Problem with CollectRnaSeqMetrics

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Nahdia Afiifah Abdul Jalil

• April 03, 2024 11:24
• Edited

Hi,

Currently i am running CollectRnaSeqMetrics tool.

 

First, I created the rRNA interval list using this command

 

picard CreateSequenceDictionary R=Homo_sapiens.GRCh38.dna.primary_assembly.fa O=Homo_sapiens.GRCh38.dna.primary_assembly.fa.dict

grep 'rRNA' Homo_sapiens.GRCh38.111.gtf  > Homo_sapiens.GRCh38.111.rRNA.gtf 

gtfToGenePred -genePredExt -geneNameAsName2 Homo_sapiens.GRCh38.111.rRNA.gtf Homo_sapiens.GRCh38.111.rRNA.gtf.genePred

awk '{print $2, $4, $5, $3, $12}' Homo_sapiens.GRCh38.111.rRNA.gtf.genePred > Homo_sapiens.GRCh38.111.rRNA.gtf.genePred.shortlist

cat homo_sapiens.GRCh38.dna.primary_assembly.fa.dict Homo_sapiens.GRCh38.111.rRNA.gtf.genePred.shortlist > Homo_sapiens.GRCh38.111.rRNA.intervals.list

 

Then, I ran CollectRnaSeqMetrics using this command

 

java -jar $PICARD_JAR/picard.jar CollectRnaSeqMetrics \

      I=/mnt/iusers01/jw01/c02544na/training/dummy/AB1_gencode44_trimmed_TruSeq3_readcountAligned.sortedByCoord.out.bam \

      O=AB1_output.RNA_Metrics \

      REF_FLAT=/mnt/iusers01/jw01/c02544na/refFlat.txt \

      STRAND=SECOND_READ_TRANSCRIPTION_STRAND \

      RIBOSOMAL_INTERVALS=/mnt/iusers01/jw01/c02544na/training/dummy/psa_transcriptomic_training/Homo_sapiens.GRCh38.111.rRNA.intervals.list

 

Then, I received error message from picard

 

Exception in thread "main" htsjdk.tribble.TribbleException: Invalid interval record contains 1 fields: 1 9437668 9437778 - RNA5SP40

at htsjdk.tribble.IntervalList.IntervalListCodec.parseIntervalString(IntervalListCodec.java:73)

at htsjdk.tribble.IntervalList.IntervalListCodec.decode(IntervalListCodec.java:130)

at htsjdk.samtools.util.IntervalList.fromReader(IntervalList.java:529)

at htsjdk.samtools.util.IntervalList.fromPath(IntervalList.java:458)

at htsjdk.samtools.util.IntervalList.fromFile(IntervalList.java:447)

at picard.analysis.directed.RnaSeqMetricsCollector.makeOverlapDetector(RnaSeqMetricsCollector.java:79)

at picard.analysis.CollectRnaSeqMetrics.setup(CollectRnaSeqMetrics.java:168)

at picard.analysis.SinglePassSamProgram.makeItSo(SinglePassSamProgram.java:145)

at picard.analysis.SinglePassSamProgram.doWork(SinglePassSamProgram.java:94)

at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:280)

at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:105)

at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:115)

 

What should I do to fix this?

Many thanks

 

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• 

  Gökalp Çelik

  • April 05, 2024 13:50

  Hi Nahdia Afiifah Abdul Jalil

  Your interval files seem to be missing required fields. Can you check whether your fields are separated by tabs?. If not you need to add {OFS="\t"} to your awk command to make it so. 

  I hope this helps. 

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