Roary is a high speed pan genome pipeline, which takes annotated assemblies in GFF3 format (produced by Prokka) and calculates the pan genome.

References:

• Andrew J. Page et al., Roary: Rapid large-scale prokaryote pan genome analysis. Bioinformatics 2015, 31:3691-3693. Pubmed  |  PMC  |  Journal

• Home page
• GitHub
• Tutorial

• Module Name: roary (see the modules page for more information)
• roary is a multithreaded application. Make sure to match the number of cpus requested with the number of threads.
• Example files in ROARY_TEST_DATA

For this interactive session we will follow the roary Tutorial. In addition to roary we will also need prokka for annotating the bacterial genomes

[user@biowulf]$ sinteractive --cpus-per-task=6 --mem=20g --gres=lscratch:20
salloc.exe: Pending job allocation 46116226
salloc.exe: job 46116226 queued and waiting for resources
salloc.exe: job 46116226 has been allocated resources
salloc.exe: Granted job allocation 46116226
salloc.exe: Waiting for resource configuration
salloc.exe: Nodes cn3144 are ready for job

[user@cn3144]$ module load roary prokka
[user@cn3144]$ cd /lscratch/$SLURM_JOB_ID
[user@cn3144]$ cp ${ROARY_TEST_DATA:-none}/* .
[user@cn3144]$ ls -1
GCA_000008285.1_ASM828v1_genomic.fna
GCA_000021185.1_ASM2118v1_genomic.fna
GCA_000026705.1_ASM2670v1_genomic.fna
GCA_000168635.2_ASM16863v2_genomic.fna
GCA_000168815.1_ASM16881v1_genomic.fna
GCA_000196035.1_ASM19603v1_genomic.fna

Annotate the genomes with prokka and create a pan genome with roary.

[user@cn3144]$ fastas=( *.fna )
[user@cn3144]$ for fasta in ${fastas[@]}; do
                      acc=${fasta:0:13}
                      prokka --kingdom Bacteria --outdir prokka_$acc --genus Listeria \
                        --locustag $acc --prefix $acc --cpus $SLURM_CPUS_PER_TASK $fasta
                  done

[user@cn3144]$ roary -p $SLURM_CPUS_PER_TASK -f ./roary_demo -e -n -v -r */*.gff
[...snip...]
[user@cn3144]$ exit
salloc.exe: Relinquishing job allocation 46116226
[user@biowulf]$

Create a batch input file (e.g. roary.sh), which uses the input file 'roary.in'. For example:

#! /bin/bash
# this file is roary.batch

function die {
    echo "$@" >&2
    exit 1
}

wd=$PWD
module load roary/3.12.0 || die "Could not load modules"
cd /lscratch/$SLURM_JOB_ID || die "no lscratch"
roary -p ${SLURM_CPUS_PER_TASK} \
  -f roary_out -e -n -r -v ./gff/*.gff \
&& mv roary_out $wd

Submit this job using the Slurm sbatch command.

sbatch --gres=lscratch:10 --cpus-per-task=6 --mem=6g roary.sh

Create a swarmfile (e.g. roary.swarm). For example:

roary -p $SLURM_CPUS_PER_TASK -f ./roary_species1 -e -n -v -r species1/*.gff
roary -p $SLURM_CPUS_PER_TASK -f ./roary_species2 -e -n -v -r species2/*.gff
roary -p $SLURM_CPUS_PER_TASK -f ./roary_species3 -e -n -v -r species3/*.gff

Submit this job using the swarm command.

swarm -f roary.swarm -g 6 -t 6 --module roary/3.12.0