STAR + Cufflinks. Cufflinks hanging. BAM XS error?

[gogodidi]

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!

[dpryan]

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.

[ccros]

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.

[zhkzhou]

4.2.3 Compatibility with Cufflinks
For unstranded RNA-seq data, Cuinks/Cudi 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 undened strand (i.e. containing only non-canonical unannotated junctions)
will be suppressed.