6. Annotating your metagenome with Prokka

Note

You can start from this point by taking the assembly from the data on snapshot snap-8f89b092. Just do:

mkdir /mnt/assembly
cd /mnt/assembly
cp /data/final-assembly.fa .

Installing Prokka

We’re going to use the Prokka software to annotate your newly assembled metagenome.

We have to download and install a lot of stuff, though – estimated ~15 -20 minutes.

First, we need to install BioPerl and NCBI BLAST+; for this we’ll use the Debian Linux package installer, ‘apt-get’:

apt-get update
apt-get -y install bioperl ncbi-blast+

Now download and unpack Prokka:

cd /mnt
curl -O http://www.vicbioinformatics.com/prokka-1.7.tar.gz
tar xzf prokka-1.7.tar.gz
curl -O http://www.vicbioinformatics.com/prokka-1.7.2
cp prokka-1.7.2 prokka-1.7/bin/prokka

Prokka depends on a lot of other software, too; so we’ll need to install all of that.

Install HMMER:

cd /mnt
curl -O ftp://selab.janelia.org/pub/software/hmmer3/3.1b1/hmmer-3.1b1.tar.gz
tar xzf hmmer-3.1b1.tar.gz
cd hmmer-3.1b1/
./configure --prefix=/usr && make && make install

Install Aragorn:

cd /mnt
curl -O http://mbio-serv2.mbioekol.lu.se/ARAGORN/Downloads/aragorn1.2.36.tgz
tar -xvzf aragorn1.2.36.tgz
cd aragorn1.2.36/
gcc -O3 -ffast-math -finline-functions -o aragorn aragorn1.2.36.c
cp aragorn /usr/local/bin

Install Prodigal:

cd /mnt
curl -O http://prodigal.googlecode.com/files/prodigal.v2_60.tar.gz
tar xzf prodigal.v2_60.tar.gz
cd prodigal.v2_60/
make
cp prodigal /usr/local/bin

Install tbl2asn:

cd /mnt
curl -O ftp://ftp.ncbi.nih.gov/toolbox/ncbi_tools/converters/by_program/tbl2asn/linux64.tbl2asn.gz
gunzip linux64.tbl2asn.gz
mv linux64.tbl2asn tbl2asn
chmod +x tbl2asn
cp tbl2asn /usr/local/bin

Install GNU Parallel:

cd /mnt
curl -O http://ftp.gnu.org/gnu/parallel/parallel-20130822.tar.bz2
tar xjvf parallel-20130822.tar.bz2
cd parallel-20130822/
ls
./configure && make && make install

Install Infernal:

cd /mnt
curl -O http://selab.janelia.org/software/infernal/infernal-1.1rc4.tar.gz
tar xzf infernal-1.1rc4.tar.gz
cd infernal-1.1rc4/
ls
./configure && make && make install

Running Prokka

Now, make a new directory:

cd /mnt
mkdir annot
cd annot

Copy in the assembly and remove all the sequences with ‘N’s in them (since prodigal fails if there are too many, and prokka uses prodigal):

python /usr/local/share/khmer/sandbox/remove-N.py /mnt/assembly/final-assembly.fa metagenome.fa

Now, run Prokka:

/mnt/prokka-1.7/bin/prokka metagenome.fa --outdir metag --prefix testasm --metagenome

There will be a bunch of files in the dierctory ‘metag/’. Probably the most interesting is ‘metag/testasm.faa’, which will contain a set of annotated protein sequences derived from the metagenome.

Next: 7. BLASTing your assembled data

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