ApplyBQSR doesn't recognise my arguments?

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Ali Hudson

• November 10, 2021 15:55
• Edited

I'm trying to recalibrate some bam files using the following code. The first section is working fine and producing the table, but the ApplyBQSR command is giving the error below. 

#!/bin/bash

module purge
module load picard/2.6.0-Java-1.8.0_131
module load GATK/4.0.5.1-foss-2018a-Python-3.6.4

## Set your master path
MASTER=/lustre/projects/Research_Project-T109423/ali/FIBR_transfer/ftp1.sequencing.exeter.ac.uk/V0085/11_trimmed/practiceSNP_calling

## Also add path to indexed reference and recalibration file
reference=/lustre/projects/Research_Project-XXXX/STAR/STAR.chromosomes.release.fasta
recal=/lustre/projects/Research_Project-XXXX/STAR/STAR_intersect_recal.vcf

bam_in=$MASTER/SNP_calling/bams/interim_bams
bam_out=$MASTER/SNP_calling/bams/clean_bams 

metadata=$MASTER/SNP_calling/metaCA10.tsv

## This just catches the array in case it's running for a value with no individual (this screws with the outputs)
IND_N=$(cut -f2 $metadata | tail -n+2 | uniq | awk 'NF > 0' | wc -l)

if [ $SLURM_ARRAY_TASK_ID -le $IND_N ]
then
    ## In array ##
    insampleID_array=( `cat $metadata | cut -f 2` )
    insampleID=$bam_in/${insampleID_array[(($SLURM_ARRAY_TASK_ID))]}

    ## Out array
    outsampleID_array=( `cat $metadata | cut -f 2` )
    outsampleID=$bam_out/${outsampleID_array[(($SLURM_ARRAY_TASK_ID))]}

    ## Apply base quality score recalibration
    ## BaseRecal

    gatk --java-options "-Xmx8g" BaseRecalibrator \
    -I $insampleID.sorted.dups.bam \
    -R $reference --known-sites $recal \
    -O $outsampleID.table

    ## ApplyBQSR
    gatk --java-options "-Xmx8g" ApplyBQSR    
    -I $insampleID.sorted.dups.bam \
    -R $reference --bqsr-recal-file $outsampleID.table \ 
    -O $outsampleID.recal.bam
    
    ## Index the recalibrated bam files
    java -Xmx10g -jar $EBROOTPICARD/picard.jar BuildBamIndex \
    I=$outsampleID.recal.bam VALIDATION_STRINGENCY=LENIENT

fi

The error is this: 

***********************************************************************

A USER ERROR has occurred: Argument output was missing: Argument 'output' is required.

***********************************************************************
Set the system property GATK_STACKTRACE_ON_USER_EXCEPTION (--java-options '-DGATK_STACKTRACE_ON_USER_EXCEPTION=true') to print the stack trace.
Using GATK jar /lustre/shared/software/GATK/4.0.5.1-foss-2018a-Python-3.6.4/gatk-package-4.0.5.1-local.jar
Running:
java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -Xmx8g -jar /lustre/shared/software/GATK/4.0.5.1-foss-2018a-Python-3.6.4/gatk-package-4.0.5.1-local.jar ApplyBQSR
/var/spool/slurmd/job866408/slurm_script: line 127: -I: command not found
/var/spool/slurmd/job866408/slurm_script: line 129: -O: command not found

If I add a \ after "gatk --java-options "-Xmx8g" ApplyBQSR" I get the following error instead:

 

***********************************************************************

A USER ERROR has occurred: Invalid argument ' '.

***********************************************************************
Set the system property GATK_STACKTRACE_ON_USER_EXCEPTION (--java-options '-DGATK_STACKTRACE_ON_USER_EXCEPTION=true') to print the stack trace.
Using GATK jar /lustre/shared/software/GATK/4.0.5.1-foss-2018a-Python-3.6.4/gatk-package-4.0.5.1-local.jar
Running:
java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -Xmx8g -jar /lustre/shared/software/GATK/4.0.5.1-foss-2018a-Python-3.6.4/gatk-package-4.0.5.1-local.jar ApplyBQSR 
/var/spool/slurmd/job866367/slurm_script: line 108: -I: command not found

Can anyone help me out?  I'm not sure what I'm doing wrong at this point. 

 

 

(Edit to add: I've also tried formatting this in a variety of ways, e.g. indenting and reording the arguments to look more like they do in others examples - but it always gives one or the other of these errors, or simply doesn't produce a file or an error file)

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• 

  Genevieve Brandt (she/her)

  • November 10, 2021 20:12

  I think this is an issue in your bash or slurm code. Could you try having the entire ApplyBQSR command on the same line and see if it works?

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• 

  Ali Hudson

  • November 11, 2021 09:26

  Thanks! I tried this yesterday but somehow an extra character must have still snuck in - I did it again just now and it seems to be working! 

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  Genevieve Brandt (she/her)

  • November 12, 2021 18:03

  Great news! Thanks for the update!

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