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Issues running RNAseqc on my bam file

Hello , I have been using rseqc for a while now. I wanted to try out RNAseqc and I am running into multiple errors. I have a genome bam sorted by coordinate from STAR. I also have a indexed ref genome fasta file and also its dictionary ready. Later I added the read group information using picard AddOrReplaceReadGroups. I ran the following command.

java -jar /usr/local/apps/rnaseqc/1.1.8/RNA-SeQC_v1.1.8.jar  -r ucsc.hg19.fasta -o xyz -s "xyz|xyz.bam|xyz" -t hg19.gtf

I got the following error.
[-] Unloading BWA 0.7.15 ...
[-] Unloading samtools 1.5 ...
[-] Unloading java 1.7.0_25 ...
[+] Loading BWA 0.7.15 ...
[+] Loading samtools 1.5 ...
[+] Loading java 1.7.0_25 ...
RNA-SeQC v1.1.8.1 07/11/14
Creating rRNA Interval List based on given GTF annotations
Retriving contig names from reference
     contig names in reference: 93
Loading GTF for Read Counting
Converting to refGene
Transcript objects to RefGen format:    1 s
java.lang.RuntimeException: No rRNA found in GTF transcript_type field
    at org.broadinstitute.cga.rnaseq.TranscriptList.toRRNAIntervalList(TranscriptList.java:414)
    at org.broadinstitute.cga.rnaseq.RNASeqMetrics.createRefGeneAndRRNAFiles(RNASeqMetrics.java:1306)
    at org.broadinstitute.cga.rnaseq.RNASeqMetrics.prepareFiles(RNASeqMetrics.java:196)
    at org.broadinstitute.cga.rnaseq.RNASeqMetrics.execute(RNASeqMetrics.java:170)
    at org.broadinstitute.cga.rnaseq.RNASeqMetrics.main(RNASeqMetrics.java:139)
No information for rRNA available. Continuing without rRNA calculations. (Using the -BWArRNA flag for best results)
Running IntronicExpressionReadBlock Walker ....
Arguments: [-T, IntronicExpressionReadBlock, --outfile_metrics, xyz/xyz/xyz.metrics.tmp.txt, -R, ucsc.hg19.fasta, -I, xyz.bam, -refseq, xyz/refGene.txt, -l, ERROR]
/var/spool/slurm/slurmd/job51966571/slurm_script: line 6: 18690 Killed                  java -jar /usr/local/apps/rnaseqc/1.1.8/RNA-SeQC_v1.1.8.jar -r ucsc.hg19.fasta -o xyz -s "xyz|xyz.bam|xyz" -t $1

I have seen a similar post on biostars but there was no solution. I would really appreciate any inputs to my problem.

Thanks.

rnaseqc rna qc bam picard
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updated by Pierre Lindenbaum Pierre Lindenbaum • written by vineelagangalapudi • vineelagangalapudi

Does hg19.gtf contain rRNA in the GTF transcript_type field?

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written by WouterDeCoster WouterDeCoster

no, that is why this is bypassing the rRNA calculation step and moving on to the next steps and then it is failing in the intronicexpressionreadblock walker step.

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written by vineelagangalapudi • vineelagangalapudi

Hello,

I am working on RNASeQC and came across the same error.

RNA-SeQC v1.1.9 06/26/16 Creating rRNA Interval List based on given GTF annotations Retriving contig names from reference contig names in reference: 639 Loading GTF for Read Counting Converting to refGene Transcript objects to RefGen format: 9 s java.lang.RuntimeException: No rRNA found in GTF transcript_type field at org.broadinstitute.cga.rnaseq.TranscriptList.toRRNAIntervalList(TranscriptList.java:318) at org.broadinstitute.cga.rnaseq.RNASeqMetrics.createRefGeneAndRRNAFiles(RNASeqMetrics.java:1167) at org.broadinstitute.cga.rnaseq.RNASeqMetrics.prepareFiles(RNASeqMetrics.java:184) at org.broadinstitute.cga.rnaseq.RNASeqMetrics.execute(RNASeqMetrics.java:161) at org.broadinstitute.cga.rnaseq.RNASeqMetrics.main(RNASeqMetrics.java:133) No information for rRNA available. Continuing without rRNA calculations. (Using the -BWArRNA flag for best results) Running IntronicExpressionReadBlock Walker .... Exception in thread "main" java.lang.ExceptionInInitializerError at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.<init>(GenomeAnalysisEngine.java:160) at org.broadinstitute.sting.gatk.CommandLineExecutable.<init>(CommandLineExecutable.java:53) at org.broadinstitute.sting.gatk.CommandLineGATK.<init>(CommandLineGATK.java:54) at org.broadinstitute.cga.rnaseq.gatk.GATKTools.runIntronReadCount(GATKTools.java:203) at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runRegionCounting(ReadCountMetrics.java:220) at org.broadinstitute.cga.rnaseq.ReadCountMetrics.runReadCountMetrics(ReadCountMetrics.java:59) at org.broadinstitute.cga.rnaseq.RNASeqMetrics.runMetrics(RNASeqMetrics.java:211) at org.broadinstitute.cga.rnaseq.RNASeqMetrics.execute(RNASeqMetrics.java:162) at org.broadinstitute.cga.rnaseq.RNASeqMetrics.main(RNASeqMetrics.java:133) Caused by: java.lang.NullPointerException at org.reflections.Reflections.scan(Reflections.java:220) at org.reflections.Reflections.scan(Reflections.java:166) at org.reflections.Reflections.<init>(Reflections.java:94) at org.broadinstitute.sting.utils.classloader.PluginManager.<clinit>(PluginManager.java:77) ... 9 more

It would be great if any inputs provided to solve the error.

Thanks.

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written by anoo • anoo

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