Computational protocol: Members of Gammaproteobacteria as indicator species of healthy banana plants on Fusarium wilt-infested fields in Central America

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[…] For a deep-sequencing analysis of the banana-associated Gammaproteobacteria community, the hypervariable V4 region of the 16 S rRNA gene was amplified in a nested PCR approach with the Gammaproteobacteria-specific primer pair Gamma395f/Gamma871r and the universal primer pair 515 f/806r, which carried sample-specific tags. The reaction mixture for the first PCR (20 μl) contained 1 × Taq&Go (MP Biomedicals, Eschwege, Germany), 2 mM MgCl2, 0.1 μM of each primer and 1 μl (~10 ng) of template DNA dilution (96 °C, 4 min; 30 cycles of 96 °C, 1 min; 54 °C, 1 min; 74 °C, 1 min; and elongation at 74 °C, 10 min). The second PCR (30 μl) was performed by using 1 × Taq&Go, 0.2 μM of each primer and 1,2 μl from dilutions (1:1000) of the first PCR mixtures (94 °C, 3 min; 32 cycles of 94 °C, 45 sections; 60 °C, 1 min; 72 °C, 18 sec; and elongation at 72 °C, 10 min). PCR products of three independent reactions were pooled in equal volumes and purified by employing the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). Amplicon libraries were generated and sequenced by a paired-end approach using the Illumina MiSeq platform (Eurofins Genomics, Ebersberg, Germany). The nucleotide sequences are available in the European Nucleotide Archive (www.ebi.ac.uk/ena) under the BioProject accession number PRJEB12550.Data were analyzed using the open source software package QIIME 1.8. Sequencing reads with more than three consecutive low quality base calls (Phred quality score ≤25) were truncated at the position where their quality began to drop, and only reads with >75% consecutive high quality base calls, without any ambiguous characters, and longer than 200 nucleotides in length were retained for further analyses. All quality sequences were adjusted in the same orientation and clustered into operational taxonomic units (OTUs) with uclust, using 3%, 5% and 10% dissimilarity thresholds. From each OTU the most abundant sequence was selected as the representative one, and the taxonomy of the representative set was assigned with the uclust-based consensus taxonomy assigner using an 80% confidence threshold. The representative sequence set was aligned with PyNAST. Based on a check with ChimeraSlayer, potential chimeric sequences were discarded. OTU tables at the different dissimilarity levels were constructed, and OTUs not assigned to the class of Gammaproteobacteria and singletons were removed from the dataset. For alpha and beta diversity analyses, OTU tables were rarefied at 4,820 reads. Diversity indices Shannon and Chao1 were determined based on the normalized clustering data. Significant differences were calculated with PASW Statistics 18 (SPSS Inc., Chicago, IL, USA) using Tukey-HSD and Games-Howell post hoc tests, depending on the homogeneity of variances, and the independent samples t-test for differences between healthy and Fusarium wilt-infested plants. Beta diversity was analyzed based on weighted UniFrac distances and ten jackknife replicates of the total rarefied datasets. Statistical analyses were performed using the adonis test with 999 permutations. [...] Profile clustering network analyses were performed to highlight single taxonomic groups corresponding to genus level (OTUs at a dissimilarity level of 3% summarized at taxonomic level 6) with considerable differences between healthy and infested state. The network analyses were carried out with taxa exhibiting a mean read change of more than 0.5% of the data set. If the ratio of relative mean abundances exceeded 1.5, the taxa were regarded as altered and assigned to the respective profile. Only taxonomic groups featuring the same pattern in at least two farms of the respective country were considered. Visualization of the networks was carried out using Cytoscape 2.8.3. Significant differences were ascertained with Metastats, where p values were computed using a combination of the nonparametric t-test, exact Fisher’s test, and the false discovery rate with 103 permutations. For the determination of the rhizosphere core microbiome of healthy banana plants, a reduced OTU table was normalized at 19,892 reads. The core microbiome was defined as those OTUs at a dissimilarity level of 3% that were present in at least 50% of the respective samples. METAGENassist was employed to visualize co-occurrence patterns of taxa within the normalized core microbiome based on Spearman’s rank correlation. For visualizing relationships, core OTUs were summarized at genus level, and unassigned reads were excluded from the dataset. […]

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