Question: Running cufflinks - gtf file not recognised
0
gravatar for snp87
3.2 years ago by
snp8740
snp8740 wrote:

Hi, I am trying to run cufflinks on my data with following command,

cufflinks -p 4 -N –g Mus_musculus.GRCm38.84.gtf -b –u Mus_musculus.GRCm38.dna.toplevel.fa accepted_hits_1.bam

but keep getting this error open: No such file or directory File –g doesn't appear to be a valid BAM file, trying SAM... Error: cannot open alignment file –g for reading

I copied the gtf file to the tophat_output folder where I am running the command and also tried referencing to the original file but neither seems to work. Anyone have any suggestions?

Thank you!

UPDATE: I changed the hyphens but it still didn't work - now the error is as follows:

cufflinks -p 4 -N -g Mus_musculus.GRCm38.84.gtf -b -u Mus_musculus.GRCm38.dna.toplevel.fa accepted_hits_1.bam 

You are using Cufflinks v2.2.1, which is the most recent release.
[bam_header_read] EOF marker is absent. The input is probably truncated.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File Mus_musculus.GRCm38.dna.toplevel.fa doesn't appear to be a valid BAM file, trying SAM...
[13:59:10] Loading reference annotation.
Error: file/directory -u does not exist!

I was using -b -u for bias correction. When I take the -b and -u commands off it seemed to run okay but would this cause problems downstream?

[bam_header_read] EOF marker is absent. The input is probably truncated.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File Mus_musculus.GRCm38.dna.toplevel.fa doesn't appear to be a valid BAM file, trying SAM...
[14:05:15] Loading reference annotation.
[14:06:04] Inspecting reads and determining fragment length distribution.
> Processed 33953 loci.                        [*************************] 100%
> Map Properties:
>   Normalized Map Mass: 0.00
>   Raw Map Mass: 0.00
>   Fragment Length Distribution: Truncated Gaussian (default)
>                 Default Mean: 200
>              Default Std Dev: 80
[14:07:30] Assembling transcripts and estimating abundances.

8:119910359-124345724   Warning: Skipping large bundle.
> Processed 33952 loci.                        [*************************] 100%
rna-seq cufflinks • 1.7k views
ADD COMMENTlink modified 3.2 years ago by genomax71k • written 3.2 years ago by snp8740

The hyphens you use for -g and -u doesn't seem to be the same you are using for other option ... did you cut and past them somehow from a different place ? I suggest to just rewrite these hyphen correctly...

ADD REPLYlink modified 3.2 years ago • written 3.2 years ago by Juke-342.6k

Thanks for noticing that (didn't realise word was automatically changing that). Is still didn't work though, now the error is as follows:

cufflinks -p 4 -N -g Mus_musculus.GRCm38.84.gtf -b -u Mus_musculus.GRCm38.dna.toplevel.fa accepted_hits_1.bam 

You are using Cufflinks v2.2.1, which is the most recent release.
[bam_header_read] EOF marker is absent. The input is probably truncated.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File Mus_musculus.GRCm38.dna.toplevel.fa doesn't appear to be a valid BAM file, trying SAM...
[13:59:10] Loading reference annotation.
Error: file/directory -u does not exist!

I was using -b -u for bias correction. When I take the -b and -u commands off it runs fine but would this cause problems downstream?

ADD REPLYlink modified 3.2 years ago by genomax71k • written 3.2 years ago by snp8740

Use this,

cufflinks -p 4 -N -u -g Mus_musculus.GRCm38.84.gtf -b Mus_musculus.GRCm38.dna.toplevel.fa accepted_hits_1.bam

ADD REPLYlink modified 3.2 years ago • written 3.2 years ago by EagleEye6.5k

Thanks for the suggestion. I'm getting a segmentation fault 11 error when using that command.

[14:59:11] Loading reference annotation. No fasta index found for Mus_musculus.GRCm38.dna.toplevel.fa. Rebuilding, please wait.. Fasta index rebuilt. [15:04:36] Inspecting reads and determining fragment length distribution. Segmentation fault: 11

ADD REPLYlink modified 3.2 years ago • written 3.2 years ago by snp8740

This might be problem associated with GTF file,

http://seqanswers.com/forums/showthread.php?t=27951

ADD REPLYlink modified 3.2 years ago • written 3.2 years ago by EagleEye6.5k

can u post the screenshot of the command and error?

ADD REPLYlink written 3.2 years ago by bioinfo_ga20

use single hyphen '-g' instead of double hyphen '--g' as Juke-34 pointed out. Same goes for '-u' option too. Clearly there is some difference between hyphens used for N,p,b and g,u options. Delete those hyphens and try to manually enter the hyphens on terminal.

ADD REPLYlink modified 3.2 years ago • written 3.2 years ago by EagleEye6.5k

If you are copying and pasting command lines from a PC/Mac (in Word etc) into unix terminal they you need to be careful of automatic conversion of hyphens to dashes etc. They look like they are hyphens to eye but will generate odd errors. You can turn off the "smart quotes and dashes" in keyboard preference panel to prevent this from happening.

ADD REPLYlink modified 3.2 years ago • written 3.2 years ago by genomax71k
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