riboFrame/HOWTO at master · matteoramazzotti/riboFrame · GitHub

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#Tested on Ubuntu 20.04

#environment preparation
git clone https://github.com/matteoramazzotti/riboFrame
sudo apt update
apt install hmmer axel
#go to https://sourceforge.net/projects/rdp-classifier/files/latest/download
unzip rdp_classifier_2.14.zip

#download of data from HMP
mkdir samples
cd samples
axel -n 10 http://downloads.hmpdacc.org/data/Illumina/stool/SRS011061.tar.bz2
tar -xjf SRS011061.tar.bz2
cd ..

#extract and split fastas
grep -A1 -E "^@.+\/1$" SRS011061/SRS011061.denovo_duplicates_marked.trimmed.1.fastq | grep -v -e "--" | sed 's/^@/>/' > samples/SRS011061.1.fasta
grep -A1 -E "^@.+\/2$" SRS011061/SRS011061.denovo_duplicates_marked.trimmed.2.fastq | grep -v -e "--" | sed 's/^@/>/' > samples/SRS011061.2.fasta

#catch ribosomal reads with hmmer
hmmsearch -E 0.00001 --domtblout samples/SRS011061.1.fwd.bact.ribosomal.table --noali --cpu 4 -o /dev/null hmms/16S_bact_for3.hmm samples/SRS011061.1.fasta
hmmsearch -E 0.00001 --domtblout samples/SRS011061.1.rev.bact.ribosomal.table --noali --cpu 4 -o /dev/null hmms/16S_bact_rev3.hmm samples/SRS011061.1.fasta
hmmsearch -E 0.00001 --domtblout samples/SRS011061.1.fwd.arch.ribosomal.table --noali --cpu 4 -o /dev/null hmms/16S_arch_for3.hmm samples/SRS011061.1.fasta
hmmsearch -E 0.00001 --domtblout samples/SRS011061.1.rev.arch.ribosomal.table --noali --cpu 4 -o /dev/null hmms/16S_arch_rev3.hmm samples/SRS011061.1.fasta

hmmsearch -E 0.00001 --domtblout samples/SRS011061.2.fwd.bact.ribosomal.table --noali --cpu 4 -o /dev/null hmms/16S_bact_for3.hmm samples/SRS011061.2.fasta
hmmsearch -E 0.00001 --domtblout samples/SRS011061.2.rev.bact.ribosomal.table --noali --cpu 4 -o /dev/null hmms/16S_bact_rev3.hmm samples/SRS011061.2.fasta
hmmsearch -E 0.00001 --domtblout samples/SRS011061.2.fwd.arch.ribosomal.table --noali --cpu 4 -o /dev/null hmms/16S_arch_for3.hmm samples/SRS011061.2.fasta
hmmsearch -E 0.00001 --domtblout samples/SRS011061.2.rev.arch.ribosomal.table --noali --cpu 4 -o /dev/null hmms/16S_arch_rev3.hmm samples/SRS011061.2.fasta

#aggregate 16S reads and plot the 16S coverage profile
perl riboTrap.pl samples/SRS011061 covplot=1

#classify 16S reads
java -Xmx1g -jar rdp_classifier_2.14/dist/classifier.jar -q samples/SRS011061.16S.fasta -o samples/SRS011061.16S.rdp

#analyze reads and create abundance analysis (some examples)
perl riboMap.pl file=samples/SRS011061.16S.rdp var=full thr=0.8 cross=over percmin=0.5 covplot=1 outplot=1 out=samples/SRS011061.ab_full
perl riboMap.pl file=samples/SRS011061.16S.rdp var=all thr=0.8 cross=over percmin=0.5 tol=10% covplot=1 outplot=1 out=samples/SRS011061.ab_all
perl riboMap.pl file=samples/SRS011061.16S.rdp var=V2,V3 thr=0.8 cross=over percmin=0.5 tol=10% covplot=1 outplot=1 out=samples/SRS011061.ab_V2,V3
perl riboMap.pl file=samples/SRS011061.16S.rdp var=V5-V6 thr=0.8 cross=over percmin=0.5 tol=10% covplot=1 outplot=1 out=samples/SRS011061.ab_V5-V6