This pipeline performs a mobilome analyses on previously selected contigs, those harboring antimicrobial genes from ResFinder database (https://bitbucket.org/genomicepidemiology/resfinder_db/downloads/), to confirm or discard the location of such genes within plasmid or chromosomal, or even within integron or lateral gene transfer (lgt) regions. It can be adapted to other purposes (databases), according to user requeriments.
Blast need to be installed (http://www.metagenomics.wiki/tools/blast/installhttp://www.metagenomics.wiki/tools/blast/install) and the ResFinder database has to be transformed to blast-format by:
cat resfinder_db/*.fsa > resfinder.fasta
makeblastdb -in resfinder.fasta -dbtype nucl -out ResFinder
If the contigs has not the metaSPAdes header format (>samplename_NODE_XXX_length_XX_cov_XX.XX) it is necessary to add contig-length information on fasta headers for further steps, using bioawk tools (https://github.com/lh3/bioawk) by:
bioawk -c fastx '{ print $name, length($seq) }' < contigs_ARGs.fna > contigs_length.txt
ruby 01.add_length_to_fasta.rb
being contigs_ARGs.fna the input file.
The integron_Finder (https://github.com/gem-pasteur/Integron_Finder), plasflow (https://github.com/smaegol/PlasFlow) and waafle (https://github.com/biobakery/waafle) softwares have to been installed, and they will be run on the contigs_ARGs.fna file by:
source ~/Integron_Finder/bin/activate
integron_finder --cpu 1 contigs_ARGs.fna
conda activate plasflow
mkdir plasflow_contigs
PlasFlow.py --input contigs_ARGs.fna --output plasflow_contigs/plasflow.csv
rm plasflow_contigs/*.fasta
mkdir waafle_contigs
waafle_search --threads 11 --out waafle_contigs/blast.txt contigs_ARGs.fna ~/waafledb
waafle_genecaller --gff waafle_contigs/genecall.gff waafle_contigs/blast.txt
waafle_orgscorer --outdir waafle_contigs/ contigs_ARGs.fna waafle_contigs/blast.txt waafle_contigs/genecall.gff ~/waafledb_taxonomy.tsv
rm waafle_contigs/blast.txt
rm waafle_contigs/genecall.gff
where the ~/ has to be substituted by the correct pathway to waafledb folder and waafledb_taxonomy.tsv file.
And the following scripts are employed to obtain the integrons and lgts regions coordenates within the contigs, and to extract this regions in fasta file and blast them versus ResFinder database (previously transformed to blastdb format):
ruby 02.extract_integron_regions.rb
ruby 03.extract_lgt_regions.rb
ruby 04.bedtools_blast.rb