rMATS on Biowulf

Quick Links

MATS is a computational tool to detect differential alternative splicing events from RNA-Seq data. The statistical model of MATS calculates the P-value and false discovery rate that the difference in the isoform ratio of a gene between two conditions exceeds a given user-defined threshold. The replicate MATS (rMATS) is designed for detection of differential alternative splicing from replicate RNA-Seq data.

References:

Shen S., Park JW., Lu ZX., Lin L., Henry MD., Wu YN., Zhou Q., Xing Y. rMATS: Robust and Flexible Detection of Differential Alternative Splicing from Replicate RNA-Seq Data. PNAS, 111(51):E5593-601. doi: 10.1073/pnas.1419161111

Documentation

Important Notes

Module Name: rmats (see the modules page for more information)
Multithreaded app (set --nthread $SLURM_CPUS_PER_TASK)
Example files in /usr/local/apps/rmats/TEST_DATA
Reference data in /fdb/STAR_indices
Unusual environment variables set
- RMATS_HOME installation directory
- RMATS_BIN executable directory
- RMATS_SRC source code directory
- RMATS_DATA sample data directory

Interactive job

Interactive jobs should be used for debugging, graphics, or applications that cannot be run as batch jobs.

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

[user@biowulf]$ sinteractive -c 16 --mem 45g --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 ~]$ mkdir -p /data/$USER/rmats && cd /data/$USER/rmats

Here is how one can use the most recent rMATS version 4.3.0:

[user@cn3144 ~]$ module load rmats/4.3.0
[+] Loading singularity  3.8.5-1  on cn3144
[+] Loading rMATS  4.3.0
[user@cn3144 ~]$ cp -r $RMATS_SRC/* . 
[user@cn3144 ~]$ make
...

-- Configuring done
-- Generating done
-- Build files have been written to: /usr/local/apps/rMATS/4.3.0/rmats-turbo-4.3.0/bamtools/build
make[2]: Entering directory '/vf/users/denisovga/rMATS/bamtools/build'
make[3]: Entering directory '/vf/users/denisovga/rMATS/bamtools/build'
make[3]: Leaving directory '/vf/users/denisovga/rMATS/bamtools/build'
make[3]: Entering directory '/vf/users/denisovga/rMATS/bamtools/build'
make[3]: Leaving directory '/vf/users/denisovga/rMATS/bamtools/build'
[  0%] Built target SharedHeaders
make[3]: Entering directory '/vf/users/denisovga/rMATS/bamtools/build'
Consolidate compiler generated dependencies of target BamTools-static
make[3]: Leaving directory '/vf/users/denisovga/rMATS/bamtools/build'
make[3]: Entering directory '/vf/users/denisovga/rMATS/bamtools/build'
[  1%] Building CXX object src/api/CMakeFiles/BamTools-static.dir/BamAlignment.cpp.o
[  2%] Building CXX object src/api/CMakeFiles/BamTools-static.dir/BamMultiReader.cpp.o
[  3%] Building CXX object src/api/CMakeFiles/BamTools-static.dir/BamReader.cpp.o
[  4%] Building CXX object src/api/CMakeFiles/BamTools-static.dir/BamWriter.cpp.o
...
[ 95%] Building CXX object src/toolkit/CMakeFiles/bamtools_cmd.dir/bamtools_revert.cpp.o
[ 96%] Building CXX object src/toolkit/CMakeFiles/bamtools_cmd.dir/bamtools_sort.cpp.o
[ 97%] Building CXX object src/toolkit/CMakeFiles/bamtools_cmd.dir/bamtools_split.cpp.o
[ 98%] Building CXX object src/toolkit/CMakeFiles/bamtools_cmd.dir/bamtools_stats.cpp.o
[ 99%] Building CXX object src/toolkit/CMakeFiles/bamtools_cmd.dir/bamtools.cpp.o
make[3]: Leaving directory '/vf/users/denisovga/rMATS/bamtools/build'
[100%] Built target bamtools_cmd
make[2]: Leaving directory '/vf/users/denisovga/rMATS/bamtools/build'
make[1]: Leaving directory '/vf/users/denisovga/rMATS/bamtools/build'
# rm -f to ignore nonexistent files since *.dylib will only exist for mac
cd bamtools/lib; rm -f *.so *.so.* *.dylib
cd rMATS_C; make;
make[1]: Entering directory '/vf/users/denisovga/rMATS/rMATS_C'
cd lbfgs_scipy && make
make[2]: Entering directory '/vf/users/denisovga/rMATS/rMATS_C/lbfgs_scipy'
f77 -c -O2  -c -o lbfgsb.o lbfgsb.f
f77 -c -O2  -c -o linpack.o linpack.f
f77 -c -O2  -c -o timer.o timer.f
make[2]: Leaving directory '/vf/users/denisovga/rMATS/rMATS_C/lbfgs_scipy'
cc -Wall -O2 -msse2 -funroll-loops -fopenmp -fPIE -o rMATSexe src/main.c src/myfunc.c src/util.c lbfgs_scipy/lbfgsb.o lbfgs_scipy/linpack.o lbfgs_scipy/timer.o -lm -lgfortran -lgsl -lgslcblas -lgomp -fPIE -L/usr/lib/x86_64-linux-gnu -lblas -llapack
make clean
make[2]: Entering directory '/vf/users/denisovga/rMATS/rMATS_C'
rm -rf util.o myfunc.o *.o src/*.o lbfgs_scipy/*.o
make[2]: Leaving directory '/vf/users/denisovga/rMATS/rMATS_C'
make[1]: Leaving directory '/vf/users/denisovga/rMATS/rMATS_C'
cd rMATS_pipeline; python setup.py build_ext;
Compiling rmatspipeline/rmatspipeline.pyx because it changed.
[1/1] Cythonizing rmatspipeline/rmatspipeline.pyx
running build_ext
building 'rmats.rmatspipeline' extension
gcc -pthread -B /opt/conda/envs/rmats/compiler_compat -Wl,--sysroot=/ -Wsign-compare -DNDEBUG -g -fwrapv -O3 -Wall -Wstrict-prototypes -fPIC -I/opt/conda/envs/rmats/rmats-turbo-4.3.0/rMATS_pipeline/rmatspipeline -I/vf/users/denisovga/rMATS/bamtools/include -I/opt/conda/envs/rmats/include/python3.6m -c rmatspipeline/rmatspipeline.cpp -o build/temp.linux-x86_64-3.6/rmatspipeline/rmatspipeline.o -O3 -funroll-loops -std=c++11 -fopenmp -D__STDC_CONSTANT_MACROS -D__STDC_LIMIT_MACROS -w -Wl,-static
cc1plus: warning: command line option ‘-Wstrict-prototypes’ is valid for C/ObjC but not for C++
g++ -pthread -shared -B /opt/conda/envs/rmats/compiler_compat -L/opt/conda/envs/rmats/lib -Wl,-rpath=/opt/conda/envs/rmats/lib -Wl,--no-as-needed -Wl,--sysroot=/ build/temp.linux-x86_64-3.6/rmatspipeline/rmatspipeline.o -L/vf/users/denisovga/rMATS/bamtools/lib -lm -lstdc++ -lbamtools -lz -o build/lib.linux-x86_64-3.6/rmats/rmatspipeline.cpython-36m-x86_64-linux-gnu.so -lbamtools -lm -std=c++11 -lz -fopenmp
cp `find ./rMATS_pipeline/build | grep so` .;

[user@cn3144 ~]$ ./test_rmats
test (tests.allow_clipping.test.ClippingAllowedTest) ... ok
test (tests.allow_clipping.test.ClippingNotAllowedTest) ... ok
test (tests.alternative_3_splice_site_novel.test.NovelJunction) ... ok
test (tests.alternative_3_splice_site_novel.test.NovelSpliceSite) ... ok
test (tests.alternative_5_splice_site_novel.test.NovelJunction) ... ok
test (tests.alternative_5_splice_site_novel.test.NovelSpliceSite) ... ok
test (tests.fixed_event_set.test.Test) ... ok
test (tests.individual_counts.test.FilteredTest) ... ok
test (tests.individual_counts.test.NotFilteredTest) ... ok
test (tests.mutually_exclusive_exons_novel.test.NovelJunction) ... ok
test (tests.mutually_exclusive_exons_novel.test.NovelSpliceSite) ... ok
test (tests.only_one_sample.test.StatOffTest) ... ok
test (tests.only_one_sample.test.StatOnTest) ... ok
test (tests.overlapped_gene.test.Test) ... ok
test (tests.prep_post.test.Test) ... ok
test (tests.read_count_edge_cases.test.Test) ... ok
test (tests.retained_intron_novel.test.NovelJunction) ... ok
test (tests.retained_intron_novel.test.NovelSpliceSite) ... ok
test (tests.skipped_exon_basic.test.Test) ... ok
test (tests.skipped_exon_novel.test.NovelJunction) ... ok
test (tests.skipped_exon_novel.test.NovelSpliceSite) ... ok
test (tests.skipped_exon_novel.test.NovelSpliceSiteNotNovelJunction) ... ok
test (tests.stat_large_file.test.Test) ... ok
test (tests.stranded.test.PairedFirstStrandTest) ... ok
test (tests.stranded.test.PairedSecondStrandTest) ... ok
test (tests.stranded.test.PairedUnstrandedTest) ... ok
test (tests.stranded.test.SingleEndFirstStrandTest) ... ok
test (tests.stranded.test.SingleEndSecondStrandTest) ... ok
test (tests.stranded.test.SingleEndUnstrandedTest) ... ok
test (tests.task_stat.test.Test) ... ok
test (tests.variable_read_length.test.Length1Test) ... ok
test (tests.variable_read_length.test.Length1VariableTest) ... ok
test (tests.variable_read_length.test.Length2Test) ... ok
test (tests.variable_read_length.test.Length2VariableTest) ... ok
test (tests.variable_read_length.test.NoLengthTest) ... ok

----------------------------------------------------------------------
Ran 35 tests in 114.831s

OK

[user@cn3144 ~]$ cp -r $RMATS_DATA/* . 

[user@cn3144 ~]$ rmats.py --s1 $PWD/s1.txt \
                          --s2 $PWD/s2.txt \
                          --gtf gtf/Homo_sapiens.Ensembl.GRCh37.72.gtf \
                          --bi /fdb/STAR_indices/2.7.8a/GENCODE/Gencode_human/release_27/genes-100 \
                          --od out_test \
                          -t paired \
                          --nthread $SLURM_CPUS_PER_TASK \
                          --readLength 50 \
                          --tophatAnchor 8 \
                          --cstat 0.0001 \
                          --tstat 6 \
                          --statoff \
                          --tmp /lscratch/${SLURM_JOB_ID}  
...

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

Batch job

Most jobs should be run as batch jobs.

Create a batch input file (e.g. submit.sh). For example:

#!/bin/bash
set -e
module load rmats
export TMPDIR=/lscratch/$SLURM_JOBID
rmats.py  --s1 $PWD/s1.txt --s2 $PWD/s2.txt --gtf $PWD/gtf/Homo_sapiens.Ensembl.GRCh37.72.gtf --bi /fdb/STAR_indices/2.7.8a/GENCODE/Gencode_human/release_27/genes-100 --od out_test -t paired --nthread $SLURM_CPUS_PER_TASK --readLength 50 --tophatAnchor 8 --cstat 0.0001 --tstat 6 --tmp /lscratch/${SLURM_JOB_ID}

Submit this job using the Slurm sbatch command.

sbatch --cpus-per-task=16 --mem=30g --gres=lscratch:20 submit.sh

Swarm of Jobs

A swarm of jobs is an easy way to submit a set of independent commands requiring identical resources.

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

rmats.py  --s1 $PWD/s1.txt --s2 $PWD/s2.txt --gtf $PWDgtf/Homo_sapiens.Ensembl.GRCh37.72.gtf --bi /fdb/STAR_indices/2.7.8a/GENCODE/Gencode_human/release_27/genes-100 --od out_test1 -t paired --nthread $SLURM_CPUS_PER_TASK --readLength 50  --tophatAnchor 8 --cstat 0.0001 --tstat 6 --tmp /lscratch/${SLURM_JOB_ID}
rmats.py  --s1 $PWD s3.txt --s2 $PWD s4.txt --gtf $PWDgtf/Homo_sapiens.Ensembl.GRCh37.72.gtf --bi /fdb/STAR_indices/2.7.8a/GENCODE/Gencode_human/release_27/genes-100 --od out_test2 -t paired --nthread $SLURM_CPUS_PER_TASK --readLength 50  --tophatAnchor 8 --cstat 0.0001 --tstat 6 --tmp /lscratch/${SLURM_JOB_ID}
rmats.py  --s1 $PWD s5.txt --s2 $PWD s6.txt --gtf $PWDgtf/Homo_sapiens.Ensembl.GRCh37.72.gtf --bi /fdb/STAR_indices/2.7.8a/GENCODE/Gencode_human/release_27/genes-100 --od out_test3 -t paired --nthread $SLURM_CPUS_PER_TASK --readLength 50  --tophatAnchor 8 --cstat 0.0001 --tstat 6 --tmp /lscratch/${SLURM_JOB_ID}

Submit this job using the swarm command.

swarm -f rmats.swarm [-g 30] [-t 16] --module rmats

where

`-g #`	Number of Gigabytes of memory required for each process (1 line in the swarm command file)
`-t #`	Number of threads/CPUs required for each process (1 line in the swarm command file).
`--module TEMPLATE`	Loads the TEMPLATE module for each subjob in the swarm