Computational protocol: Effect of Streptomycin Treatment on Bacterial Community Structure in the Apple Phyllosphere

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Protocol publication

[…] The sequences were screened against chloroplast and other non-ribosomal DNA using BLASTn and the Ribosomal Database Project Classifier , . Potential chimeras were identified using Mallard and omitted from further study after manual verification . Short sequences that did not cover the reference E. coli base positions 103 to 920 were also omitted from further study. The remaining sequences were aligned using the SILVA aligner (25) and imported into ARB . Using a filter to exclude the nonoverlapping ends beyond positions 103 to 920, a neighbor-joining matrix was generated in ARB and exported. The fasta file from these same sequences was also exported and used for taxonomic classification of the bacterial communities using the RDP Classifier.Mothur was used to assign operational taxonomic units (OTUs) at the 97% sequence similarity level and to calculate Shannon and Simpson’s diversity indices, Pielou’s index of evenness, and rarefaction curves. For beta diversity analyses, PhyML was used to generate a maximum likelihood tree of all the sequences . The settings used were: the general time reversible (GTR) correction model; optimized equilibrium frequencies; estimated proportion of invariable sites, four substitution rate categories; estimated gamma shape parameter; and SPR tree topology search operations. The validity of using the GTR correction model was also confirmed by applying jModelTest2 on smaller subsets of the 16S rRNA gene dataset . Due to the computer intensive nature of the software, the full dataset was not processed. The tree generated by PhyML was then imported into mothur, and the weighted UniFrac metric that accounts for phylogenetic relationships among taxa was calculated and visualized in a Principle Coordinate Analysis (PCoA) plot. Hypothesis testing to determine which variables influenced bacterial community structure at the different orchards was performed on weighted UniFrac using the analysis of similarity (ANOSIM) with 10,000 iterations. A Mantel test with the Pearson correlation coefficient and 1000 iterations was used to determine the effect of relative distances between orchard sites on the bacterial communities at each sampling site. The hypothesis tests were repeated using Bray-Curtis distance (non-phylogenetic) defining OTUs at the 97%, 99%, and 100% (i.e., unique sequences) similarity thresholds. The Bonferroni correction was applied to correct for multiple comparisons. […]

Pipeline specifications

Software tools BLASTN, RDP Classifier, mothur, PhyML, jModelTest
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Malus domestica
Chemicals Streptomycin