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  • Mate-pair information damaged on file merge

    Hi all,

    I'm trying to merge WGS data for one sample that's been run across 6 lanes.
    Now the six individual files all validate (with Picard ValidateSamFile) correctly; the problem comes when I try to merge them.

    I originally used Picard MergeSamFiles to merge them but this screwed with the mate-pairs ; then I recently found that most of the GATK pipeline will take multiple input files and merge them into a single output, so I tried this at the step of the GATK IndelRealigner, which I understand corrects mate-pair errors.

    Both Picard MergeSamFiles and GATK IndelRealigner have messed up the mate-pair information during the merge. So I wondered if anyone knew of a way to merge the files whilst preserving the mate pair information.

    I should point out that I've tried correcting this (4 times) with Picard FixMateInformation and each time it's run into various problems after about 18 hours.

    the commands I used were:

    java -Xmx24g -Xms24g -jar ~/apps/GATK/GenomeAnalysisTK.jar \
    -I nodup17_01.bam \
    -I nodup17_02.bam \
    -I nodup17_03.bam \
    -I nodup17_04.bam \
    -I nodup17_05.bam \
    -I nodup17_06.bam \
    -R ~/gatk/ucsc.hg19.fasta \
    -T IndelRealigner \
    -targetIntervals nodup17.intervals \
    -o realign17.bam \
    -nt 12 \
    -known ~/gatk/Mills_and_1000G_gold_standard.indels.hg19.vcf \
    -known ~/gatk/1000G_phase1.indels.hg19.vcf \
    -known ~/gatk/dbsnp_135.hg19.vcf

    java -Xmx24g -Xms24g -jar ~/apps/picard/MergeSamFiles.jar \
    I=nodup17_01.bam \
    I=nodup17_02.bam \
    I=nodup17_03.bam \
    I=nodup17_04.bam \
    I=nodup17_05.bam \
    I=nodup17_06.bam \
    o=realign17.bam \
    USE_THREADING=true \
    SO=coordinate \
    TMP_DIR=~/local17/ \
    CREATE_INDEX=true \
    CREATE_MD5_FILE=true

    java -Xmx24g -Xms24g -jar ~/apps/picard/FixMateInformation.jar \
    INPUT=realign17.bam \
    OUTPUT=realign17_FM.bam \
    SO=coordinate \
    TMP_DIR=local17/ \
    CREATE_INDEX=true \
    CREATE_MD5_FILE=true

    Thanks
    Richard

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