Computational protocol: Energy Gradients Structure Microbial Communities Across Sediment Horizons in Deep Marine Sediments of the South China Sea

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Protocol publication

[…] Sequences were processed using mothur (v 1.38.0; Kozich et al., ). Reads were clustered at 97% and classified against the SILVA database (v. 119). Singleton OTUs were removed prior to analysis. OTUs that were present in greater than 1% relative abundance in the negative (sediment-free) control and classified as common human or kit contaminants (Salter et al., ) were removed from the dataset. To confirm their identity, phylogenetic trees were generated for all OTUs classified as anaerobic methanotrophic archaea (ANME) and for the 8 most abundant OTUs classified to known sulfate-reducing bacteria lineages with Fasttree (v. 2.1.10; Price et al., ) using the Jukes-Cantor model (Figures S1, S2).Diversity metrics (Shannon-Weiner index Magurran, , Pielou's evenness Pielou, ), and number of OTUs present) were calculated prior to rarefaction. Samples were rarefied to 4,093 sequences, and the community dissimilarity matrix was computed using weighted UniFrac distances (Lozupone et al., ) from both untransformed and square root-transformed OTU tables; results from the former are reported unless square root transformation altered the significance of a statistical test. All statistical analyses were performed using the vegan package for R (v. 2.3-3; Oksanen et al., ) and the PERMANOVA+ package in PRIMER 6 (Gorley and Clarke, ). Distance-based linear modeling (Wasmund et al., ) was used to determine the best subsets of measured environmental parameters for predicting microbial community structure, while environmental fitting (Carr et al., ) was used to determine the correlation between individual environmental parameters and microbial community structure.Sequences were deposited to NCBI under accession number PRJNA362622. […]

Pipeline specifications

Software tools mothur, FastTree, UniFrac
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Escherichia coli, Homo sapiens