Computational protocol: Transient Changes in Bacterioplankton Communities Induced by the Submarine Volcanic Eruption of El Hierro (Canary Islands)

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Protocol publication

[…] The Sterivex units (Millipore) were filled with 1.8 ml of lysis buffer (50 mM Tris-HCl pH 8.3, 40 mM EDTA pH 8.0, 0.75 M sucrose) treated with lysozyme, proteinase K and sodium dodecyl sulfate. Nucleic acids were extracted with phenol and concentrated in an Amicon 100 (Millipore) as described in Massana et al. []. DNA was quantified spectrophotometrically (Nanodrop, Thermo Scientific) and a subsample was used for pyrosequencing at the Research and Testing Laboratory (Lubbock, TX, USA; using the bTEFAP method by 454 GL FLX technology as described previously []. Primers 28F (5’-GAGTTTGATCNTGGCTCAG-3’) and 519R (5’- GTNTTACNGCGGCKGCTG-3’) generated amplicons spanning the V1 to V3 regions of the bacterial 16S rRNA gene (∼500 bp), and primers 341F (5′-GYGCASCAGKCGMGAAW-3′) and 958R (5′-GGACTACVSGGGTATCTAAT-3′) were used to amplify archaeal fragments spanning the V3 to V5 regions (∼600 bp). The generated pyrosequencing data were processed using the QIIME (Quantitative Insights Into Microbial Ecology) pipeline [] as described in Sánchez et al. []. After an ID was assigned to each sample using a bar code, a sequence filtration step was performed before denoising. Sequences were removed from the subsequent analyses if they were shorter than 150 bp, had an average quality score < 25 calculated in sliding windows of 50 bp, or had an uncorrectable barcode or > 3 ambiguous bases. The remaining sequences were run through Denoiser to reduce the impact of pyrosequencing errors []. Curated sequences were then grouped into operational taxonomic units (OTUs) or phylotypes using UCLUST [] with a minimum identity of 97%. A representative sequence from each phylotype was chosen by selecting the most abundant sequence in each cluster. The resulting representative sequences were checked for chimeras using ChimeraSlayer [] in mothur []. The identity of 16S rRNA phylotypes was determined using the RDP Classifier [] implemented in QIIME. BLAST was also used for certain unclassified OTUs as some lineages were not correctly classified by RDP. OTUs represented by one single tag (singletons) were discarded to avoid potential artifacts in diversity estimates. Likewise, OTUs assigned to chloroplasts or mitochondria were removed. Venn Diagram Plotter ( was used to generate area-proportional Venn Diagrams. Chao1 diversity metrics and rarefaction curves were computed in QIIME and plotted in Kaleidagraph (v.4.1). Non-metric multidimensional scaling (nMDS) plots were performed and plotted in R (Vegan package) []. Phylogenetic trees were constructed with RAxML [] using the GTR substitution matrix (implemented as GTRGAMMA) and an alignment made with MUSCLE [] that was previously trimmed using the Gblocks software [] to eliminate highly diverged regions. Sequence data has been deposited in the MG-RAST public database ( under ID numbers 4600697–4600744 (Project Name: Hierro submarine volcano). […]

Pipeline specifications

Software tools QIIME, UCLUST, ChimeraSlayer, mothur, RDP Classifier, vegan, RAxML, MUSCLE, Gblocks
Databases MG-RAST
Applications Phylogenetics, Metagenomic sequencing analysis, 16S rRNA-seq analysis, Nucleotide sequence alignment
Organisms Serinus canaria, Bacteria, Bacteroidetes
Diseases Drug Eruptions
Chemicals Oxygen, Sulfur