Computational protocol: Culture Media and Individual Hosts Affect the Recovery of Culturable Bacterial Diversity from Amphibian Skin

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Protocol publication

[…] Following DNA extraction, assessment of the cultured communities was done as for the initial culture-independent swab, following methods of Walke et al. (), with the exception that we used 2 μl of DNA template in the PCR. Out of the 48 experimental samples, five (two from the TSA culture media and one from each of the other culture media) were removed from the dataset because they would not amplify. In addition, out of the four control plates, only two could be included because most contained too little DNA for sequencing (as was expected), and even the two that were included had very little DNA. From each of the 45 remaining samples, 200 ng of PCR product was pooled to make a composite sample, which was then cleaned with the QIAquick PCR Purification Kit (Qiagen, Valencia, California). The final pooled sample was sent for sequencing on an Illumina Mi-Seq instrument at the Dana-Farber Cancer Institute of Harvard University following Caporaso et al. () using a 250 bp single-end strategy.Raw forward 16S rRNA amplicon sequences were demultiplexed and quality-filtered using the default parameters of the Quantitative Insight into Microbial Ecology (QIIME) pipeline (Caporaso et al., ), with a few exceptions: we allowed for no errors in the barcodes, increased the number of minimum consecutive low-quality base calls allowed before truncating a read (r) to 10, and decreased the fraction of the minimum number of consecutive high-quality base calls to include a read (p) to 0.5. Sequences matching PhiX, added to increase base diversity in Illumina sequencing runs, were removed from the dataset using Geneious (Biomatters, Ltd, version 8.1.8). For remaining sequences, a 97% similarity threshold was used to cluster sequences into operational taxonomic units (OTUs, ~bacterial species) using the UCLUST method (Edgar, ). Each OTU was represented by the most abundant sequence clustered within it, which was aligned to the Greengenes 13_8 reference database (DeSantis et al., ) using PyNAST (Caporaso et al., ) and assigned taxonomy using the RDP classifier (Wang et al., ). OTUs assigned to chloroplast or mitochondria were removed, and then OTUs with fewer than 0.01% (524 sequences) of the total reads were removed (Bokulich et al., ; Hughey et al., ). The sequencing depth per sample ranged from 3,991 to 164,519, including the two controls, which, as expected, had the lowest read counts at 3,991 and 8,299 reads. We removed the controls from the set of samples, given that their removal did not change the total number of OTUs, but would have reduced substantially the cut-off for the standardization of the sampling effort across samples. Thus, we rarefied the sample set to a depth of 59,000 sequences/sample. This final dataset for the culture plates consisted of 347 OTUs across the 43 samples, with 93–277 OTUs per sample (mean ± SD = 174 ± 44). 16S rRNA amplicon sequences are deposited in the NCBI's SRA under the accession number SRP112779. [...] Alpha diversity estimates were calculated with QIIME for the metrics: richness (OTUs/culture plate), Faith's phylogenetic diversity (measure of diversity based on the branch length of the phylogenetic tree) and the Shannon Index (H', which assesses community evenness). We fitted the diversity metrics to GLMMs. We considered the predictor variable “Media” as a fixed factor and “Individual toad” as a random factor given the nestedness of the media types within individuals. The GLMMs were performed using appropriate error distributions for the diversity metrics to account for heteroscedasticity. For richness, a negative binomial error distribution was applied to the model using the log link function. For phylogenetic diversity and the Shannon index, which was transformed to Hill number (effective number of species; MacArthur, ), we used a Gamma error distribution with the inverse link function. The models were run using the R functions glmer.nb for richness, and glmer for phylogenetic diversity and the Shannon Index, from the package lmer4 (Bates et al., ). Multiple comparisons were conducted with Tukey tests using the function glht in the package multcomp (Hothorn et al., ), which includes multiple comparisons for GLMs. Although not an explicit goal of this study, we also compared the alpha diversity estimates among individual toads since variation in total bacterial diversity across individuals can influence the culturable diversity. For instance, individual toads with high bacterial diversity had higher alpha diversity estimates in their plated communities relative to that from other individuals (Walke et al., ). We used GLMs for this purpose. GLMs were performed as described above, with a negative binomial error distribution for richness estimates and a Gamma error distribution for Shannon and phylogenetic diversity.Changes in the structure of the bacterial communities across media types were determined by the calculation of dissimilarity distances based on the Bray-Curtis distance measure, which takes into account OTU relative abundance in each community. We are only including the results based on Bray-Curtis since the Jaccard distance measure, which takes into account only the presence/absence composition of the communities, produced consistent results. A statistical comparison of the communities across media types based on the Bray-Curtis dissimilarities was done with a permutational multivariate analysis of variance (PERMANOVA, Anderson, ). In addition, we compared the bacterial communities of the media types based on nutrient level, where LB and TSA were grouped as high nutrient concentration media types, and R2A and dR2A as low. When performing the PERMANOVAs, the argument “strata” was used to define the group within which to limit the permutations (i.e., individual toad) due to the nestedness of the replicates of the media types and the potential variation in the skin bacterial communities among toads. Importantly, an analysis excluding those toads that did not have replicates of all media types (toads 4, 11, and 12) was consistent with results from the inclusive analysis that is presented. Lastly, due to potential influence of the individual toads on the plated bacterial communities, as shown in the ordination (Figure 2), we also compared the communities across toads where the argument “strata” was not used when performing the PERMANOVAs. Bray-Curtis dissimilarity distances were calculated with the function vegdist, and PERMANOVAs were performed with the function adonis, both functions from the vegan package (Okansen et al., ). To visualize the results, we used principal coordinate analysis (PCoA).Reduced variation in community structure across samples could occur on high nutrient media due to fast-growing bacteria out-competing slow-growing bacteria, and among samples from the same individual toad. As a way to test this, we compared the multivariate homogeneity of dispersion (i.e., distance from objects to cluster centroid) across media types based on Bray-Curtis dissimilarity distances. We used the function betadisper from the vegan package and conducted a hypothesis test to determine whether there are statistical differences in dispersion among groups using the function anova from the default R package stats. […]

Pipeline specifications

Software tools QIIME, Geneious, UCLUST, PyNAST, RDP Classifier, multcomp, vegan
Databases Greengenes
Applications Phylogenetics, 16S rRNA-seq analysis, GWAS
Organisms Batrachochytrium dendrobatidis, Bacteria
Diseases Infection