Computational protocol: Microbial Diversity and Structure Are Drivers of the Biological Barrier Effect against Listeria monocytogenes in Soil

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Protocol publication

[…] The generated sequence data were processed by the quantitative insights into microbial ecology (QIIME) pipeline []. Briefly, sequences that were less than 300 bp or greater than 500 bp in length, that presented mismatches in the primer sequences, or ambiguous bases were discarded. The remaining sequences were assigned to samples according to their unique MIDs. Chimeric sequences were removed and sequences were clustered into Operational Taxonomic Units (OTUs) (97% sequence similarity) using Usearch quality filter (http://www.drive5.com/usearch/) [] and the gold. fa reference set. A representative sequence for each OTU was selected based on the most abundant sequence in each OTU. This representative sequence was used for taxonomic identification using the Ribosomal Database Project (RDP) and the reference data sets from Greengenes. Phylogenetic alignment of sequences was done with the PyNAST program with a minimum length of 150 bp and a minimum percent identity of 75.0 []. Once the number of sequence reads had been homogenised between microcosms, alpha diversity was used to describe the microbial richness, diversity and evenness within the constructed microcosms using Chao1 (a nonparametric richness estimator based on distribution of singletons and doubletons), Abundance-based Coverage Estimator (ACE; a nonparametric richness estimator based on distribution of abundant (≥10) and rare (<10) OTUs), Shannon, Inverse Simpson and equitability metrics. β-diversity (diversity between groups of samples) was used to test phylogeny-based community composition among the constructed microcosms using weighted Unique Fraction of branches shared (UniFrac) distances [,]. The UniFrac distance matrix was analysed by principal coordinate plots (PCoA) and uncertainty in PCoA plots was estimated using jackknife analysis (jackknife replicate = 10). 3D PCoA plots were used to visualize the similarities or dissimilarities of variables that best represent the pair-wise distances between sample groups. […]

Pipeline specifications

Software tools QIIME, USEARCH, PyNAST, UniFrac
Databases Greengenes
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Listeria monocytogenes