deFuse uses clusters of discordant paired end reads to guide split read alignments across gene-gene fusion boundaries in RNA-Seq data. Filters are applied to reduce false positives and results are annotated.

Reference data sets required by deFuse are stored under

Note that the versions of gmap/gsnap available on biowulf make use of a new index format. Only reference data sets ending in _newgmap are compatible with these versions of gmap.

deFuse runs are set up using a configuration file which may change between defuse versions. Use the configuration file included with the version you are using as a starting point for your analysis. They can be found under

deFuse is a pipeline that makes use of internal and external tools. Pipeline steps can be run on the same machine as the main driver script (defuse.pl -s direct ...) or submitted to compute nodes ( defuse.pl -s slurm ...). The -p option determines how many jobs are run in parallel.

We do not recommend to use "-s slurm" on biowulf because it will submit multiple short subjobs, which will take longer queue time.

defuse.pl option summary (for version 0.8.0):

Usage: defuse.pl [options]
Run the deFuse pipeline for fusion discovery.
  -h, --help      Displays this information
  -c, --config    Configuration Filename
  -d, --dataset   Dataset Directory
  -o, --output    Output Directory
  -r, --res       Main results filename (default: results.tsv 
                  in Output Directory)
  -a, --rescla    Results with a probability column filename 
                  (default: results.classify.tsv in Output Directory)
  -b, --resfil    Filtered by the probability threshold results filename 
                  (default: results.filtered.tsv in Output Directory)
  -1, --1fastq    Fastq filename 1
  -2, --2fastq    Fastq filename 2
  -n, --name      Library Name (default: Output Directory Suffix)
  -l, --local     Job Local Directory (default: Output Directory)
  -s, --submit    Submitter Type (default: direct)
  -p, --parallel  Maximum Number of Parallel Jobs (default: 1)

Note that the driver script was renamed to defuse_run.pl in version 0.8.0. However, defuse.pl is still available as a symbolic link. Note also that starting with version 0.8.0 the dataset directory has to be provided on the command line with -d

References:

• Andrew McPherson et al. deFuse: an algorithm for gene fusion discovery in tumor RNA-Seq data. PLoS Comput Biol. 2011, 7:e1001138. Pubmed |  PMC |  Journal.

• Bitbucket

• Module Name: defuse (see the modules page for more information)
• defuse can be run in local or slurm mode. In local mode it can use multiple CPUs
• Example files in $DEFUSE_TEST_DATA
• Reference data in /fdb/defuse/

Allocate an interactive session and run the program. Sample session:

[user@biowulf]$ sinteractive --cpus-per-task=16 --mem=20g
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 ~]$ DATA=/usr/local/apps/defuse/TEST_DATA/small
[user@cn3144 ~]$ module load defuse
[user@cn3144 ~]$ cp /usr/local/apps/defuse/config_hg19_ens69.txt config.txt
[user@cn3144 ~]$ defuse.pl -c config.txt -o small.out \
  -d /fdb/defuse/hg19_ens69_newgmap \
  -1 $DATA/rna/spiked.1.fastq -2 $DATA/rna/spiked.2.fastq \
  -s direct -p 12
[user@cn3144 ~]$

[user@cn3144 ~]$ exit
salloc.exe: Relinquishing job allocation 46116226
[user@biowulf ~]$

A deFuse batch job can run in two different ways - either all jobs started by the main defuse.pl are run on the same compute node, or they are submitted to other nodes via slurm.

Here is an example script that will run all jobs on the same node as the node running the main deFuse script. This makes use of a small data set of simulated RNASeq reads. Note that bowtie is allowed 2 threads in the sample config file, so the number of parallel jobs is limited to half the number of CPUs.

#! /bin/bash
# filename: small.sh
set -e

DATA=/usr/local/apps/defuse/TEST_DATA/small
DEFUSE_VER=0.8.1
module load defuse/$DEFUSE_VER || exit 1

cp -r $DATA . || exit 1
cp /usr/local/apps/defuse/$DEFUSE_VER/config_hg19_ens69.txt config.txt

defuse.pl -c config.txt -o small.out \
    -d /fdb/defuse/hg19_ens69_newgmap \
    -1 small/rna/spiked.1.fastq -2 small/rna/spiked.2.fastq \
    -s direct -p $SLURM_CPUS_PER_TASK

The batch file is submitted to the queue with a command similar to the following:

biowulf$ sbatch --cpus-per-task=20 small.sh

The profile of a local defuse job with 24 CPUs processing 60M 125nt paired end reads:

The other approach is shown in the following batch script with runs the main script on a compute node with just 2 CPUs allocated. The main script in turn submits subjobs via slurm. This example uses data obtained from the Gerstein lab for cell line NCI-H660, which contains a known TMPRSS2-ERG fusion.

#! /bin/bash
# this file is large.sh

# defuse version
DEFUSE_VER=0.8.1
module load defuse/$DEFUSE_VER || exit 1
cp /usr/local/apps/defuse/$DEFUSE_VER/config_hg19_ens69.txt config.txt

# large test data - copy if it doesn't already exist
if [[ ! -d large ]]; then
    cd large
    DATA=/usr/local/apps/defuse/TEST_DATA/NCIH660
    cp $DATA/NCIH660.fastq.tar.gz .
    tar -xzf NCIH660.fastq.tar.gz
    rm NCIH660.fastq.tar.gz
    cd ..
fi

defuse.pl -c config.txt -o ncih660.out \
    -d /fdb/defuse/hg19_ens69_newgmap \
    -1 large/NCIH660_1.fastq \
    -2 large/NCIH660_2.fastq \
    -s slurm -p 25

Which is submitted as follows

biowulf$ sbatch large.sh

To set up a swarm of defuse jobs, each running the subjobs in local mode, use a swarm file like this:

defuse.pl -c config.txt -o defuse1.out \
  -d /fdb/defuse/hg19_ens69_newgmap \
  -1 defuse1.1.fastq \
  -2 defuse1.2.fastq \
  -s direct -p 12 
defuse.pl -c config.txt -o defuse2.out \
  -d /fdb/defuse/hg19_ens69_newgmap \
  -1 defuse2.1.fastq \
  -2 defuse2.2.fastq \
  -s direct -p 12 
[...]

Then submit the swarm, requesting 24 CPUs and 10GB memory for each task

biowulf$ swarm -g 10 -t 24 swarmfile --module=defuse/0.8.1