Computational protocol: Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics

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Protocol publication

[…] To assign genes to TIGRFams [], we used HMMer 3.1b1 44 [] and the trusted score cutoff for each family in TIGRFam 15.0. TIGRFam assigns enzyme commision numbers to some of its families.To assign genes to enzyme commission numbers via KEGG [], we downloaded the last public release of KEGG (from 2011) and we searched for a best hit with over 30% identity and above 80% coverage using RapSearch v2.22 []. If the best hit was assigned an enzyme commission number by KEGG, then we transferred that annotation to the gene.To assign genes to enzyme commision numbers via SEED and RAST [], we used the SEED server, based on code from http://servers.nmpdr.org/sapling/server.cgi?code=server_paper_example6.pl. These results were viewed using the Fitness Browser (http://fit.genomics.lbl.gov/).To compute the phylogenetic profile of DUF1638 and the RamA-like protein, we used MicrobesOnline []. For DUF1638, we used the presence or absence of PF07796. For the RamA-like protein, we used the presence of an ortholog of the RamA-like protein (VIMSS 5050244). However in Roseobacter sp. SK209-2-6, the RamA-like protein is split into two proteins (RSK20926_19262 and RSK20926_19267), and we manually classified the RamA-like protein as present in this bacterium. […]

Pipeline specifications

Software tools HMMER, RAPSearch, RAST
Databases TIGRFAMs KEGG NMPDR
Application Phylogenetics
Organisms Escherichia coli, Bacteria, Desulfovibrio vulgaris
Chemicals Amino Acids, Homocysteine, Methionine