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STAR + Cufflinks. Cufflinks hanging. BAM XS error?

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  • STAR + Cufflinks. Cufflinks hanging. BAM XS error?

    Hello All,

    I have an RNA-Seq pipeline, using the STAR aligner and Cufflinks.
    I am running cufflinks in the 'Reference Annotation Based Transcript' assemvbly mode to predict alternative splicing variants.
    I have been using Cufflinks on our compute servers for a while now, and I didn't have any problems until recently.
    For the past few weeks, Cufflinks always hangs after a few hours of running.
    I can't seem to identify the problem. It doesn't seem to be a network issue, as I have installed cufflinks locally and also have the data locally.

    I have tried this with cufflinks 2.2.1 and 2.1.1, and both hang. Using STAR version 2.4.0f1.

    The only error I get is associated with the STAR output (and I didn't have this problem before either)
    BAM record error: found spliced alignment without XS attribute
    I find it strange because I do specify the '--outSAMstrandField intronMotif' parameter for STAR.

    Why do I get this error even though I have specified the correct parameters for STAR?
    Could this be the reason Cufflinks is hanging?

    Thank you!

  • #2
    Perhaps STAR 2.4.0f1 has a bug. Try something like this with one of the BAM files that's causing a problem:
    samtools view foo.bam | awk -F '\t' '{if($6 ~"N") print $0}' | grep -v "XS:A:"
    If something like that prints any lines then the file will cause an error in cufflinks. This would then likely be a bug in STAR.


    • #3
      I have been using STAR and a de novo assembler called IsoSCM to identify 3'UTR. IsoSCM requested the XS tag to work, so I set the same --outSAMStrandField option and it went well.
      I note that using "--outFilterIntronMotifs RemoveNonCanonical" is also recommended with Cufflinks in the STAR manual.


      • #4
        4.2.3 Compatibility with Cufflinks
        For unstranded RNA-seq data, Cuinks/Cu di require spliced alignments with XS strand attribute,which STAR will generate with --outSAMstrandField intronMotif option. As required, the XS strand attribute will be generated for all alignments that contain splice junctions. The spliced alignments that have unde ned strand (i.e. containing only non-canonical unannotated junctions)
        will be suppressed.