Hey Tom,
my first thought is that there is a record with a contig/chromosome not
found in your reference Fasta. Could you help identify the SAM record that
is causing the problem?
N
On Wed, Oct 29, 2014 at 6:13 AM, Tom Harrop <thomas.har...@ird.fr> wrote:
> Hi,
>
> I'm running CollectRnaSeqMetrics with the following command:
>
> > java -jar /usr/local/bin/picard/CollectRnaSeqMetrics.jar
> > REF_FLAT=../../genome/genePredFixed
> > CHART_OUTPUT=subset.seqmetrics.chart INPUT=subset.bam
> > OUTPUT=subset.seqmetrics
> > REFERENCE_SEQUENCE=../../genome/Osativa_204_v7.0.fa STRAND=NONE
>
> This results in a Java error:
>
> > [Wed Oct 29 10:55:47 CET 2014] picard.analysis.CollectRnaSeqMetrics
> > REF_FLAT=../../genome/genePredFixed STRAND_SPECIFICITY=NONE
> > CHART_OUTPUT=subset.seqmetrics.chart INPUT=subset.bam
> > OUTPUT=subset.seqmetrics
> > REFERENCE_SEQUENCE=../../genome/Osativa_204_v7.0.fa MINIMUM_LENGTH=500
> > RRNA_FRAGMENT_PERCENTAGE=0.8 METRIC_ACCUMULATION_LEVEL=[ALL_READS]
> > ASSUME_SORTED=true STOP_AFTER=0 VERBOSITY=INFO QUIET=false
> > VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5
> > MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false
> > [Wed Oct 29 10:55:47 CET 2014] Executing as tom@Tom on Linux
> > 3.13.0-39-generic amd64; OpenJDK 64-Bit Server VM 1.7.0_65-b32; Picard
> > version: 1.123(286a232caea2fdc8fdd88574c09c460b46386fff_1413818736)
> > IntelDeflater
> > INFO 2014-10-29 10:55:48 CollectRnaSeqMetrics Loaded 44975
> genes.
> > [Wed Oct 29 10:55:48 CET 2014] picard.analysis.CollectRnaSeqMetrics
> > done. Elapsed time: 0.02 minutes.
> > Runtime.totalMemory()=504889344
> > To get help, see
> > http://broadinstitute.github.io/picard/index.html#GettingHelp
> > 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:185)
> > at
> >
> picard.cmdline.CommandLineProgram.instanceMainWithExit(CommandLineProgram.java:125)
> > at
> > picard.analysis.CollectRnaSeqMetrics.main(CollectRnaSeqMetrics.java:97)
>
> I have Java 1.7 installed, but I also tried 1.6 and got the same error.
>
> ValidateSamFile gives the following output:
>
> > Error Type Count
> > ERROR:MISSING_READ_GROUP 1
> > WARNING:RECORD_MISSING_READ_GROUP 179364
>
> But running ValidateSamFile with a reference fasta also gives a Java error:
>
> > [Wed Oct 29 10:57:57 CET 2014] picard.sam.ValidateSamFile
> > INPUT=subset.bam REFERENCE_SEQUENCE=../../genome/Osativa_204_v7.0.fa
> > MODE=VERBOSE MAX_OUTPUT=100 IGNORE_WARNINGS=false VALIDATE_INDEX=true
> > IS_BISULFITE_SEQUENCED=false MAX_OPEN_TEMP_FILES=8000 VERBOSITY=INFO
> > QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5
> > MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false
> > [Wed Oct 29 10:57:57 CET 2014] Executing as tom@Tom on Linux
> > 3.13.0-39-generic amd64; OpenJDK 64-Bit Server VM 1.7.0_65-b32; Picard
> > version: 1.123(286a232caea2fdc8fdd88574c09c460b46386fff_1413818736)
> > IntelDeflater
> > ERROR: Read groups is empty
> > WARNING: Read name SEQILMN01:272:C509GACXX:5:2313:9949:22409, A record
> > is missing a read group
> > [Wed Oct 29 10:57:57 CET 2014] picard.sam.ValidateSamFile done.
> > Elapsed time: 0.00 minutes.
> > Runtime.totalMemory()=504889344
> > To get help, see
> > http://broadinstitute.github.io/picard/index.html#GettingHelp
> > Exception in thread "main" java.lang.NullPointerException
> > at
> >
> htsjdk.samtools.reference.ReferenceSequenceFileWalker.get(ReferenceSequenceFileWalker.java:87)
> > at
> > htsjdk.samtools.SamFileValidator.validateNmTag(SamFileValidator.java:445)
> > at
> >
> htsjdk.samtools.SamFileValidator.validateSamRecordsAndQualityFormat(SamFileValidator.java:283)
> > at
> >
> htsjdk.samtools.SamFileValidator.validateSamFile(SamFileValidator.java:201)
> > at
> >
> htsjdk.samtools.SamFileValidator.validateSamFileVerbose(SamFileValidator.java:160)
> > at picard.sam.ValidateSamFile.doWork(ValidateSamFile.java:169)
> > at
> >
> picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:185)
> > at picard.sam.ValidateSamFile.main(ValidateSamFile.java:102)
>
> Thanks,
>
> Tom
>
>
>
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