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Metagenome assembly and binning (continued)

In the previous tutorial we have seen how to recover draft genomes from raw metagenomic data. But we do not know what kind of organisms we have yet!

Identyfying an unknown species from a genome is not always easy, especially if the species has not been described before. In this tutorial, you'll learn a few ways of identyfying (or attempting to identidy) an unkown genome

Getting the Data

This tutorial focuses on one particular genome bin but you can download the necessary data by executing the code block below.

mkdir -p ~/mag/data
cd ~/mag/data
curl -O -J -L https://osf.io/d65jm/download
chmod -w *.fa

Ribosomal RNA

The simplest thing we can do is search our draft genome for rRNA genes. Since these genes are usually quite conserved across species/genera, it could give us a broad idea of our organism

barrnap -o bin2_rrna.fa bin.2.fa
cat bin2_rrna.fa

Now, Use the online Blast service to search similar sequences to the rRNA we obtained.

Assigning taxonomy to each contig

We'll use diamond against the swissprot database for quickly assigning taxonomy to our contigs.

First, we download and build the database

curl -O ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/complete/uniprot_sprot.fasta.gz
diamond makedb --in uniprot_sprot.fasta.gz --db uniprot_sprot -p 4

then we run diamond

diamond blastx -p 4 -q bin.2.fa -f 6 -d uniprot_sprot.dmnd -o bin2_diamond.txt

Question

How did that go? Did we find anything meaningful?

It is very possible the swissprot database is too small for finding meaningful hits for undersequenced / poorly known organisms.

Let us try with another piece of software, kraken2

sudo mkdir /mnt/kraken2
cd /mnt/kraken2
sudo curl -O https://ccb.jhu.edu/software/kraken2/dl/minikraken2_v1_8GB.tgz
sudo tar xf minikraken2_v1_8GB.tgz 
sudo chmod +r *
cd -

and then (don't forget to move back to your results directory if you haven't)

kraken2 --memory-mapping --db /mnt/kraken2 --threads 4 --output bin2_kraken.txt --report bin2_kraken_report.txt metabat/bin.2.fa

Question

Does this confirm our initial diagnostic? What kind of organism do we have?

Functional annotation

Now that we have at least a genus for our organism, let us try to look at what it does:

curl -O -J -L https://osf.io/7k5tv/download

then we add functional categories to our genes using eggnog-mapper:

emapper.py --data_dir /mnt/nog/ -d actNOG -i annotation/bin.2/bin.2.faa --no_refine -o bin2_NOG

and we download bin2_NOG.emapper.annotations to our own computers for downstream analysis

rpoB phylogeny

Warning

We're about to produce a gene tree. Phylogenetic trees based on one gene must be interpreted very carefully, and may not reflect the actual phylogeny of the species

Coming soon