Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • 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

  • #2
    Is this just a typo? It should be -singleEnd not -singelEnd.

    Comment


    • #3
      sry, actually it is a typo....
      Problem still exists, though

      Comment


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

        Comment


        • #5
          No, but I would recommend that you use RSeQC (formerly EVER-seq) instead.

          Comment


          • #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


            • #7
              I have the same question as you are!

              Comment


              • #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

                Latest Articles

                Collapse

                • seqadmin
                  Strategies for Sequencing Challenging Samples
                  by seqadmin


                  Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
                  03-22-2024, 06:39 AM
                • seqadmin
                  Techniques and Challenges in Conservation Genomics
                  by seqadmin



                  The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

                  Avian Conservation
                  Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
                  03-08-2024, 10:41 AM

                ad_right_rmr

                Collapse

                News

                Collapse

                Topics Statistics Last Post
                Started by seqadmin, Yesterday, 06:37 PM
                0 responses
                8 views
                0 likes
                Last Post seqadmin  
                Started by seqadmin, Yesterday, 06:07 PM
                0 responses
                8 views
                0 likes
                Last Post seqadmin  
                Started by seqadmin, 03-22-2024, 10:03 AM
                0 responses
                49 views
                0 likes
                Last Post seqadmin  
                Started by seqadmin, 03-21-2024, 07:32 AM
                0 responses
                66 views
                0 likes
                Last Post seqadmin  
                Working...
                X