Computational protocol: 16SrDNA Pyrosequencing of the Mediterranean Gorgonian Paramuricea clavata Reveals a Link among Alterations in Bacterial Holobiont Members, Anthropogenic Influence and Disease Outbreaks

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Protocol publication

[…] The open-source, platform-independent, community-supported software program, mothur , was used to process and analyze the sequence data. Sequence reads were trimmed as described (see Results and Discussion) and chimeric sequences were removed. To assess abundance and phylogenetic identity, each trimmed read sequence was BLASTed against a reference database of nearly 400.000 rRNA genes for the bacterial domain available from the SILVA rRNA database project (http://www.arb-silva.de/). MEGAN (Metagenome Analysis Software, version 3.8, University of Tübingen, Tübingen, Germany) analysis was then carried out using a bit-score threshold of 35 and retaining only hits whose bit scores were within 10% of the best score. In addition all assignments hit by <20 reads were discarded.To assess taxonomic diversity sequences were clustered as operational taxonomic units (OTUs) at 97% similarity. OTU diversity was examined using biodiversity indices and rarefaction analysis by the mothur software. […]

Pipeline specifications

Software tools mothur, MEGAN
Application Phylogenetics
Organisms Bacteria, Pristis clavata, Homo sapiens, Vibrio coralliilyticus