Could it be because we search for the associated sequence dictionary using
the fasta file name, and the dictionary it finds for one is missing or
mismatching? You could search for *.dict or *.fai files for each path.
N
On Thu, Aug 28, 2014 at 3:50 PM, Jeremy Volkening <volken...@wisc.edu>
wrote:
> 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
> > >
> > >
> > >
> > >
> > >
> > >
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> > >
> > >
>
>
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