Well, that did the trick. I had given up on providing the reference
sequence because it always crashed picard with:
Exception in thread "main" java.lang.NullPointerException
at
htsjdk.samtools.reference.ReferenceSequenceFileWalker.get(ReferenceSequenceFileWalker.java:87)
at
picard.analysis.SinglePassSamProgram.makeItSo(SinglePassSamProgram.java:113)
at
picard.analysis.SinglePassSamProgram.doWork(SinglePassSamProgram.java:53)
at
picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:183)
at
picard.cmdline.CommandLineProgram.instanceMainWithExit(CommandLineProgram.java:124)
at
picard.analysis.CollectAlignmentSummaryMetrics.main(CollectAlignmentSummaryMetrics.java:94)
and it wasn't clear to me why it would need the full reference when
all of the relevant info should be in the BAM. After your suggestion,
however, I kept trying and it seems picard didn't like the path I was
providing:
R=../oar_3.1/Ovis_aries.Oar_v3.1.dna.toplevel.fa
It's interesting that following symlinks worked just fine:
R=../oar_3.1/ref.fa
R=./Ovis_aries.Oar_v3.1.dna.toplevel.fa
Is this perhaps a bug, feature, or user error? Maybe a length
limitation on the path? In any case, thanks for the help and the
easy fix.
Jeremy
On Thu, 28 Aug 2014 14:54:01 -0400
Nils Homer <nho...@broadinstitute.org> wrote:
> Try giving it a reference sequence (R=...) to see if the
> alignment-based metrics are output.
>
> N
>
>
> On Thu, Aug 28, 2014 at 2:35 PM, Jeremy Volkening <volken...@wisc.edu>
> wrote:
>
> > Hello,
> >
> > I have a set of ~ 1 billion gDNA paired reads mapped to a reference
> > genome with BWA-MEM. I tried to use picard's
> > CollectAlignmentSummaryMetrics to generate a summary of the mapping,
> > but it reports zero aligned reads:
> >
> > FIRST_OF_PAIR 549154663 549154663 1 0 0 0 0 0 0
> > 0 0 0 0 0 99.936337 0 0 0 0 0 0.000001
> > SECOND_OF_PAIR 549154663 549154663 1 0 0 0 0 0 0
> > 0 0 0 0 0 98.611736 0 0 0 0 0 0
> > PAIR 1098309326 1098309326 1 0 0 0 0 0 0 0 0
> > 0 0 0 99.274036 0 0 0 0 0 0
> >
> > A look at the SAM flags manually seems to indicate that most pairs
> > are properly aligned,
> > and the output of samtools flagstat seems to agree:
> >
> > 1102426754 + 0 in total (QC-passed reads + QC-failed reads)
> > 0 + 0 duplicates
> > 1097354292 + 0 mapped (99.54%:-nan%)
> > 1102426754 + 0 paired in sequencing
> > 551276790 + 0 read1
> > 551149964 + 0 read2
> > 1042528490 + 0 properly paired (94.57%:-nan%)
> > 1096261806 + 0 with itself and mate mapped
> > 1092486 + 0 singletons (0.10%:-nan%)
> > 36064341 + 0 with mate mapped to a different chr
> > 14493831 + 0 with mate mapped to a different chr (mapQ>=5)
> >
> > I don't mind using samtools flagstat to evaluate the mapping, but
> > I'm puzzled by this
> > and concerned that the problems with the picard output signal
> > further potential issues
> > using picard for duplicate removal, etc. I've seen a few users
> > report similar behavior
> > in the past, and one was able to pin it to their combination of OS
> > and java, but I've
> > tried multiple combinations of java package (Sun, OpenJDK) and
> > picard version and get
> > the same results.
> >
> > To generate the above results I used:
> >
> > Debian 'Wheezy'
> > java version 1.7.0_67
> > picard version 1.119
> > samtools version 0.1.18
> >
> > and the picard command was:
> >
> > java -jar /opt/picard/CollectAlignmentSummaryMetrics.jar
> > MAX_INSERT_SIZE=500 I=bwa.map.w_rg.bam O=bwa.picard.summary
> >
> > As per BWA the library insert size distribution has mean and sd of
> > ~ 180 and 20, respectively.
> > I am attaching a pruned SAM file with just a pair of reads that
> > appear to be mapped correctly
> > but which picard reports as unaligned as per above. Any suggestions
> > or help would be much
> > appreciated.
> >
> > Thanks,
> > Jeremy
> >
> >
> >
> >
> >
> > ------------------------------------------------------------------------------
> > Slashdot TV.
> > Video for Nerds. Stuff that matters.
> > http://tv.slashdot.org/
> > _______________________________________________
> > Samtools-help mailing list
> > Samtools-help@lists.sourceforge.net
> > https://lists.sourceforge.net/lists/listinfo/samtools-help
> >
> >
------------------------------------------------------------------------------
Slashdot TV.
Video for Nerds. Stuff that matters.
http://tv.slashdot.org/
_______________________________________________
Samtools-help mailing list
Samtools-help@lists.sourceforge.net
https://lists.sourceforge.net/lists/listinfo/samtools-help
