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This page shows you how to run GATK4 using our recently installed Singularity GATK4 container. The runAsPipeline script, accessible through the rcbio/1.0 module, converts the bash script into a pipeline that easily submits jobs to the Slurm scheduler for you.

Features of the this pipeline:

Given a sample sheet, generate folder structure for data processing
Submit each step as a cluster job using sbatch.
Automatically arrange dependencies among jobs.
Email notifications are sent when each job fails or succeeds.
If a job fails, all its downstream jobs automatically are killed.
When re-running the pipeline on the same data folder, if there are any unfinished jobs, the user is asked to kill them or not.
When re-running the pipeline on the same data folder, the user is asked to confirm to re-run or not if a step was done successfully earlier.

Please read below for an example.

The workflows are downloaded from: https://github.com/gatk-workflows/gatk4-data-processing and and https://github.com/gatk-workflows/gatk4-somatic-snvs-indels

Jump startJumpstart

Here are the commands to test out the workflow using example data. The whole run need needs a few hours if the cluster is not busy.

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ssh user123@o2.hms.harvard.edu

# set up screen software: https://wiki.rc.hms.harvard.edu/pages/viewpage.action?pageId=20676715
cp /n/shared_db/misc/rcbio/data/screenrc.template.txt ~/.screenrc

screen

srun --pty -p interactive -t 0-12:0:0 --mem 16000MB -n 2 /bin/bash

mkdir -p /n/scratch3/users/${USER:0:1}/$USER/testGATK4  

cd /n/scratch3/users/${USER:0:1}/$USER/testGATK4

module load gcc/6.2.0 python/2.7.12

export PATH=/n/shared_db/singularity/hmsrc-gatk/bin:/home/ld32/rcbioDev/bin:/opt/singularity/bin:$PATH

# setup database. Only need run this once. It will setup database in home, so make sure you have at least 5G free space at home.
setupDB.sh


cp /n/shared_db/singularity/hmsrc-gatk/scripts/* .

buildSampleFoldersFromSampleSheet.py sampleSheet.xlsx

runAsPipeline fastqToBam.sh "sbatch -p short --mem 4G -t 1:0:0 -n 1" noTmp run 2>&1 | tee output.log

# check email or use this command to see the workflow progress
squeue -u $USER -o "%.18i %.9P %.28j %.8u %.8T %.10M %.9l %.6D %R %S"

# After all jobs finish run, run this command to start database, and keep it running in background
runDB.sh  &     

java -XX:+UseSerialGC -Dconfig.file=your.conf -jar /n/shared_db/singularity/hmsrc-gatk/cromwell-43.jar run processing-for-variant-discovery-gatk4.wdl -i unmappedBams/group1/in.json 2>&1 | tee -a group1.log

java -XX:+UseSerialGC -Dconfig.file=your.conf -jar /n/shared_db/singularity/hmsrc-gatk/cromwell-43.jar run processing-for-variant-discovery-gatk4.wdl -i unmappedBams/group2/in.json 2>&1 | tee -a group2.log

setupJson.sh

java -XX:+UseSerialGC -Dconfig.file=your.conf -jar /n/shared_db/singularity/hmsrc-gatk/cromwell-43.jar run mutect2.wdl -i unmappedBams/exon.json && findVCF.sh

# Stop database
killall runDB.sh

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If you need help connecting to O2, please review the Using Slurm Basic wiki page.

From Windows, use the graphical PuTTY program to connect to o2.hms.harvard.edu and make sure the port is set to the default value of 22.

From a Mac Terminal, use the ssh command, inserting your eCommons ID instead of user123:

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ssh user123@o2.hms.harvard.edu

# set up screen software: https://wiki.rc.hms.harvard.edu/pages/viewpage.action?pageId=20676715
cp /n/shared_db/misc/rcbio/data/screenrc.template.txt ~/.screenrc

screen  # start screen session. For detail: https://wiki.rc.hms.harvard.edu/pages/viewpage.action?pageId=20676715

Start an interactive job, and create a working folder

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srun --pty -p interactive -t 0-12:0:0 --mem 16000MB -n 2 /bin/bash mkdir -p /n/scratch3/users/${USER:0:1}/$USER/testGATK4 cd /n/scratch3/users/${USER:0:1}/$USER/testGATK4

Load the pipeline-related modules

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# This will setup the path and environmental variables for the pipeline
module load gcc/6.2.0 python/2.7.12
export PATH=/n/shared_db/singularity/hmsrc-gatk/bin:/home/ld32/rcbioDev/bin:$PATH



# setup database. Only need run this once. It will setup database in home, so make sure you have at least 5G free space at home.
setupDB.sh

Build some testing data in the current folder

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true	cp /n/shared_db/singularity/hmsrc-gatk/scripts/* . buildSampleFoldersFromSampleSheet.py sampleSheet.xlsx

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Take a look at the example files

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# this command shows the content of newly created folders
ls -l group*/*

# Below is out of the ls command
group1/tumor1:

total 74K
lrwxrwxrwx 1 ld32 rccg 120 Jul 19 08:43 lib1_lane1_1.fq -> /n/groups/shared_databases/gatk/ftp.broadinstitute.org/bundle/2.8/b37/3.3-0-in/group1/patient1/ERR166302_ch14_16_1.fastq
lrwxrwxrwx 1 ld32 rccg 120 Jul 19 08:43 lib1_lane1_2.fq -> /n/groups/shared_databases/gatk/ftp.broadinstitute.org/bundle/2.8/b37/3.3-0-in/group1/patient1/ERR166302_ch14_16_2.fastq
lrwxrwxrwx 1 ld32 rccg 120 Jul 19 08:43 lib1_lane2_1.fq -> /n/groups/shared_databases/gatk/ftp.broadinstitute.org/bundle/2.8/b37/3.3-0-in/group1/patient1/ERR166303_ch14_16_1.fastq
lrwxrwxrwx 1 ld32 rccg 120 Jul 19 08:43 lib1_lane2_2.fq -> /n/groups/shared_databases/gatk/ftp.broadinstitute.org/bundle/2.8/b37/3.3-0-in/group1/patient1/ERR166303_ch14_16_2.fastq

group2/normal1:
total 8.0K
lrwxrwxrwx 1 ld32 rccg 121 Jul 19 08:43 lib2_lane1_1.fq -> /n/groups/shared_databases/gatk/ftp.broadinstitute.org/bundle/2.8/b37/3.3-0-in/group2/control1//ERR166316_ch14_16_1.fastq
lrwxrwxrwx 1 ld32 rccg 121 Jul 19 08:43 lib2_lane1_2.fq -> /n/groups/shared_databases/gatk/ftp.broadinstitute.org/bundle/2.8/b37/3.3-0-in/group2/control1//ERR166316_ch14_16_2.fastq
lrwxrwxrwx 1 ld32 rccg 121 Jul 19 08:43 lib2_lane2_1.fq -> /n/groups/shared_databases/gatk/ftp.broadinstitute.org/bundle/2.8/b37/3.3-0-in/group2/control1//ERR166317_ch14_16_1.fastq
lrwxrwxrwx 1 ld32 rccg 121 Jul 19 08:43 lib2_lane2_2.fq -> /n/groups/shared_databases/gatk/ftp.broadinstitute.org/bundle/2.8/b37/3.3-0-in/group2/control1//ERR166317_ch14_16_1.fastq

The original bash script

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less fastqToBam.sh


# Here is the content of fastqToBam.sh
#!/bin/sh

module load gcc/6.2.0 python/2.7.12 java/jdk-1.8u112 samtools/1.9 bwa/0.7.17

[ -d group2 ] || { echo group2 is not found. You need at least two groups to run this pipeline; exit 1; }

pwdhere=`pwd`

for group in `ls -d group*/|sed 's|[/]||g'`; do
    echo working on group:  $group
    cd $pwdhere/$group
    for sample in `ls -d */|sed 's|[/]||g'`; do
        rm $pwdhere/unmappedBams/$group/$sample.inputBamList.txt 2>/dev/null
        mkdir -p $pwdhere/unmappedBams/$group/$sample
        echo working on sample: $sample
        for r1 in `ls $sample/*_1.fastq $sample/*_1.fq 2>/dev/null`; do
            readgroup=${r1#*/}
            readgroup=${readgroup%_*}
            r2=${r1%_*}_2${r1##*_1}
            echo working on readgroup: $readgroup
                        [[ -f $r2 ]] || { echo -e "\n\n!!!Warning: read2 file '$r2' not exist, ignore this warning if you are working with single-end data\n\n"; r2=""; }

            #@1,0,fastqToSam,,sbatch -p short -t 0-4 --mem 4G
            java -XX:+UseSerialGC -Xmx8G -jar /n/app/picard/2.8.0/bin/picard-2.8.0.jar FastqToSam \
               	FASTQ=$r1 \
                FASTQ2=$r2 \
                OUTPUT=$pwdhere/unmappedBams/$group/$sample/$readgroup.bam \
                READ_GROUP_NAME=${readgroup##*_} \
                SAMPLE_NAME=$sample \
                LIBRARY_NAME=${readgroup%%_*} \
                PLATFORM_UNIT=H0164ALXX140820.2 \
                PLATFORM=illumina \
                SEQUENCING_CENTER=BI \
                RUN_DATE=2014-08-20T00:00:00-0400

				echo $pwdhere/unmappedBams/$group/$sample/$readgroup.bam >> $pwdhere/unmappedBams/$group/$sample.inputBamList.txt
        done
        sed "s/sampleName/$sample/; s/inputBamList.txt/${pwdhere//\//\\/}\/unmappedBams\/$group\/$sample.inputBamList.txt/" $pwdhere/processing-for-variant-discovery-gatk4-template.json > $pwdhere/unmappedBams/$group/in.json

        break;
    done
done

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These comments are recgnized by our pipeline runner and the command following them are submitted as slurm jobs:

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#@1,0,fastqToSam,,sbatch -p short -t 0-4 --mem 4G # means this is step 1, depends on nothing, and use 4 hours and 4G memory

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Test run the modified bash script as a pipeline

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runAsPipeline fastqToBam.sh "sbatch -p short -t 10:0 -n 1" useTmp

This command will generate new bash script named slurmPipeLine.201907200946.sh in flag folder (201907200946 is 201907200946 is the timestamp that runAsPipeline was invoked at). Then test run it, meaning does not really submit jobs, but only create a fake job id, 123 for each step. If you were to append run at the end of the command, the pipeline would actually be submitted to the Slurm scheduler.

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Thus far in the example, we have not actually submitted any jobs to the scheduler. To submit the pipeline, you will need to append the run parameter to the command. If run is not specified, test mode will be used, which does not submit jobs and gives theplaceholder the placeholder of 123 for jobids in the command's output.

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You can use the command:

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squeue -u $USER -o "%.18i %.9P %.28j %.8u %.8T %.10M %.9l %.6D %R %S"

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You can use the command:

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ls -l flag

This command list all the logs created by the pipeline runner. *.sh files are the slurm scripts for eash step, *.out files are output files for each step, *.success files means job successfully finished for each step and *.failed means job failed for each steps.

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You can rerun this command in the same folder

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true	runAsPipeline fastqToBam.sh "sbatch -p short --mem 4G -t 1:0:0 -n 1" noTmp run 2>&1 \| tee output.log

This command will check if the earlier run is finished or not. If not, ask user to kill the running jobs or not, then ask user to rerun the successfully finished steps or not. Click 'y', it will rerun, directly press 'enter' key, it will not rerun.

Call variance:

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# start database and let it run in background (& allow the command keep running in background) 
runDB.sh &
java -XX:+UseSerialGC -Dconfig.file=your.conf -jar /n/shared_db/singularity/hmsrc-gatk/cromwell-43.jar run processing-for-variant-discovery-gatk4.wdl -i unmappedBams/group1/in.json 2>&1 | tee -a group1.log



java -XX:+UseSerialGC -Dconfig.file=your.conf -jar /n/shared_db/singularity/hmsrc-gatk/cromwell-43.jar run processing-for-variant-discovery-gatk4.wdl -i unmappedBams/group2/in.json 2>&1 | tee -a group2.log



setupJson.sh


java -XX:+UseSerialGC -Dconfig.file=your.conf -jar /n/shared_db/singularity/hmsrc-gatk/cromwell-43.jar run mutect2.wdl -i unmappedBams/exon.json && findVCF.sh

# Stop the database
killall runDB.sh

To run the workflow on your own data

To instead run a workflow on your own data, transfer the sample sheet to your local machine following this wiki page and modify the sample sheet. Then you can transfer it back to O2 under your account, then go to the build folder structure step.

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Let us know if you have any questions. Please include your working folder and commands used in your email. Any comment comments and suggestion suggestions are welcome!

Versions Compared

Old Version 29

New Version 30

Key

Jump startJumpstart

Start an interactive job, and create a working folder

Load the pipeline-related modules

Build some testing data in the current folder

Take a look at the example files

The original bash script

Call variance:

To run the workflow on your own data