Computational protocol: Temporal stability of bacterial symbionts in a temperate ascidian

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Protocol publication

[…] To compare bacterial community structure among samples, we constructed Bray–Curtis similarity matrices using square root transformations of relative T-RF abundance data and visualized the results in non-metric multi-dimensional scaling (nMDS) plots. To determine whether the bacterial community in D. fulgens was stable over time, similarity matrices were analyzed with nested permutational multivariate analysis of variance (PERMANOVA) with the factors season and month (within season). In addition, PERMANOVA with the single factor month was performed to ensure that non-seasonal temporal patterns were not missed by forcing monthly data into ‘seasonal’ categories. PERMANOVA pairwise comparisons were corrected based on the Benjamini-Yekutieli (B-Y) false discovery rate control () and an experiment-wise error rate of 0.05. Finally, permutational multivariate analyses of dispersion (PERMDISP) were conducted to test for heterogeneity of dispersion among seasons and months. PERMANOVA and PERMDISP analyses were conducted using PERMANOVA+ implemented in Primer v. 6 (Plymouth Marine Laboratory, UK).To determine the putative identity of T-RFLP profile peaks, in silico digestions of 16S rRNA gene sequences (see below) were performed in Geneious v. 8 (). Cut sites at the 5′-end of each sequence were identified based on the recognition sequence of the restriction endonucleases HaeIII (GG’CC) and MspI (C’CGG) and utilized to predict the corresponding length of sequences in T-RFLP profiles. In addition to T-RF peak identification, comparing clone libraries and T-RFLP profiles help to determine the specificity of individual T-RFs. Identical or closely related sequences that match to a single T-RF provide evidence for a phylotype-specific peak, while unrelated sequences that match to a same T-RF indicate a multiple phylotype peak. [...] Clone libraries based on a fragment of the 16S rRNA gene sequence were constructed for two colonies of D. fulgens, collected in April 2011 (DF2) and May 2011 (DF7), to recover near full-length 16S rRNA gene sequences. Clone libraries were built with the same primer pair used for T-RFLP analysis (without the 6-FAM label) as described previously (). All clones obtained were sent for purification and sequencing to Macrogen, Inc. (Seoul, Korea). Raw sequence reads were processed in Geneious v. 8 () by aligning forward and reverse reads to yield a final consensus sequence for each clone. Quality-checked sequences are archived in GenBank under accession numbers KR348488-KR348508.Bacterial sequences were ascribed to operational taxonomic units (OTUs) based on 99% sequence identity (nearest-neighbor algorithm). Representative sequences from each 99% OTU were analyzed using the Ribosomal Database Project II () sequence classifier and the BLASTn tool from GenBank to assess taxonomic affiliations and check for sequencing artifacts (e.g., chimeras). Sampling coverage of clone libraries was calculated using the bootstrap estimator (). For phylogenetic analyses, all recovered sequences herein and reference sequences from GenBank were aligned using Clustal W v. 2 () with a gap opening penalty of 24 and a gap extension penalty of 4, values appropriate for aligning gene sequences (e.g., 16S rRNA) with multiple variable and conserved regions (). To build phylogenetic trees, neighbor-joining (NJ) and maximum likelihood (ML) analyses were conducted in MEGA v. 5.2.2 (). For NJ analyses, the Jukes-Cantor model of nucleotide substitution was used and data were re-sampled using 1,000 bootstrap replicates (). The ML tree was built based on the GTR+I+G () model with substitution rates varying among sites according to an invariant and gamma distribution and 100 bootstrap replicates. […]

Pipeline specifications

Software tools Geneious, BLASTN, Clustal W, MEGA-V
Applications Phylogenetics, GWAS
Organisms Bacteria, Caenorhabditis elegans