Now that we have extracted the genes/proteins we want to functionally annotate those. There are a bunch of ways of doing this. We will use webMGA to do do rpsBLAST searches against the COG database. COGs are clusters of orthologs genes, i.e. evolutionary counterparts in different species, usually with the same function (http://www.ncbi.nlm.nih.gov/COG/). Many COGs have known functions and the COGs are also grouped at a higher level with functional classes.
To download the protein sequences that Prodigal generated, open a local terminal and type:
mkdir -p ~/metagenomics/cfa/prodigal cd ~/metagenomics/cfa/prodigal scp email@example.com:~/metagenomics/cfa/prodigal/baltic-sea-ray-noscaf-41.1000.aa.fa .
To get COG classifications of your proteins, go to webMGA http://weizhong-lab.ucsd.edu/metagenomic-analysis/ and select Server / Function annotation / COG. Upload the protein file (baltic-sea-ray-noscaf-41.1000.aa.fa) and use the default -e value cutoff. rpsBLAST is used, which is a BLAST based on position specific scoring matrices (pssm). For each COG, one such pssm has been constructed. These are compiled into a database of profiles that is searched against. http://www.ncbi.nlm.nih.gov/staff/tao/URLAPI/wwwblast/node20.html. rpsBLAST is more sensitive than a normal BLAST, which is important if genomes in your metagenome are distant from existing sequences in databases. It is also faster than searching against all proteins out there.
When the search is done you get a zipped folder. On milou, create the directory:
mkdir -p ~/metagenomics/cfa/wmga-cog
Use wget or curl to download the zip file on uppmax or use scp to upload it to that folder i.e.:
scp output.zip firstname.lastname@example.org:~/metagenomics/cfa/wmga-cog
Then unzip the file on kalkyl:
cd ~/metagenomics/cfa/wmga-cog unzip output.zip
Have a look at the README.txt to see what all the files represent. The file output.2 includes detailed information on the classifications for every protein with a hit below the -e value cutoff. View them with:
less README.txt less -S output.2
NOTE: If the queueing takes too much time you can also just copy the results from the project dir:
cp -r /proj/g2014113/metagenomics/cfa/wmga-cog/ ~/metagenomics/cfa/
Question: What seem to be the 3 most abundant COG classes in our combined sample (not taking coverage into account)?