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Pipeline publication

[…] nto operational taxonomy units (OTUs) at 0.97 cut-off with UCLUST. Representative sequence of each OTU was then sent to RDP Classifier 2.1 for taxonomy identification. QIIME was used to generate the OTUs table for all samples. OTUs were filtered by abundance and frequencies, any OTU with less than 5 pyrotags and frequency lower than 50% (present in <50% of samples) were removed. Then remaining OTUs were used to calculate Spearman correlation with BFB. Those with Spearman coefficient value > 0.6 or <−0.6 (P-value < 0.01) were retained as bacterial species which were strongly correlated with BFB., Time series heat-map of BFB was generated with function ‘heatmap.2’ in R3.0 package ‘gplots’. One way ANOVA and post-hoc Tukey HSD tests on summer-autumn and winter-spring were conducted with R package. Spearman and Pearson correlation analysis were conducted to identify those operational parameters and water/sludge quality parameters which showed strong relationships with BFB. Pearson and Spearman coefficient index were calculated with function ‘rcorr’ in R3.0 package ‘Hmisc’.The Cytoscape3.0 was applied to generate the network between BFB and their correlated bacterial OTUs. The Spring-Embedded layout algorithm on edge value was used to cluster OTUs and BFB in the network. Canonical corresponding analysis (CCA) was generated by Canoco4.5., An EIN was a Bayesian network (BN) with both environmental parameters and microbial interactions as proposed in a study using EIN to predict the microbial community of ocean with time series data. To construct the EIN, all environmental parameters, selected OTUs and BFB were merged into one matrix; then this matrix was sent to learn the BN by Bayesian Network Inference with Java Objects (BANJO) v2.1 (http://www.cs.duke.edu/~amink/software/banjo/). Due to different units for environmental parameters, all the environmental parameters were transformed to 1 to 100 by the following equation for normalization, , where is the normalized value for parameter j at time i, is the observed value, MAX and MIN give the maximum and minimum values for parameter j across all time points., OTUs and BFB were all using relative abundance in the matrix. OTUs and BFB were selected by a standard that the average abundance should be larger than 0.01% and the presence across samples should be larger than 75%. After filtering, only the most abundant […]

Pipeline specifications

Software tools gplots, Cytoscape, BANJO