Extracting rRNA encoding reads and annotating them

Taxonomic composition of a sample can be based on e.g. BLASTing the contigs against a database of reference genomes, or by utilising rRNA sequences. Usually assembly doesn’t work well for rRNA genes due to their highly conserved regions, therefore extracting rRNA from contigs will miss a lot of the taxonomic information that can be obtained by analysing the reads directly. Analysing the reads also has the advantage of being quantitative, i.e. we don’t need to calculate coverages by the mapping procedure we applied for the functional genes above. We will extract rRNA encoding reads with the program sortmeRNA which is one of the fastest software solutions for this. The program sortmeRNA has built-in multithreading support so this time we use that for parallelization. These are the commands to run:

mkdir -p ~/metagenomics/cta/sortmerna
cd ~/metagenomics/cta/sortmerna
samplenames=(0328 0403 0423 0531 0619 0705 0709 1001 1004 1028 1123)
for s in ${samplenames[*]}
    do sortmerna -n 2 --db \
    /proj/g2014113/src/sortmerna-1.9/rRNA_databases/silva-arc-16s-database-id95.fasta \
    /proj/g2014113/src/sortmerna-1.9/rRNA_databases/silva-bac-16s-database-id85.fasta \
    --I /proj/g2014113/metagenomics/cta/reads/${s}_pe.fasta \
    --accept ${s}_rrna \
    --other ${s}_nonrrna  \
    --bydbs  \
    -a 8  \
    --log ${s}_bilan  \
    -m 5242880

Again, this command takes rather long to run (~5m per sample) so just copy the results if you don’t feel like waiting:

cp /proj/g2014113/metagenomics/cta/sortmerna/* ~/metagenomics/cta/sortmerna

It outputs the reads or part of reads that encode rRNA in a fasta file. These rRNA sequences can be classified in many ways, and again blasting them against a suitable database is one option. Here we use a simple and fast method (unless you have too many samples), the classifier tool at RDP (ribosomal database project). This uses a naive bayesian classifier trained on many sequences of defined taxonomies. It gives bootstrap support values for each taxonomic level; usually the support gets lower the further down the hierarchy you go. Genus level is the lowest level provided. You can use the web service if you prefer, and upload each file individually, or you can use the uppmax installation of RDP classifier like this (~4m):

mkdir -p ~/metagenomics/cta/rdp
cd ~/metagenomics/cta/rdp
for s in ../sortmerna/*_rrna*.fasta
    do java -Xmx1g -jar /glob/inod/src/rdp_classifier_2.6/dist/classifier.jar \
    classify \
    -g 16srrna  \
    -b `basename ${s}`.bootstrap  \
    -h `basename ${s}`.hier.tsv  \
    -o `basename ${s}`.class.tsv  \