Hi Ryan,
Can I ask you to please post your question on the GATK forum? That's where
we'll address Picard questions from now on:
http://gatkforums.broadinstitute.org/
In the meantime I'll hunt down the answer to your question.
Best,
Geraldine
On Tue, Mar 17, 2015 at 3:25 PM, Nils Homer <nho...@broadinstitute.org>
wrote:
>
> ---------- Forwarded message ----------
> From: Ryan Davis <rrda...@gmail.com>
> Date: Tue, Mar 17, 2015 at 1:32 PM
> Subject: [Samtools-help] MergeBamAlignment issue
> To: samtools-help@lists.sourceforge.net
>
>
> Hi,
>
> I am attempting to merge the output of tophat in order to run some RNASeq
> QC metrics. This is a single read 50 bp on a Hiseq. In order to get past
> the fact that topphat gives a MAPQ of 255 to unmapped reads (not 0 as
> expected by Picard) I used the following tool (
> https://github.com/cbrueffer/tophat-recondition) to change it.
>
> Once completed, I added read groups using picard and then sorted the
> accepted_hits.bam by coordinate and sorted the unmapped reads by queryname.
>
> tophat-recondition.py /home/ryan/NGS_Data/No_Dox
>
> java -Xmx2g -jar /home/ryan/jar/picard-tools-1.129/picard.jar \
> AddOrReplaceReadGroups \
> I=/home/ryan/NGS_Data/unmapped_fixup.bam \
> O=/home/ryan/NGS_Data/unmapped_fixup-RG.bam \
> RGID=No_Dox RGLB=No_Dox RGPL=illumina RGPU=GCCAAT RGSM=No_Dox
>
>
> java -Xmx2g -jar /home/ryan/jar/picard-tools-1.129/picard.jar \
> AddOrReplaceReadGroups \
> I=/home/ryan/NGS_Data/accepted_hits.bam \
> O=/home/ryan/NGS_Data/accepted_hits-RG.bam \
> SORT_ORDER=coordinate \
> RGID=No_Dox RGLB=No_Dox RGPL=illumina RGPU=GCCAAT RGSM=No_Dox \
> CREATE_INDEX=true
>
> java -Xmx2g -jar /home/ryan/jar/picard-tools-1.129/picard.jar \
> SortSam \
> I=/home/ryan/NGS_Data/unmapped_fixup-RG.bam \
> O=/home/ryan/NGS_Data/unmapped_fixup-RG-sorted.bam \
> SORT_ORDER=queryname
>
> java -Xmx2g -jar /home/ryan/jar/picard-tools-1.129/picard.jar \
> SortSam \
> I=/home/ryan/NGS_Data/accepted_hits-RG.bam \
> O=/home/ryan/NGS_Data/accepted_hits-RG-sorted.bam \
> SORT_ORDER=coordinate \
> CREATE_INDEX=true
>
> java -Xmx2g -jar /home/ryan/jar/picard-tools-1.129/picard.jar \
> MergeBamAlignment \
> UNMAPPED_BAM=/home/ryan/NGS_Data/unmapped_fixup-RG-sorted.bam \
> ALIGNED_BAM=/home/ryan/NGS_Data/accepted_hits-RG-sorted.bam \
> O=/home/ryan/NGS_Data/merge_unmapped_accepted_hits_No_Dox.bam \
> SORT_ORDER=coordinate \
> REFERENCE_SEQUENCE=/home/ryan/Reference/human_spikein/hg19_spikein.fa \
> PROGRAM_RECORD_ID=Tophat \
> PROGRAM_GROUP_VERSION=0.1 \
> PROGRAM_GROUP_COMMAND_LINE=tophat/dummy \
> PAIRED_RUN=false \
> VALIDATION_STRINGENCY=LENIENT \
> CREATE_INDEX=true
>
> I then get the following warning followed by the error:
>
> WARNING 2015-03-17 09:44:22 SamAlignmentMerger Exception merging
> bam alignment - attempting to sort aligned reads and try again: Aligned
> record iterator (HS3:608:C6LNLACXX:7:2101:9946:63417) is behind the
> unmapped reads (HS3:608:C6LNLACXX:7:2101:9947:11009)
> INFO 2015-03-17 09:44:33 SamAlignmentMerger Read 1000000 records
> from alignment SAM/BAM.
> INFO 2015-03-17 09:44:43 SamAlignmentMerger Read 2000000 records
> from alignment SAM/BAM.
> ....
> INFO 2015-03-17 09:58:01 SamAlignmentMerger Read 96000000 records
> from alignment SAM/BAM.
> INFO 2015-03-17 09:58:09 SamAlignmentMerger Read 97000000 records
> from alignment SAM/BAM.
> INFO 2015-03-17 09:58:15 SamAlignmentMerger Finished reading
> 97571897 total records from alignment SAM/BAM.
> [Tue Mar 17 09:58:16 PDT 2015] picard.sam.MergeBamAlignment done. Elapsed
> time: 14.32 minutes.
> Runtime.totalMemory()=1908932608
> To get help, see
> http://broadinstitute.github.io/picard/index.html#GettingHelp
> Exception in thread "main" java.lang.IllegalStateException: Aligned record
> iterator (HS3:608:C6LNLACXX:7:1101:10000:11036) is behind the unmapped
> reads (HS3:608:C6LNLACXX:7:1101:10000:48402)
> at
> picard.sam.AbstractAlignmentMerger.mergeAlignment(AbstractAlignmentMerger.java:383)
> at
> picard.sam.SamAlignmentMerger.mergeAlignment(SamAlignmentMerger.java:153)
> at picard.sam.MergeBamAlignment.doWork(MergeBamAlignment.java:248)
> at
> picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:187)
> at
> picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:95)
> at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:105)
>
> I have searched and have been unsuccessful at resolving this problem. Any
> ideas?
>
> I am running picard 1.129 using java version "1.7.0_60"
>
> ValidateSamFile on both inputs into MergeBamAlignment returns no errors in
> those files. I am at a lost and seeking for help!
>
> Thanks,
>
> Ryan
>
>
>
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>
--
Geraldine A. Van der Auwera, Ph.D.
Bioinformatics Scientist II
GATK Support & Outreach
Broad Institute
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by Intel and developed in partnership with Slashdot Media, is your hub for all
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news, videos, case studies, tutorials and more. Take a look and join the
conversation now. http://goparallel.sourceforge.net/
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