Computational protocol: Microbial community composition of deep-sea corals from the Red Sea provides insight into functional adaption to a unique environment

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Protocol publication

[…] Bacterial community analysis was conducted using mothur (v.1.36.1). Briefly, sequence reads were trimmed and read pairs were merged into contigs. Contigs longer than 306 bp and those with ambiguously called bases were discarded. Sequences that occurred only once were discarded. The sequences were then pre-clustered, allowing for up to a 2 base pair difference between sequences. Chimeras were removed using UCHIME, as implemented in mothur. Sequences were classified with the Greengenes database using a 60% bootstrap cutoff, and mitochondria, chloroplasts, archaea, eukaryote, and unknown sequences were removed. All samples were subsampled to 4,600 sequences, corresponding to the lowest number of sequences available for any sample. Bacterial community compositions were visualized using pie charts and a stack column plot based on annotated sequences. Subsampled sequences were clustered into operational taxonomic units (OTUs) with a similarity cutoff of 97%. Alpha diversity indices (Chao1, Simpson Evenness, and Inverse Simpson Index) were calculated using mothur. Beta diversity differences were visualized in a principal coordinate analysis (PCoA) based on a Bray-Curtis dissimilarity matrix and Pearson correlation. Differences between coral and seawater samples were assessed using Analysis of Molecular Variance (AMOVA) in mothur. A one-factorial permutational MANOVA (PERMANOVA) analysis was conducted using PRIMER v6 software (PRIMER–E Ltd, Ivybridge, UK) on square root transformed OTU abundance counts. The test design was based on Bray-Curtis similarities, partial sum of squares type III, 9,999 permutations of residuals under a reduced model using Monte-Carlo simulations, and followed by pair-wise tests. Core microbiomes were determined following a conservative approach, considering only OTUs that occurred in every sample of a given coral species. […]

Pipeline specifications

Software tools mothur, UCHIME
Application 16S rRNA-seq analysis
Chemicals Carbon, Oxygen