The V1-V3 region of the 16S rRNA gene were amplified using primers 5′-AGAGTTTGATCCTGGCTCAG-3′ and 5′-ATTACCGCGGCTGCTGG-3′ as previously described44 (link), 45 . The primers contained an adapter sequence and unique barcodes. The positive controls for all runs were purified. Anaerotruncus colhominis DNA, reagent controls and non-template controls were set up for each PCR run. Following amplification and purification, the amplicons were pooled at equimolar concentrations and sequenced on the 454 GS FLX Titanium Sequencing Platform (Roche, USA) as described elsewhere44 (link), 45 at the Genome Institute (University of Washington in St. Louis, MO, USA).
Quality control and data processing were performed using in-house pipelines at the Jackson Laboratory. Briefly, reads with length <200 bp and/or with more than a single ambiguous base call were discarded. Chimeric sequences and reads without the adapter sequences were also removed. Reads from the same samples were binned based on barcode and then the barcode, adapter and primer sequences at both terminals were trimmed. Samples with read depth less than 500 were discarded based on rarefaction analysis of all the samples. Alignment and taxonomic classification (Phylum to Genus) of the reads was carried out with the Ribosomal Database Project Naïve Bayesian Classifier using a 0.5 filter.
Quality control and data processing were performed using in-house pipelines at the Jackson Laboratory. Briefly, reads with length <200 bp and/or with more than a single ambiguous base call were discarded. Chimeric sequences and reads without the adapter sequences were also removed. Reads from the same samples were binned based on barcode and then the barcode, adapter and primer sequences at both terminals were trimmed. Samples with read depth less than 500 were discarded based on rarefaction analysis of all the samples. Alignment and taxonomic classification (Phylum to Genus) of the reads was carried out with the Ribosomal Database Project Naïve Bayesian Classifier using a 0.5 filter.