GATK4 RNA-Seq Germline using Singularity Container

 

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 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.

The workflows are downloaded from: https://github.com/gatk-workflows/gatk4-rnaseq-germline-snps-indels and modified to work on O2 slurm cluster.

Notice the original workflow uses reference and annotation files listed in this file: 

https://github.com/gatk-workflows/gatk4-rnaseq-germline-snps-indels/blob/master/gatk4-rna-germline-variant-calling.inputs.json 

We download the genome reference and all annotation files from: 

https://console.cloud.google.com/storage/browser/genomics-public-data/references/Homo_sapiens_assembly19_1000genomes_decoy/ except for the gtf file, which is downloaded from here: https://console.cloud.google.com/storage/browser/gatk-test-data/intervals?project=broad-dsde-outreach

We then modified the json file to this one: 

1 /n/shared_db/singularity/hmsrc-gatk/scripts/gatk4-rna-germline-variant-calling.inputs.template.json
1 Please modified the file to use your own reference and annotation files as you like.

Jump start

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

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 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 /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 java/jdk-1.8u112 star/2.5.4a 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 fastqToBamGermline.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 & for json in unmappedBams/group*/*.json; do echo working on $json [ -f $json.done ] && continue java -XX:+UseSerialGC -Dconfig.file=your.conf -jar /n/shared_db/singularity/hmsrc-gatk/cromwell-43.jar run gatk4-rna-germline-variant-calling.wdl -i $json 2>&1 | tee -a $json.log && touch $json.done done # rerun the above command if anything does not work # find all .vcf files and create sym links in final folder. If there are dulplicate files: mkdir finalVCFs ln -s $PWD/cromwell-executions/RNAseq/*/call-VariantFiltration/execution/*.variant_filtered.vcf.gz finalVCFs/ 2>/dev/null #merge all CVFs to single VCF file vcfFiles=`ls finalVCFs/*.variant_filtered.vcf.gz` module load bcftools/1.9 bcftools merge -o final.vcf --force-samples $vcfFiles # Stop database killall runDB.sh

Details

Log on to O2

If you need help connecting to O2, please review the How to login to O2 wiki page.



From Windows, use MobaXterm or PuTTY 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:

1 2 3 4 5 6 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 interactive job, and create working folder

1 2 3 4 srun --pty -p interactive -t 0-12:0:0 --mem 16000MB -n 2 /bin/bash mkdir /n/scratch3/users/${USER:0:1}/$USER/testGATK4 cd /n/scratch3/users/${USER:0:1}/$USER/testGATK4
1 2 3 4 5 6 7 # This will setup the path and environmental variables for the pipeline module load gcc/6.2.0 python/2.7.12 java/jdk-1.8u112 star/2.5.4a 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

1 2 cp /n/shared_db/singularity/hmsrc-gatk/scripts/* . buildSampleFoldersFromSampleSheet.py sampleSheet.xlsx


Take a look at the example files

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 # 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

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 less fastqToBamGermline.sh # Here is the content of fastqToBamGermline.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 inputsams="" 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=${readgroup%%_*} \ PLATFORM_UNIT=H0164ALXX140820.2 \ PLATFORM=illumina \ SEQUENCING_CENTER=BI \ RUN_DATE=2014-08-20T00:00:00-0400 inputsams="$inputsams INPUT=$pwdhere/unmappedBams/$group/$sample/$readgroup.bam" done sed "s/inputBamFileNamePath/${pwdhere//\//\\/}\/unmappedBams\/$group\/$sample.bam/" $pwdhere/gatk4-rna-germline-variant-calling.inputs.template.json > $pwdhere/unmappedBams/$group/$sample.json #@2,1,merge,,sbatch -p short -n 1 -t 0-4:0 --mem 12G java -Xmx12g -jar $PICARD/picard-2.8.0.jar MergeSamFiles VALIDATION_STRINGENCY=SILENT OUTPUT=$pwdhere/unmappedBams/$group/$sample.bam $inputsams SORT_ORDER=coordinate && samtools index $pwdhere/unmappedBams/$group/$sample.bam done done


How does this bash script work?

There is a loop that goes through the two group folders and samples, convert the fastq files into unmapped bam files

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

1 #@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
1 Test run the modified bash script as a pipeline
1 runAsPipeline fastqToBamGermline.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 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.

Ideally, with 'useTmp', the software should run faster using local /tmp disk space for database/reference than the network storage. For this workflow, we don't need it.

Sample output from the test run

Note that only step 2 used -t 50:0, and all other steps used the default -t 10:0. The default walltime limit was set in the runAsPipeline command, and the walltime parameter for step 2 was set in the bash_script_v2.sh

Run the pipeline

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 of 123 for jobids in the command's output. 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 runAsPipeline fastqToBamGermline.sh "sbatch -p short --mem 4G -t 1:0:0 -n 1" noTmp run 2>&1 | tee output.log # You should see something similar as above. But this time, the pipeline runner really submitted all the jobs # Below is the output Fri Jul 19 10:07:29 EDT 2019 Runing: /home/ld32/rcbioDev/bin/runAsPipeline fastqToBamGermline.sh sbatch -p short --mem 4G -t 1:0:0 -n 1 noTmp run module list: Currently Loaded Modules: 1) java/jdk-1.8u112 2) gcc/6.2.0 3) python/2.7.12 converting fastqToBamGermline.sh to flag/slurmPipeLine.201907191007.run.sh find loop start: for group in `ls -d group*/|sed 's|[/]||g'`; do find loop start: for sample in `ls -d */|sed 's|[/]||g'`; do find loop start: for r1 in `ls $sample/*_1.fastq $sample/*_1.fq 2>/dev/null`; do find job marker: #@1,0,fastqToSam,,sbatch -p short -t 0-4 --mem 4G sbatch options: sbatch -p short -t 0-4 --mem 4G find job: java -Xmx8G -jar /n/app/picard/2.8.0/bin/picard-2.8.0.jar FastqToSam \ ... step: 1, depends on: 0, job name: fastqToSam, flag: fastqToSam.group2.normal1.normal1/lib2_lane2_1.fq depend on no job sbatch -p short -t 0-4 --mem 4G --mail-type=FAIL --nodes=1 -J 1.0.fastqToSam.group2.normal1.normal1_lib2_lane2_1.fq -o /n/groups/rccg/ld32/SingularityGATK1/flag/1.0.fastqToSam.group2.normal1.normal1_lib2_lane2_1.fq.out -e /n/groups/rccg/ld32/SingularityGATK1/flag/1.0.fastqToSam.group2.normal1.normal1_lib2_lane2_1.fq.out /n/groups/rccg/ld32/SingularityGATK1/flag/1.0.fastqToSam.group2.normal1.normal1_lib2_lane2_1.fq.sh # Submitted batch job 46108236 ... All submitted jobs: job_id depend_on job_flag 46108220 null 1.0.fastqToSam.group1.tumor1.tumor1_lib1_lane1_1.fq 46108224 null 1.0.fastqToSam.group1.tumor1.tumor1_lib1_lane2_1.fq 46108232 null 1.0.fastqToSam.group2.normal1.normal1_lib2_lane1_1.fq 46108236 null 1.0.fastqToSam.group2.normal1.normal1_lib2_lane2_1.fq ... ---------------------------------------------------------

Monitoring the jobs

You can use the command:

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

To see the job status (running, pending, etc.). You also get two emails for each step, one at the start of the step, one at the end of the step.

Check job logs

You can use the command:

1 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.

You also get two emails for each step, one at the start of the step, one at the end of the step.

Re-run the pipeline in case some jobs fail

You can rerun this command in the same folder

1 runAsPipeline fastqToBamGermline.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:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 # start database and let it run in background (& allow the command keep running in background) runDB.sh & for json in unmappedBams/group*/*.json; do echo working on $json [ -f $json.done ] && continue java -XX:+UseSerialGC -Dconfig.file=your.conf -jar /n/shared_db/singularity/hmsrc-gatk/cromwell-43.jar run gatk4-rna-germline-variant-calling.wdl -i $json 2>&1 | tee -a $json.log && touch $json.done done # rerun the above command if anything does not work # find all .vcf files and create sym links in final folder. If there are dulplicate files: mkdir finalVCFs ln -s $PWD/cromwell-executions/RNAseq/*/call-VariantFiltration/execution/*.variant_filtered.vcf.gz finalVCFs/ 2>/dev/null #merge all CVFs to single VCF file vcfFiles=`ls finalVCFs/*.variant_filtered.vcf.gz` module load bcftools/1.9 bcftools merge -o final.vcf --force-samples $vcfFiles # Stop database killall runDB.sh


To run the workflow on your own data

To instead run 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.



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