Computational protocol: Microbial Community Structure of Relict Niter-Beds Previously Used for Saltpeter Production

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[…] Raw 16S rRNA gene pyrotag sequences were qualitatively screened and trimmed with QIIME 1.7.0 , maintaining the sequence length at ≥300 nt. The sequences had an average quality score of ≥25. A denoising function was applied to the filtered sequence data . The denoised sequence data were grouped into OTUs with the UCLUST algorithm . The sequences exhibiting >97% identities were grouped into one OTU. The representative sequences of each OTU were aligned with PyNAST , and chimeric sequences were detected and removed using ChimeraSlayer . The taxonomic assignment of each OTU was performed with the RDP classifier trained by a dataset from Greengenes at a minimum confidence level of 0.8 . The sequence data were compared with the 16S rRNA database of RDP release 10 using the BLAST+ program . [...] The procedures for quality screening, trimming, denoising, and OTU selection were same as those described above for 16S rRNA gene pyrotag analysis, except that sequence identity (>85%) was determined according to the threshold of Pester et al. . The filtered sequences were manually checked after aligning with MEGA program . Chimeric sequences were detected and removed using the UCHIME algorithm on the FunGene platform . Sequence data were compared with the GenBank database using the BLAST+ program . [...] After taxonomy assignments, α-diversity indices (e.g., Chao1, Shannon, and Simpson) of the 16S rRNA and amoA genes pyrotag libraries were calculated by QIIME and EstimateS (version 9.1.0) software, respectively. The coverage values were calculated using equation [1– (n/N)], where n is the number of OTUs in a single read (singleton) and N is the total number of reads analyzed . The weighted UniFrac distances were calculated and used for principal coordinate analysis (PCoA) . Phylogenetic trees for the bacterial and archaeal amoA libraries for Fast UniFrac analysis were constructed with the MEGA program based on the neighbor-joining algorithm . PCoA and jackknife clustering analyses were performed on weighted and normalized data with QIIME and the Fast UniFrac program , respectively. To identify OTUs that exhibited significant differences in abundance between different soil samples, Welch’s t-test (confidence interval method: Welch’s inverted, [p<0.05] for two groups [definition of each group was described in the Results and Discussion]) having possibly unequal variances was performed with STAMP software , .The distance matrix tree of the bacterial and archaeal amoA genes, which is based on the maximum likelihood method, was constructed with the MEGA program . The topology of the trees was estimated by 1,000 bootstrap replicates . Previously reported amoA pyrotag sequences retrieved from soil environments in Austria, Costa Rica, Greenland, and Namibia were downloaded from the Sequence Read Archive (SRA) at NCBI (accession no. SRA047303) and used for the statistical and phylogenetic comparisons . Subclusters within the “Ca. Nitrososphaera” and “Ca. Nitrosopumilus” were defined according to Pester et al. . […]

Pipeline specifications