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  • sphil
    Senior Member
    • Apr 2010
    • 192
    #1

    RNA-SeQC input error

    Hey guys,

    I want to use RNA-SeQC for my Rna-seq data. Preparing it like it's done on the website I get the following error:

    java.lang.IllegalStateException: Inappropriate call if not paired read
    at net.sf.samtools.SAMRecord.requireReadPaired(SAMRecord.java:642)
    at net.sf.samtools.SAMRecord.getMateUnmappedFlag(SAMRecord.java:669)
    at org.broadinstitute.sting.utils.sam.GATKSAMRecord.getMateUnmappedFlag(GATKSAMRecord.java:307)
    at org.broadinstitute.cga.rnaseq.gatk.CountReadMetricsWalker.map(CountReadMetricsWalker.java:188)
    at org.broadinstitute.cga.rnaseq.gatk.CountReadMetricsWalker.map(CountReadMetricsWalker.java:37)
    at org.broadinstitute.sting.gatk.traversals.TraverseReads.traverse(TraverseReads.java:103)
    at org.broadinstitute.sting.gatk.traversals.TraverseReads.traverse(TraverseReads.java:51)
    at org.broadinstitute.sting.gatk.executive.LinearMicroScheduler.execute(LinearMicroScheduler.java:69)
    at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:225)
    at org.broadinstitute.sting.gatk.CommandLineExecutable.execute(CommandLineExecutable.java:104)
    at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:227)
    at org.broadinstitute.cga.rnaseq.gatk.GATKTools.runIntronReadCount(GATKTools.java:225)
    at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runRegionCounting(ReadCountMetrics.java:236)
    at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runReadCountMetrics(ReadCountMetrics.java:57)
    at org.broadinstitute.cga.rnaseq.RNASeqMetrics.runMetrics(RNASeqMetrics.java:207)
    at org.broadinstitute.cga.rnaseq.RNASeqMetrics.execute(RNASeqMetrics.java:158)
    at org.broadinstitute.cga.rnaseq.RNASeqMetrics.main(RNASeqMetrics.java:127)


    It proposes that the reads are :
    Inappropriate call if not paired read

    however i call with the '-singelEnd' flag such that it should be fine. Any guesses how to fix it?


    Best,

    Phil
    Tags: None
    • simonandrews
      Simon Andrews
      • May 2009
      • 870
      #2
      Is this just a typo? It should be -singleEnd not -singelEnd.

        Comment

        • sphil
          Senior Member
          • Apr 2010
          • 192
          #3
          sry, actually it is a typo....
          Problem still exists, though

            Comment

            • aparna
              Member
              • Feb 2009
              • 17
              #4
              Does anybody know how to fix this? Apparently I emailed the developers - no response.

                Comment

                • kopi-o
                  Senior Member
                  • Feb 2008
                  • 319
                  #5
                  No, but I would recommend that you use RSeQC (formerly EVER-seq) instead.

                    Comment

                    • sschavan
                      Junior Member
                      • Oct 2012
                      • 7
                      #6
                      Hi all,

                      I was running RNA-SeQC software in Windows with command line options. I tried with the example sets provided on https://confluence.broadinstitute.or...Tools/RNA-SeQC and following the instructions as stated.

                      The command was:
                      "java -jar RNA-SeQC_v1.1.7.jar -n 1000 -s "TestId|ThousandReads.bam|TestDesc" -t gencode.v7.annotation_goodContig.gtf -r Homo_sapiens_assembly19.fasta -o ./testReport/ -strat gc -gc gencode.v7.gc.txt"

                      However I ran into the below error, and I am trying to see if anyone else faced the same error or knows the fix:

                      C:\example_files_RNASeQC>java -jar RNA-SeQC_v1.1.7.jar -n 1000 -s "TestId|ThousandReads.bam|TestDesc" -t gencode.v
                      7.annotation_goodContig.gtf -r Homo_sapiens_assembly19.fasta -o C:\example_files_RNASeQC\testReport -strat gc -gc
                      gencode.v7.gc.txt
                      RNA-SeQC v1.1.7 05/14/12
                      Creating rRNA Interval List based on given GTF annotations
                      Retriving contig names from reference
                      contig names in reference: 85
                      Loading GTF for Read Counting
                      Converting to refGene
                      Transcript objects to RefGen format: 1 s
                      Running IntronicExpressionReadBlock Walker ....
                      Arguments: [-T, IntronicExpressionReadBlock, --outfile_metrics, C:\example_files_RNASeQC\testReport/TestId/TestId.
                      metrics.tmp.txt, -R, Homo_sapiens_assembly19.fasta, -I, ThousandReads.bam, -refseq, C:\example_files_RNASeQC\testR
                      eport/refGene.txt, -l, ERROR]
                      org.broadinstitute.sting.utils.exceptions.UserException: Unable to read index file, for input source: C:\example_f
                      iles_RNASeQC\testReport\refGene.txt.idx
                      at org.broadinstitute.sting.gatk.refdata.tracks.RMDTrackBuilder.getFeatureSource(RMDTrackBuilder.java:224)
                      at org.broadinstitute.sting.gatk.refdata.tracks.RMDTrackBuilder.createInstanceOfTrack(RMDTrackBuilder.java:128)
                      at org.broadinstitute.sting.gatk.refdata.tracks.RMDTrackBuilder.createInstanceOfTrack(RMDTrackBuilder.java:145)
                      at org.broadinstitute.cga.rnaseq.gatk.CountReadMetricsWalker.initialize(CountReadMetricsWalker.java:521)
                      at org.broadinstitute.cga.rnaseq.gatk.IntronicExpressionReadBlockWalker.initialize(IntronicExpressionReadBlockWalker.java:40)
                      at org.broadinstitute.sting.gatk.executive.LinearMicroScheduler.execute(LinearMicroScheduler.java:48)
                      at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:248)
                      at org.broadinstitute.sting.gatk.CommandLineExecutable.execute(CommandLineExecutable.java:113)
                      at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:236)
                      at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:146)
                      at org.broadinstitute.cga.rnaseq.gatk.GATKTools.runIntronReadCount(GATKTools.java:226)
                      at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runRegionCounting(ReadCountMetrics.java:243)
                      at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runReadCountMetrics(ReadCountMetrics.java:58)
                      at org.broadinstitute.cga.rnaseq.RNASeqMetrics.runMetrics(RNASeqMetrics.java:220)
                      at org.broadinstitute.cga.rnaseq.RNASeqMetrics.execute(RNASeqMetrics.java:166)
                      at org.broadinstitute.cga.rnaseq.RNASeqMetrics.main(RNASeqMetrics.java:135)
                      RNA-SeQC Total Runtime: 0 min

                      Any comments are highly appreciated.

                      Thanks
                      Shweta

                        Comment

                        • kove
                          Junior Member
                          • Jul 2012
                          • 1
                          #7
                          I have the same question as you are!

                            Comment

                            • uqfgaiti
                              Member
                              • Nov 2012
                              • 13
                              #8
                              Hi all,

                              as kove and sschavan above, I was running RNA-SeQC software in command line. I tried withto follow the example sets provided on https://confluence.broadinstitute.or...Tools/RNA-SeQC and following the instructions as stated.

                              The command was:
                              "java -jar /hox/u/uqfgaiti/Tools/picard-tools-1.84/RNA-SeQC_v1.1.7.jar -r ampQue1.fasta -s "TestID|CEL-Seq.final.sorted.bam|TestDesc" -t 2507_Intergenic+Intronic.gtf -n 1000 -BWArRNA rRNA.fasta -o RNA-SeQC_out"

                              However I ran into the below error, and I am trying to see if anyone else faced the same error or knows the fix:

                              RNA-SeQC v1.1.7 05/14/12
                              Retriving contig names from reference
                              contig names in reference: 13397
                              Loading GTF for Read Counting
                              Converting to refGene
                              Transcript objects to RefGen format: 2 s
                              Running IntronicExpressionReadBlock Walker ....
                              Arguments: [-T, IntronicExpressionReadBlock, --outfile_metrics, RNA-SeQC_out/TestID/TestID.metrics.tmp.txt, -R, ampQue1.fasta, -I, CEL-Seq.final.sorted.bam, -refseq, RNA-SeQC_out/refGene.txt, -l, ERROR]
                              net.sf.samtools.util.RuntimeEOFException: Premature EOF; BinaryCodec in readmode; streamed file (filename not available)
                              at net.sf.samtools.util.BinaryCodec.readBytes(BinaryCodec.java:373)
                              at net.sf.samtools.util.BinaryCodec.readByteBuffer(BinaryCodec.java:480)
                              at net.sf.samtools.util.BinaryCodec.readInt(BinaryCodec.java:491)
                              at net.sf.samtools.BAMFileReader.readSequenceRecord(BAMFileReader.java:429)
                              at net.sf.samtools.BAMFileReader.readHeader(BAMFileReader.java:403)
                              at net.sf.samtools.BAMFileReader.<init>(BAMFileReader.java:144)
                              at net.sf.samtools.BAMFileReader.<init>(BAMFileReader.java:114)
                              at net.sf.samtools.SAMFileReader.init(SAMFileReader.java:514)
                              at net.sf.samtools.SAMFileReader.<init>(SAMFileReader.java:167)
                              at org.broadinstitute.sting.gatk.datasources.reads.SAMDataSource$ReaderInitializer.call(SAMDataSource.java:927)
                              at org.broadinstitute.sting.gatk.datasources.reads.SAMDataSource$SAMReaders.<init>(SAMDataSource.java:788)
                              at org.broadinstitute.sting.gatk.datasources.reads.SAMDataSource$SAMResourcePool.createNewResource(SAMDataSource.java:747)
                              at org.broadinstitute.sting.gatk.datasources.reads.SAMDataSource$SAMResourcePool.getAvailableReaders(SAMDataSource.java:718)
                              at org.broadinstitute.sting.gatk.datasources.reads.SAMDataSource.<init>(SAMDataSource.java:261)
                              at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.createReadsDataSource(GenomeAnalysisEngine.java:755)
                              at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.initializeDataSources(GenomeAnalysisEngine.java:666)
                              at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:227)
                              at org.broadinstitute.sting.gatk.CommandLineExecutable.execute(CommandLineExecutable.java:113)
                              at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:236)
                              at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:146)
                              at org.broadinstitute.cga.rnaseq.gatk.GATKTools.runIntronReadCount(GATKTools.java:226)
                              at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runRegionCounting(ReadCountMetrics.java:243)
                              at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runReadCountMetrics(ReadCountMetrics.java:58)
                              at org.broadinstitute.cga.rnaseq.RNASeqMetrics.runMetrics(RNASeqMetrics.java:220)
                              at org.broadinstitute.cga.rnaseq.RNASeqMetrics.execute(RNASeqMetrics.java:166)
                              at org.broadinstitute.cga.rnaseq.RNASeqMetrics.main(RNASeqMetrics.java:135)
                              RNA-SeQC Total Runtime: 0 min

                              Any comments are highly appreciated.

                              Thanks
                              Federico

                                Comment

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