picard MergeBamAlignment fails to merge ubam and bam

Question

[Frieda]

I am following the steps recommended by GATK to preprocess my data for human data.

java -jar picard.jar FastqToSam -F1 forward.fq.gz -F2 reverse.fq.gz -O unaligned.bam -SM HG001 -RG HG2YWAFX2.1

java -Xmx8G -jar picard.jar MarkIlluminaAdapters -I unaligned.bam -O markilluminaadapters.bam -M markilluminaadapters_metrics.txt

java -Xmx8G -jar picard.jar SamToFastq -I markilluminaadapters.bam -FASTQ interleaved.fq -CLIPPING_ATTRIBUTE XT -CLIPPING_ACTION 2 -INTERLEAVE true -NON_PF true

bwa index ref.fa

bwa mem -M -t 4 ref.fa interleaved.fq > aligned.sam

java -Xmx16G -jar picard.jar MergeBamAlignment \
  -R ref.fa \
  -UNMAPPED_BAM unaligned.bam \
  -ALIGNED_BAM aligned.sam \
  -O mergebamalignment.bam \
  -CREATE_INDEX true \
  -ADD_MATE_CIGAR true \
  -CLIP_ADAPTERS false \
  -CLIP_OVERLAPPING_READS true \
  -INCLUDE_SECONDARY_ALIGNMENTS true \
  -MAX_INSERTIONS_OR_DELETIONS -1 \
  -PRIMARY_ALIGNMENT_STRATEGY MostDistant \
  -ATTRIBUTES_TO_RETAIN XS

This workflow fails at the MergeBamAlignment step with the following error:

WARNING 2021-06-22 17:48:18     SamAlignmentMerger      Exception merging bam alignment - attempting to sort aligned reads and try again: Inappropriate call if not paired read
    INFO    2021-06-22 17:48:18     SamAlignmentMerger      Finished reading 1348 total records from alignment SAM/BAM.
    [Tue Jun 22 17:48:18 CEST 2021] picard.sam.MergeBamAlignment done. Elapsed time: 0.00 minutes.
    Runtime.totalMemory()=500695040
    To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp
    Exception in thread "main" java.lang.IllegalStateException: Inappropriate call if not paired read
            at htsjdk.samtools.SAMRecord.requireReadPaired(SAMRecord.java:899)
            at htsjdk.samtools.SAMRecord.getProperPairFlag(SAMRecord.java:907)
            at picard.sam.AbstractAlignmentMerger.setValuesFromAlignment(AbstractAlignmentMerger.java:973)
            at picard.sam.AbstractAlignmentMerger.transferAlignmentInfoToFragment(AbstractAlignmentMerger.java:680)
            at picard.sam.AbstractAlignmentMerger.transferAlignmentInfoToPairedRead(AbstractAlignmentMerger.java:752)
            at picard.sam.AbstractAlignmentMerger.mergeAlignment(AbstractAlignmentMerger.java:481)
            at picard.sam.SamAlignmentMerger.mergeAlignment(SamAlignmentMerger.java:186)
            at picard.sam.MergeBamAlignment.doWork(MergeBamAlignment.java:368)
            at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:308)
            at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:103)
            at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:113)

I also checked the following:

1. The ubam and bam file have the same number of reads. (To get number of reads: samtools view -c bamfile AND to get number of mapped reads: samtools view -c -F 260)
2. I tried sorting the ubam and bam files using SortSam and then running MergeBamAlignment, but MergeBamAlignment failed with the same exception.

Does anyone know why this exception occurs and how to avoid it?

[Ram]

Hi,

There is no need to add broadinstitute as a tag - that tag makes no sense. I've removed it, please don't re-add it.

Answer 1

[h.mon]

The problem is bwa mem thinks you are aligning single reads. Therefore, Picard thinks it is dealing with single end reads:

Inappropriate call if not paired read

In case of interleaved files, you need the -p flag with bwa mem.

[Frieda]

@h.mon Your suggestion worked . Thank you very much :)