Computational protocol: Comparison of community structures of Candidatus Methylomirabilis oxyfera-like bacteria of NC10 phylum in different freshwater habitats

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Protocol publication

[…] High-throughput sequencing of bacterial 16S rRNA genes was performed on the Illumina MiSeq platform (Hangzhou Guhe Information and Technology Co., Ltd., Zhejiang, China). Briefly, the V3-V4 region of bacterial 16S rRNA genes was amplified by the primer 319f (5′-ACTCCTACGGGAGGCAGCAG-3′) and primer 806r (5′-GGACTACHVGGGTWTCTAAT-3′) as previously described. Briefly, PCRs were performed in a 30 μl mixture containing 0.5 μl Dimethyl sulfoxide, 1.0 μl forward primer (10 mM), 1.0 μl reverse primer (10 mM), 5.0 μl DNA template, and 15.0 μl Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB). The thermal program was as follows: 98 °C for 30 s; 30 cycles of 98 °C for 15 s, 58 °C for 15 s, and 72 °C for 15 s; and a final extension at 72 °C for 1 min. PCR products were purified using an agarose gel DNA purification kit (Qiagen, Chatsworth, California, USA) and then were subjected to Illumina MiSeq sequencing (2 × 300 bp). Analysis of Mi-Seq sequencing data was conducted using Quantitative Insights Into Microbial Ecology (QIIME, according to Shen et al.. High-quality NC10 phylum bacterial sequences were further confirmed by aligning these sequences with the reported NC10 sequences deposited in GenBank using the BLAST search engine. Representative sequences for each operational taxonomic unit (OTU) as defined by 97% sequence identity were obtained for further analysis. The coverage of NC10 bacteria in each sample was calculated as C = [1 − (n1/N)] × 100, where n1 is the number of unique OTUs and N is the total number of sequences in the sample. [...] Phylogenetic analysis of the recovered NC10-related 16S rRNA gene sequences was performed by Mega 5 software using the neighbour-joining method, and the evolutionary distances were computed using the Maximum Composite Likelihood method. The robustness of the tree topology was tested with a bootstrap analysis (1000 replicates), and bootstrap values >50 (500 replicates) are shown at the branches. [...] The geographical distribution of NC10 bacterial communities and their potential correlations with environmental factors were determined using principal components analysis (PCA) and canonical correspondence analysis (CCA), respectively, using the CANOCO software. Venn diagrams were built using Venny 2.0 ( Pearson moment correlation analysis (significance level α = 0.05) was used to test for potential correlations between the NC10 bacterial diversity, abundance and different environmental factors, using the SPSS 18.0 software (SPSS, Chicago, Illinois, USA). […]

Pipeline specifications

Software tools QIIME, MEGA, VENNY, SPSS
Applications Miscellaneous, Phylogenetics, 16S rRNA-seq analysis
Organisms Escherichia coli, Bacteria
Chemicals Methane