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 ------------------------------------------------------------------------------ _______________________________________________ Samtools-help mailing list Samtools-help@lists.sourceforge.net https://lists.sourceforge.net/lists/listinfo/samtools-help
