Computational protocol: Metatranscriptomic Analysis Reveals Unexpectedly Diverse Microbial Metabolism in a Biogeochemical Hot Spot in an Alluvial Aquifer

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Protocol publication

[…] Genes of interest for selected metabolic pathways were confirmed with MetaCyc (Caspi et al., ) and identified in the metatranscriptome with BLAST 2.2.30+ (Camacho et al., ), BLASTP (Altschul et al., ), and JGI IMG (Markowitz et al., ) as described by Jewell et al. (). Additional genes of interest were identified in selected bins using the Department of Energy Systems Biology Knowledgebase (KBase, “RAST Annotate Microbial Contigs v1.0.0” workflow. Sequence alignments were performed with MUSCLE (Edgar, ), neighbor-joining trees generated using FastTree (Price et al., ), and the data were integrated with expression data and visualized with the Interactive Tree Of Life (Letunic and Bork, ). Replicate RPKM data were summed for analysis, as were DNA fold coverage data. Heat maps were generated with the R ggplot2 package (Wickham, ; R Core Team, ).A data file including ORF IDs and for each ORF: scaffold ID, bin ID, fold coverage from DNA libraries for each sample, RPKM from cDNA libraries for each sample, top 10 UniRef hits, percent protein sequence identities for each UniRef hit, translated sequence of the predicted ORF, functional annotations, and additional information is available at the following doi: […]

Pipeline specifications

Software tools BLASTP, MUSCLE, FastTree, iTOL, Ggplot2
Databases UniRef
Applications Miscellaneous, Phylogenetics, Nucleotide sequence alignment
Chemicals Acetone, Amino Acids, Calcium, Carbon, Carbon Dioxide, Pyruvic Acid