Multiplexed bacterial tag-encoded FLX amplicon pyrosequencing was performed using the Titanium platform (Roche Applied Science, Indianapolis, IN, USA) as previously described20 (link) in a commercial facility (MRDNALab. Shallowater, TX, USA). Briefly, a single step PCR with broad-range universal primers and 22 cycles of amplification was used to amplify the 16S rRNA genes as well as to introduce adaptor sequences and sample-specific bar-code oligonucleotide tags into the DNA. Two regions of the 16S rRNA genes were sequenced: V1–V3 and V7–V9. The primers used for sequencing have been previously described21 (link). Adaptor sequences were trimmed from raw data with 98% or more of bases demonstrating a quality control of 30 and sequences binned into individual sample collections based on bar-code sequence tags, which were then trimmed. Sequences <300 bp were discarded and the rest were clustered into species- level operational taxonomic units (s-OTUs) at 97% sequence similarity and assigned a taxonomic identity by alignment to locally hosted version of the HOMD database using the Blastn algorithm. Analyses were conducted using the QIIME pipeline22 (link), as well as our own internally developed analysis pipeline PhyloToAST23 . Results were visualized using the Python library matplotlib. Phylogenetic tree data was visualized through the Interactive Tree Of Life webserver24 (link).
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