Why Cufflinks is not Running?

Question

[k.kathirvel93]

Hi Everyone,

I am working in RNA-seq data analysis, I tried Cufflinks for Transcript Quantification from STAR mapped BAM file, but its not showing any progress, can anyone find me solution for this ? am using version 2.2.1 .
Thanks in advance.

Here is the Command line :

 cufflinks -p 16 --library-type fr-unstranded -o STAR_Output/ 
 -G /home/bioinformatics/Documents/Kathir/Glaucoma/RNA/Reference/ensemble_GRCh38.77/Homo_sapiens.GRCh38.77.gtf 
-u /home/bioinformatics/Documents/Kathir/Glaucoma/RNA/STAR/STAR_Output--RNAAligned.sortedByCoord.out.bam

[GenoMax]

but its not showing any progress

How did you reach that conclusion? It is not possible to give you any guidance with just that. Are you able to see the process run (using top)? Is there an output file being produced. Is there a space missing in this last bit of the command line somewhere /home/bioinformatics/Documents/Kathir/Glaucoma/RNA/STAR/STAR_Output--RNAAligned.sortedByCoord.out.bam or perhaps cufflinks is confused by -- you have in that name (if it is legit).

[k.kathirvel93]

I have been waiting for more than 3 hours, No outputs created in the folder -o STAR_Output/ . and top command Doesn't showing cufflinks is running. I have been used cufflinks three months back successfully.

[GenoMax]

Are you running this directly on the command line (without a job scheduler)? Does ps -ef | grep cuff show a process that looks like cufflinks?

[k.kathirvel93]

Are you running this directly on the command line (without a job scheduler)?

Yes.

and
ps -ef | greb cuff showing this.

bioinfo+ 11218  6224  0 12:18 pts/1    00:00:00 cufflinks -p 16 --library-type fr-unstranded -o STAR_Output/ -G /home/bioinformatics/Documents/Kathir/Glaucoma/RNA/Reference/ensemble_GRCh38.77/Homo_sapiens.GRCh38.77.gtf -u /home/bioinformatics/Documents/Kathir/Glaucoma/RNA/STAR/STAR_Output--RNAAligned.sortedByCoord.out.bam

[GenoMax]

I have a feeling that the process may be a zombie, if nothing is being written to output dir. You should kill it and start over. I am still not sure about the strange -- inclusion in the file name. I would suggest replacing that with an _.

[WouterDeCoster]

You should know that the old 'Tuxedo' pipeline of Tophat(2) and Cufflinks is no longer the "advisable" tool for RNA-seq analysis. The software is deprecated/ in low maintenance and should be replaced by HISAT2, StringTie and ballgown. See this paper: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. There are also other alternatives, including alignment with STAR and bbmap, or pseudo-alignment using salmon followed by DESEq2 or edgeR.

[k.kathirvel93]

Hi WouterDeCoster, Are you suggesting me to use StringtTie instead of Cufflinks for Quantification?.

[WouterDeCoster]

Yes, see also here: How does Cufflinks compare to StringTie?