Computational protocol: Acidification increases abundances of Vibrionales and Planctomycetia associated to a seaweed-grazer system: potential consequences for disease and prey digestion efficiency

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Protocol publication

[…] A total of 3,204,094 partial 16S rRNA gene sequences were obtained from the 24 samples (i.e., two CO2 conditions X four replicates for seaweed in the presence and the absence of grazer and four grazer gut samples with two CO2 conditions). The bacterial community analyses were performed using Quantitative Insights into Microbial Ecology (QIIME version 1.8.0) software (). Sequences were screened and filtered for a minimum read length of 350 bp (after reads were paired) and less than two undetermined nucleotides. Selected high-quality sequences were clustered into Operational Taxonomic Units (OTUs) within reads using denovo OTU picking method. Representative sequences for each OTU were selected using the “most-abundant” method and OTU sequence alignment was carried out using PyNAST () and Greengenes v.13.8 (). Taxonomic assignments were done using the UCLUST () method with a 97% confidence threshold. To assign each OTU to the closest matching described taxon, searches were performed against the Greengenes taxonomy database v.13.8 for16S rRNA (), and sequences were putatively assigned to a described taxon with a minimum threshold of 0.001 (default value). Eukaryotes (i.e., chloroplasts and mitochondria) matching sequences were excluded from the OTU table in downstream analyses as well as rare OTUs (singletons and doubletons) and unassigned sequences (those sequences that did not match any of those from the Greengenes database, with a minimum threshold of 0.001).Quality filtering resulted in 2,877,493 high-quality sequences, with an average of 119,896 ± 46,294 reads per sample, which were clustered into 42,730 unique operational taxonomic units (OTUs). The OTU table was rarefied to the minimum number of sequences (66,831). As a result, a total of 41,139 unique OTUs remained. Public access to the data can be done through: the statistical and diversity (alpha and beta) analysis were done using the filtered rarefied (to the minimum number of sequences—66,831) OTU table and considered significant at P < 0.05.Alpha diversity indexes, including Chao I richness (), observed number of species (OTUs) and Shannon diversity, were calculated using QIIME software. Bacterial community structure (beta diversity) was assessed by permutational multivariate analyses of variance (PERMANOVA) using Bray–Curtis dissimilarity matrices from square-root transformed data. PERMANOVA tested for differences among samples with different levels of a priori factors: Type of sample: Seaweed vs Grazer gut; for both CO2 treatments: Ambient vs Acidified; for Seaweed: Grazed vs Non-grazed, and the interactions among these factors. The homogeneity of multivariate dispersions (based on mean distance to group centroid for all groups within each factor) was tested using a resemblance based permutation test (PERMDISP). To visualize differences and to assess dissimilarity between samples, Canonical Analysis of Principal coordinates (CAP) plots were constructed to test the assignment/clustering of treatments interaction (S.muticum XGrazingxAcidification and Grazer gutxAcidification) as a priori factor. Similarities and dissimilarities in bacterial communities between acidification treatments were explored using, similarity percentage analyses (SIMPER). For those bacterial taxonomic groups that displayed a high contribution (concerning their differential abundances in the treatments being compared) for the differences between the grazer gut and the seaweed and the two CO2 levels, two-way analyses of variance (ANOVA) were performed (with the preliminary tests for normality and homogeneity of variances being implemented). Species (S. muticum and isopod) and acidification (CO2 ambient and elevated) were tested as factors affecting the structure/composition bacterial communities. For bacterial OTUs for which significant interaction were detected, a post-hoc t-test was implemented using a Bonferroni correction and a conservative alpha (considering the comparisons made: CO2 effect in S. muticum, CO2 effect on isopod gut), effect of type of tissue (seaweed/gut) in ambient CO2 and effect of type of tissue in elevated CO2 (P (T ≤ t) two tail < 0.0125).All bacterial community structure statistical analyses were performed using the software program PRIMER-E + PERMANOVA v.6 (; ). […]

Pipeline specifications

Software tools QIIME, PyNAST, UCLUST
Databases Greengenes
Application 16S rRNA-seq analysis
Organisms Sargassum muticum, Bacteria, Escherichia coli
Chemicals Carbon