Computational protocol: Phylogenetically conserved resource partitioning in the coastal microbial loop

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[…] DNA extraction, metagenome library construction and sequencing were previously described (; ). Metagenome abundances for predicted coding sequences (CDS) were determined by mapping non-redundant sequencing reads back to the contigs using Bowtie 2 () with default settings and calculating the average coverage for each locus using the bedtools coverage tool (). Relative abundances of operational taxonomic units (OTUs) detected in the 16S rRNA amplicon libraries were highly correlated with 16S rRNA gene abundances that were assembled using EMIRGE () from the metagenomes (data not shown). [...] Probes were designed for 16S rRNA gene OTUs commonly found in coastal Pacific Ocean samples () using ARB (), as previously described (). Duplicate RNA samples were extracted, fluorescently labeled, fragmented and hybridized as previously described (), and secondary ion mass spectrometry analysis of microarrays hybridized with 13C rRNA was performed at LLNL with a Cameca NanoSIMS 50 (Cameca, Gennevilliers, France). A more detailed protocol is available in the . OTUs were considered significantly isotopically enriched if the slope minus two calculated s.e.’s was >0 and if the slope was significant based on a t-score statistic (t=slope/s.e.) with a P-value of <0.05. Data are presented for the average slope from two biological replicates. The phylogenetic tree for representative 16S rRNA genes was generated by aligning sequences with Muscle (), selecting conserved sites with Gblocks () and constructing the tree in FastTree () with default settings. […]

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