cuffmerge help for novice please!

[DrJay]

Hi all,
be gentle with me - its my first post! I have two conditions with 3 replicate files.
Ive run tophat and cufflinks successfully on the RNAseq data.
However when it comes to merging the gtf transcripts, despite following the nature protocol closely, cuffmerge is not running properly.

Ive seen a few people post this question in different forums but I haven't seen anyone answer the query so I dont know what the solution is!
Essentially after running the cuffmerge command on a compiled assemblies script file list, it looks like the operation is starting but after about 10 seconds I get returned to the command prompt. No error messages appear but the merged asm folder is empty and the program has clearly aborted. I just dont know why .

Can anyone please help? Im running linux on an ubuntu parallels system with 12GB memory allocated. The bin folder contains symbolic links to the bowtie2 genome files and the gtf gene file. Thanks again if anyone can point me to things to check! Apologies in advance for any forum rules I have probably broken!

$ cuffmerge -g genes.gtf -s genome.fa -p 4 assemblies.txt

[Wed Oct 7 03:01:46 2015] Beginning transcriptome assembly merge
-------------------------------------------

[Wed Oct 7 03:01:46 2015] Preparing output location ./merged_asm/
[Wed Oct 7 03:02:23 2015] Converting GTF files to SAM
j12@ubuntu:~/bin$

[GenoMax]

What does your assemblies.txt file look like?

[DrJay]

Hi, file is as follows:

/home/jcf1r12/rnaseq/JFcontrol1_clout/transcripts.gtf
/home/jcf1r12/rnaseq/JFcontrol2_clout/transcripts.gtf
/home/jcf1r12/rnaseq/JFcontrol3_clout/transcripts.gtf
/home/jcf1r12/rnaseq/JFct1_clout/transcripts.gtf
/home/jcf1r12/rnaseq/JFct2_clout/transcripts.gtf
/home/jcf1r12/rnaseq/JFct3_clout/transcripts.gtf

[GenoMax]

From cuffmerge help:

Note that <seq_dir> must contain one fasta file per reference chromosome, and each file must be named after the chromosome, and have a .fa or .fasta extension.

[DrJay]

Thank you so much for suggestion.

I have tried rerunning but with passing -s to the /Users/JF/Homo_sapiens/rnaseq/Ensembl/GRCh37/Sequence/Chromosomes directory which contains all individual named chromosome fa files but the same abort happens?

[GenoMax]

Have you tried to remove/move the empty "merged_asm" folder from past runs and re-running?

[DrJay]

Have you tried to remove/move the empty "merged_asm" folder from past runs and re-running?

Yes just tried that, sadly no different

[GenoMax]

Are you using the latest cuff* programs? The transcript.gtf files in the cl_out directories are not empty, correct? Does the program run if you don't provide the reference GTF file? Since you are running in a VM also remove the -p option.

[DrJay]

Are you using the latest cuff* programs? The transcript.gtf files in the cl_out directories are not empty, correct? Does the program run if you don't provide the reference GTF file? Since you are running in a VM also remove the -p option.

Yes cufflinks is the latest version. The transcript.gtf files are all about 40MB. No the same thing happens without the gtf file (after it warns me that I havent included a reference file).

And similarly doesn't run when I remove the -p option either.

I wondered if it was something to do with samtools as when I check the version installed, it is an older version. Ive downloaded, unzipped and ran 'make' in the new version folder and then copied all the files to the bin directory but when I run dpkg it still states the old version of samtools.

Not sure if this is relevent but obviously Im struggling!

[GenoMax]

Did you compile cufflinks yourself or did you download pre-compiled binaries? Last I remember TopHat suite programs need the old version of Samtools (0.1.19).

[DrJay]

Did you compile cufflinks yourself or did you download pre-compiled binaries? Last I remember TopHat suite programs need the old version of Samtools (0.1.19).

The Nature protocol paper uses version 1.3 so this is what I have used. I downloaded the binary files and used following script:

tar zxvf /media/psf/Homo_sapiens/cufflinks-1.3.0.OSX_x86_64.tar.gz
cd cufflinks-1.3.0.OSX_x86_64/
cp * $HOME/bin
export PATH=$PATH:/home/JF/bin/cufflinks-1.3.0.OSX_x86_64

[GenoMax]

I wonder if that is part of the problem.

Can you download the latest cufflinks from here: http://cole-trapnell-lab.github.io/cufflinks/install/

What version of TopHat did you use?

[DrJay]

I used tophat on an insitutional hub for speed. It runs version 2.0.14

I can try that but im pretty sure I have tried with a few cufflinks versions now. Do I just run a similar script as I posted above to install the new version of cufflinks or is there any other script I need to use to ensure I am running the up to date version? (or should i run cuffmerge from the cufflinks_2.2.1 folder?)

[DrJay]

DOH!!!! Ok. . . who's spotted it.......

[GenoMax]

Make sure you run the cuffmerge from the cufflinks_2.2.1 folder (and the chromosome option). You may need to append your $PATH so that the 2.2.1 cuff* programs are found by the system.