KWBD
uncultured bacterium
16S rRNA gene libraries were generated for each sample using end-point PCR. Briefly, 50µL reactions contained 1.25U DreamTaqTM polymerase, 0.2mM dNTP mixture (Fermentas), 0.5M Betaine, 1xDreamTaqTM Buffer (Fermentas), 250nM 27F and 125nM 338R. Cycling was carried out on a Techne thermal cycler (Techne, Burlington, NJ) using the conditions: 96°C for 3 minutes, followed by 30 cycles of 96°C for 30 seconds, 55°C for 45 seconds, 72°C for 45 seconds, and a final extension at 72°C for 10 minutes. Duplicate reactions were pooled, purified and concentrated using Amicon Ultra 30K filtration devices (Millipore) following manufacturer's instructions. Unique Titanium Fusion A and B barcodes (454 Life Sciences, Branford, CT) were added to PCR products using the primer set TiA-8nt-27F and TiB-338R from Stevenson et al. (2011), where the 8nt tag was unique to each 16S library: CAAGGTTC for KW, CAAGTCCA for PG, CAAGTCGT for KWBD, and CAAGTGCT for PGBD. The 50µL tagging reactions were performed as described above using 2µL purified PCR product and 50nM each of TiA-8nt-27F and Ti-B-338R primers with amplification for 6 cycles. Tagged 16S libraries were purified using the Wizard® PCR Preps DNA Purification System (Promega), quantified by Qubit fluorometry (Invitrogen), and equimolar amounts of each library were combined and pyrosequenced using the GS-FLX instrument (454 Life Sciences/Roche) at the Advanced Center for Genome Technology (OU, Norman, OK).    Sequences were analyzed using the bioinformatics software package, mothur (Schloss et al. 2009). An implementation of the Amplicon Noise algorithm (Quince et al. 2011) was used to reduce sequencing error; and those sequences containing ambiguities, homopolymers &gt; eight base pairs, or errors in the forward primer or barcode were removed. Unique sequences were trimmed to overlap a minimum of 200 base pairs and aligned against the SILVA reference alignment database (Pruesse et al. 2007) using the NAST-aligner (DeSantis et al. 2006). Those sequences within 2 base pairs of one another were pre-clustered to reduce the number of spurious operational taxonomic units (OTUs) resulting from PCR or sequencing errors (Huse et al. 2010) and screened for Chimeras using UChime (Edgar et al. 2011). A distance matrix was generated and used to cluster sequences into OTUs at the 97% similarity level using the average neighbour algorithm (Huse et al. 2010). A representative sequence from each OTU was assigned a taxonomic classification based on the naïve Bayesian classifier (Wang et al. 2006) and all richness and diversity measurements were calculated using the mothur software package (Schloss et al. 2009).    16S rRNA gene libraries were generated for each sample using end-point PCR. Briefly, 50µL reactions contained 1.25U DreamTaqTM polymerase, 0.2mM dNTP mixture (Fermentas), 0.5M Betaine, 1xDreamTaqTM Buffer (Fermentas), 250nM 27F and 125nM 338R. Cycling was carried out on a Techne thermal cycler (Techne, Burlington, NJ) using the conditions: 96°C for 3 minutes, followed by 30 cycles of 96°C for 30 seconds, 55°C for 45 seconds, 72°C for 45 seconds, and a final extension at 72°C for 10 minutes. Duplicate reactions were pooled, purified and concentrated using Amicon Ultra 30K filtration devices (Millipore) following manufacturer's instructions. Unique Titanium Fusion A and B barcodes (454 Life Sciences, Branford, CT) were added to PCR products using the primer set TiA-8nt-27F and TiB-338R from Stevenson et al. (2011), where the 8nt tag was unique to each 16S library: CAAGGTTC for KW, CAAGTCCA for PG, CAAGTCGT for KWBD, and CAAGTGCT for PGBD. The 50µL tagging reactions were performed as described above using 2µL purified PCR product and 50nM each of TiA-8nt-27F and Ti-B-338R primers with amplification for 6 cycles. Tagged 16S libraries were purified using the Wizard® PCR Preps DNA Purification System (Promega), quantified by Qubit fluorometry (Invitrogen), and equimolar amounts of each library were combined and pyrosequenced using the GS-FLX instrument (454 Life Sciences/Roche) at the Advanced Center for Genome Technology (OU, Norman, OK).    Sequences were analyzed using the bioinformatics software package, mothur (Schloss et al. 2009). An implementation of the Amplicon Noise algorithm (Quince et al. 2011) was used to reduce sequencing error; and those sequences containing ambiguities, homopolymers &gt; eight base pairs, or errors in the forward primer or barcode were removed. Unique sequences were trimmed to overlap a minimum of 200 base pairs and aligned against the SILVA reference alignment database (Pruesse et al. 2007) using the NAST-aligner (DeSantis et al. 2006). Those sequences within 2 base pairs of one another were pre-clustered to reduce the number of spurious operational taxonomic units (OTUs) resulting from PCR or sequencing errors (Huse et al. 2010) and screened for Chimeras using UChime (Edgar et al. 2011). A distance matrix was generated and used to cluster sequences into OTUs at the 97% similarity level using the average neighbour algorithm (Huse et al. 2010). A representative sequence from each OTU was assigned a taxonomic classification based on the naïve Bayesian classifier (Wang et al. 2006) and all richness and diversity measurements were calculated using the mothur software package (Schloss et al. 2009).