Computational protocol: Metagenomics of Hydrocarbon Resource EnvironmentsIndicates Aerobic Taxa and Genes to be Unexpectedly Common

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Protocol publication

[…] The analyses were conducted using Phoenix 2. The raw 16S rRNA sequence data are available from the Sequence Read Archive (SRA) at NCBI under accession numbers listed in Table . From these reads, operational taxonomic units (OTUs) were generated using average neighbor clustering at 5% distance cutoffs. The OTUs were assigned to taxa using the RDP classifier on the SILVA Small Subunit rRNA Database Release 108 (SSU ref NR 108; http://www.arb-silva.de/no_cache/download/archive/release_108/Exports). A bootstrap value cutoff of 60% was used for the assignments, which resulted in the taxa being at the genus or a higher taxonomic rank and the number of taxa being smaller than that of OTUs. Samples were compared via clustering and ordination methods in reduced space, using Mothur. The Bray–Curtis index was used as a measure of dissimilarity between communities. Communities were clustered into Newick-formatted trees using the UPGMA algorithm implemented in Mothur. The sample relation tree in Newick format was visualized using MEGA. Differences and similarities among amplicon libraries were also explored with the Non-Metric Multidimensional Scaling (NMDS) ordination method in Mothur using the majorization algorithm from Borg and Groenen. Sample OTUs were subsequently grouped by environment types identified in the NMDS to facilitate the comparison of alpha diversity between environment types. To determine if the spatial separations of the groups observed in the NMDS plot were statistically significant, the Analysis of Molecular Variance (AMOVA) and weighted Unifrac tools in Mothur were used. Mothur was also used to determine the pairwise correlation between each possible pair of OTUs identified in all of the 160 samples. […]

Pipeline specifications

Software tools Phoenix, RDP Classifier, mothur, MEGA
Application 16S rRNA-seq analysis