2011b (link)) using the implementation in the PhyloTOAST software suite (Dabdoub et al., 2016 (link)). Sequences with an average quality score of 30 over a sliding window of 50 bp and length >200 bp were assigned a taxonomic identity by alignment to the HOMD database (Chen et al., 2010 (link)) using the Blastn algorithm. Analyses were conducted using the QIIME (Caporaso et al., 2010 (link)) and PhyloToAST (Dabdoub et al., 2016 (link)) pipelines.