Picard MarkDuplicates error: Value was put into PairInfoMap more than once

Question

[Floydian_slip]

Hi,

I saw this error encountered by a few other users and I tried all that was suggested but nothing worked for me. I am still getting the error on Picard MarkDuplicates step:

Exception in thread "main" htsjdk.samtools.SAMException: Value was put into PairInfoMap more than once.  1: GIAB:HWI-ST1023:220:H84P1ADXX:2:2115:7649:16177

Here is what I did:

First sorted and converted bams to fastq as I did not have original fastqs:

samtools sort -n NIST-hg001-7001-b-ready.bam -o NIST-hg001-7001-b-ready.sorted.bam
bedtools bamtofastq -i NIST-hg001-7001-b-ready.sorted.bam -fq NIST-hg001-7001-b-ready_R1.fastq -fq2 NIST-hg001-7001-b-ready_R2.fastq

Then I aligned using BWA mem:

bwa mem -t 8 -M -R '@RG\tID:GIAB\tSM:NA12878\tPL:illumina\tLB:lib\tPU:7001' reference_genome/hg19 NIST-hg001-7001-ready_R1.fastq NIST-hg001-7001-ready_R2.fastq > NIST-hg001-7001.sam

Sort Sam using Picard:

java -jar picard-tools-1.119/SortSam.jar I=NIST-hg001-7001.sam O=NIST-hg001-7001_sorted.bam SO=coordinate CREATE_INDEX=true

And mark duplicates:

java -jar picard-tools-1.119/MarkDuplicates.jar I=NIST-hg001-7001_sorted.bam O=NIST-hg001-7001_dedup.bam M=NIST-hg001-7001.metric

When it gives the error:

Exception in thread "main" htsjdk.samtools.SAMException: Value was put into PairInfoMap more than once.  1: GIAB:HWI-ST1023:220:H84P1ADXX:2:2115:7649:16177

I tried to clean the bam using Samtools:

samtools view -h NIST-hg001-7001_sorted.bam | grep -v null | samtools view -bS - > cleaned.bam

And also used FixMateInformation using Picard:

java -jar /home/nitin/Bioinformatics/Toolbox/src/Misc/picard-tools-1.119/FixMateInformation.jar I=NIST-hg001-7001_sorted.bam O=NIST-hg001-7001_fixedMate.bam

But none of them worked. I still get the same error.

Does anybody have a solution. I have spent a lot of time on it without any use. Thank a lot in advance.

~N

[Pierre Lindenbaum]

Validate or comment your previous questions:

• high depth Illumina samples from 1000 Genomes
• difference between BWA mem, sampe and bwasw
• importance of known sites/resources in GATK pipeline
• EdgeR: different values in expression and differential expression tables
• Individual or population level variant calling?

Answer 1

[Pierre Lindenbaum]

version of picard is just too old. 1.119: (25 August 2014 ) it doesn't support the supplementary flag.

https://github.com/broadinstitute/picard/issues/72

[Floydian_slip]

That did not help either. Upgrading to the latest version 2.1.0 also gave the same error. Any other suggestions?

[mcff23]

I am having the same problem and I am using Picard's version 1.141. Any other suggestion?

Thanks in advance!

Mónica

Answer 2

[Peter Georgeson]

I had a similar problem and had to first use samtools to filter out secondary alignments. i.e.

bwa mem -M -t 5 -k 19 -R "..." ref.fasta in1.fastq.gz in2.fastq.gz |
samtools view -F 0x100 -bSu - | 
samtools sort -o output.bam

Then run Picard's MarkDuplicates.

Hope that helps!