Computational protocol: Analysis of Bacterial Community Composition of Corroded Steel Immersed in Sanya and Xiamen Seawaters in China via Method of Illumina MiSeq Sequencing

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[…] The raw fastq files were demultiplexed and quality-filtered using QIIME (version 1.9.1) () with the following criteria: (i) The output data (reads) were truncated at any site receiving an average quality score < 20 over a 50 base pair (bp) sliding window. (ii) Primers were matched exactly allowing a two nucleotide mismatching, and reads containing ambiguous bases were removed. (iii) Sequences whose overlap was longer than 10 bp were merged according to their overlap sequence. Operational taxonomic units (OTUs) were clustered with a 97% similarity cut-off using UPARSE version 7.1 (). The normalization process followed OTU clustering. Chimeric sequences were identified and removed using UCHIME (; ). The taxonomy of each 16S rRNA gene sequence was analyzed with RDP Classifier () against the Silva (SSU128) 16S rRNA database using a confidence threshold of 70% ().The relative abundances of the phylum, class and genus levels were plotted as a bar graph. Heatmaps based on the relative abundance of OTUs at the phylum and genus levels were also generated with R program (). A venn diagram was created using Mothur v.1.30.1 () to identify the similarities and differences of the communities in the three kinds of samples (sample SOH, samples from Sanya, and samples from Xiamen). In alpha diversity analysis, alpha diversity parameters such as Chao, Ace, Simpson, and Shannon were estimated using mothur (version v.1.30.1) with a 97% similarity cut-off (). They provided a means of evaluating the potential total number of OTU and an estimate of the level of diversity in each sample. Rarefaction curves based on these metrics were generated. In beta diversity analysis, differences in the bacterial communities among the nine samples were preformed by a hierarchical cluster tree created using the unweighted pair-group method with arithmetic mean (UPGMA). A principal co-ordinates analysis (PCoA) plot was also obtained using Mothur with the calculation of Bray–Curtis (). […]

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