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-   -   Problems with cuffmerge (http://seqanswers.com/forums/showthread.php?t=15092)

Nnogueira 10-27-2011 08:35 AM

Problems with cuffmerge
 
0



Hi everyone,

I'm trying to use cuffmerge but I get the following error message:

[Thu Oct 27 13:47:16 2011] Converting GTF files to SAM [FAILED] Error: gtf_to_sam not found on this system. Did you forget to include it in your PATH?

My command line is:

/storage/app/cufflinks-1.0.3/cufflinks-1.0.3.Linux_x86_64/cuffmerge -g /storage/data/natasha/gtf/miRNA_related_tracks_known_miRNAs_hg18.gtf -s /storage/data/natasha/chrs_fa/hg18_no_random_no_hap.fa assembles_cancer.txt

I've tested the gtf_to_sam program and it works fine.

Do I have do put something else in my command line? I've read the manual, but I couldn't find anything...

thanks in advance!

westerman 10-27-2011 10:03 AM

How did you test the gtf_to_sam program? Please give the command line you used to do the test.

ercfrtz 11-01-2011 09:57 AM

I have fixed the issue.

Nnogueira 11-01-2011 10:40 AM

Hi,

thanks for the replys! I've already fixed it. It was a problem with my bash file, nothing to do with cufflinks... sorry!

To answer the other reply, my command line for testing the gtf_to_sam program was

cufflinks-1.0.3.Linux_x86_64/gtf_to_sam transcripts.gtf SRR04006_gtf2sam.sam

and it worked fine.

thanks again for the replys!

westerman 11-01-2011 10:44 AM

Glad you got it fixed. Just to help out people who may read this topic. I suspect that gtf_to_sam was not in your PATH. Thus the fix to your bash file as well as using a given path (cufflinks-1.0.3.Linux_x86_64) to start up the program.

cewells 01-02-2012 08:58 AM

clarification
 
I am having the same issue, i.e.

"Error:gtf_to_sam not found on this system. Did you forget to include it in your PATH?"

I have included the path to gtf_to_sam in my .bashrc file PATH variable, but still I get this error.

Any thoughts? I appreciate your help --

Christina

jbrwn 01-02-2012 09:54 AM

Quote:

Originally Posted by cewells (Post 60695)
I am having the same issue, i.e.

"Error:gtf_to_sam not found on this system. Did you forget to include it in your PATH?"

I have included the path to gtf_to_sam in my .bashrc file PATH variable, but still I get this error.

Any thoughts? I appreciate your help --

Christina

what's the location of gtf_to_sam? what's your $PATH? did you source your bashrc or log out and back in?

cewells 01-02-2012 10:12 AM

With help from Cole Trapnell, I pinpointed the problem.

In case anyone runs into the same issue, gtf_to_sam should be specified in your PATH as:

PATH=$PATH:/home/cufflinks/ <or wherever you've stored gtf_to_sam>

Then I used

. ~/.bashrc

to reload the .bashrc file without logging out.

-Christina

wangli 05-30-2012 07:10 AM

Dear cewells

I defined the path in the command line.
But when I utilized .~/.bashrc, it showed that it cannot find the file or directory.
I used ls -a to see the hidden file, but no .bashrc.
Do you have an idea of this problem?
Thanks!

westerman 05-30-2012 07:31 AM

Quote:

Originally Posted by wangli (Post 74743)
Dear cewells

I defined the path in the command line.
But when I utilized .~/.bashrc, it showed that it cannot find the file or directory.
I used ls -a to see the hidden file, but no .bashrc.
Do you have an idea of this problem?
Thanks!

Did you edit your .bashrc or just put the path on the command line?

The basic idea is to
1) Edit your .bashrc or .bash_profile to have the new PATH in it.

2) Either logout/login or source a 'source' or a '.' of the .bashrc or .bash_profile in order to read the file in and make the PATH change.


I should point out that doing the above is basic UNIX 101. You will not get far unless you understand how your shell gets set up in the first place.

wanfahmi 12-03-2012 06:27 AM

Hi,

I got an error when I am at the stage of merge using cuffmerge. Below is my error

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

[Mon Dec 3 15:23:23 2012] Beginning transcriptome assembly merge
-------------------------------------------

[Mon Dec 3 15:23:23 2012] Preparing output location ./merged_asm/
Traceback (most recent call last):
File "/usr/local/cufflinks/cuffmerge", line 576, in <module>
sys.exit(main())
File "/usr/local/cufflinks/cuffmerge", line 538, in main
gtf_input_files = test_input_files(transfrag_list_file)
File "/usr/local/cufflinks/cuffmerge", line 268, in test_input_files
g = open(line,"r")
IOError: [Errno 2] No such file or directory: '1\tCufflinks\ttranscript\t89917\t91031\t1000\t-\t.\tgene_id "CUFF.1"; transcript_id "CUFF.1.1"; FPKM "0.6709083067"; frac "1.000000"; conf_lo "0.316487"; conf_hi "1.025329"; cov "2.502314";'


Please somebody help. Thank you!

westerman 12-03-2012 06:32 AM

Your 'assemblies.txt' file does not contain a list of files but rather contains data. At least that is my first guess.

wanfahmi 12-03-2012 06:50 AM

Hi Westerman,

Thanks for replying. do you mean the 'assemblies.txt' should have a list? Actually you are correct I guess as I did cat the all transcript.gtf. How should I do? Thanks

westerman 12-03-2012 07:01 AM

I do not do this too often and I am too busy at the moment to double-check but what you should have is a list of the individual files. E.g.,

cat >assemblies.txt
file1.gtf
file2.gtf
etc.

Not the contents of the files. The file names.

wanfahmi 12-03-2012 07:05 AM

Problems solved! thank you.

I did create a file that only contains the names not the contains of file. Its running.

Gonza 09-13-2014 11:09 AM

Hi all, I have a cuffmerge problem. It says "EOF marker is absent. The input is probably truncated". I have run tophat and cufflinks successfully. What does this mean??
Thanks.
G

[gonzalo@localhost trimmed_reads]$ cuffmerge -g genes.gtf -s genome.fa -p 20 assemblies.txt

[Sat Sep 13 12:33:25 2014] Beginning transcriptome assembly merge
-------------------------------------------

[Sat Sep 13 12:33:25 2014] Preparing output location ./merged_asm/
[Sat Sep 13 12:33:35 2014] Converting GTF files to SAM
[12:33:35] Loading reference annotation.
[12:33:37] Loading reference annotation.
[12:33:40] Loading reference annotation.
[12:33:43] Loading reference annotation.
[12:33:45] Loading reference annotation.
[12:33:48] Loading reference annotation.
[Sat Sep 13 12:33:51 2014] Quantitating transcripts
You are using Cufflinks v2.2.1, which is the most recent release.
Command line:
cufflinks -o ./merged_asm/ -F 0.05 -g genes.gtf -q --overhang-tolerance 200 --library-type=transfrags -A 0.0 --min-frags-per-transfrag 0 --no-5-extend -p 20 ./merged_asm/tmp/mergeSam_fileJRPl29
[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 ./merged_asm/tmp/mergeSam_fileJRPl29 doesn't appear to be a valid BAM file, trying SAM...
[12:33:51] Loading reference annotation.
[12:33:57] Inspecting reads and determining fragment length distribution.
Processed 28060 loci.
Map Properties:
Normalized Map Mass: 154834.00
Raw Map Mass: 154834.00
Fragment Length Distribution: Truncated Gaussian (default)
Default Mean: 200
Default Std Dev: 80
[12:33:59] Assembling transcripts and estimating abundances.
Processed 28060 loci.
[Sat Sep 13 12:34:11 2014] Comparing against reference file genes.gtf
You are using Cufflinks v2.2.1, which is the most recent release.
[Sat Sep 13 12:34:34 2014] Comparing against reference file genes.gtf
You are using Cufflinks v2.2.1, which is the most recent release.

Brian Bushnell 09-13-2014 11:38 AM

That message is a bug in Samtools; don't worry about it.

Gonza 09-13-2014 05:40 PM

Thanks. I ignored it and successfully run tuxedo. However, when loading cuffdiff to R i keep getting this error. I have no idea what this is. Any thiughts on solutions? thanks...

> cuff_data <- readCufflinks()
Creating database /Users/gonzalovillarino/Desktop/05_cuffdiff/cuffData.db
Reading Run Info File /Users/gonzalovillarino/Desktop/05_cuffdiff/run.info
Writing runInfo Table
Reading Read Group Info /Users/gonzalovillarino/Desktop/05_cuffdiff/read_groups.info
Writing replicates Table
Reading /Users/gonzalovillarino/Desktop/05_cuffdiff/genes.fpkm_tracking
Error in scan(file, what, nmax, sep, dec, quote, skip, nlines, na.strings, :
line 1305 did not have 17 elements

> cuff_data
Error: object 'cuff_data' not found
CuffSet instance with:
0 samples
0 genes
0 isoforms
0 TSS
0 CDS
0 promoters
0 splicing
0 relCDS

Nanu 10-14-2014 03:43 AM

When I am running the cuffmerge then i got the following error. I mentioned the .gtf file name in it which was created by cufflinks

[Tue Oct 14 17:08:29 2014] Beginning transcriptome assembly merge
-------------------------------------------

[Tue Oct 14 17:08:29 2014] Preparing output location ./merged_asm/
Warning: no reference GTF provided!
[Tue Oct 14 17:08:31 2014] Converting GTF files to SAM
[FAILED]
Error: gtf_to_sam not found on this system. Did you forget to include it in your PATH?

Rashedul Islam 12-09-2014 01:50 PM

I have solved the problem by adding this line to the .hashrc file.

"export PATH=$PATH:/home/cufflinks-2.2.1.Linux_x86_64"

aniruddha.otago 06-25-2016 04:21 PM

Hi there,

I am running cuffmerge and having this problem:

My command:
cuffmerge -g /home/aniruddha/GTF_files/Human_genome/GRCh37_build_65/Homo_sapiens.GRCh37.65.gtf -p 10 /home/aniruddha/PDL1_RNA-Seq_8_AC/Cuffmerge_8PDcelllines/assembled_tc_list.txt

[Sun Jun 26 12:02:08 2016] Beginning transcriptome assembly merge
-------------------------------------------

[Sun Jun 26 12:02:08 2016] Preparing output location ./merged_asm/
[Sun Jun 26 12:02:23 2016] Converting GTF files to SAM
[FAILED]
Error: gtf_to_sam not found on this system. Did you forget to include it in your PATH?

When I see the cufflinks executables I dont see gtf_to_sam .
am I missing something ?


cufflinks-2.2.1.Linux_x86_64]$ ls
AUTHORS README cuffdiff cuffmerge cuffquant gffread isoforms.fpkm_tracking test_data.sam
LICENSE cuffcompare cufflinks cuffnorm genes.fpkm_tracking gtf_to_sam skipped.gtf transcripts.gtf


Thanks for your help.

Brian Bushnell 06-25-2016 04:46 PM

I recommend you avoid the Tuxedo tools. You can get much better results with something else. I won't give a specific alternative because I don't know the goal of your research, but I cannot imagine a question where the Tuxedo suite is the best answer. Or even a good answer.

aniruddha.otago 06-25-2016 09:18 PM

I think I have solved the problem now. For some reason gtf_to_sam tool was missing.
Anyways, for this particular analysis at this stage all I am wanting is to identify differential expression and differentially expressed isoforms. There is hardly any consensus of the best tools to be used. Taxedo suite is off course the kost used one. Anyone have any useful data driven opinion or comments on the use of tools for indetifying DE. ? Also if you have any comments on tools or approaches you think its good for comparing DE of genes with DNA methylation profiles of the same samples. ?
Thanks in advance.

Brian Bushnell 06-25-2016 09:38 PM

DESeq and edgeR are commonly used for differential expression analysis, and both are superior to Tuxedo tools, from what I understand. Cufflinks is incapable of determining that genes specific to the Y chromosome are differentially expressed between human males and females. If that's not sufficient evidence of it being a bad tool, I can't imagine what is. I don't currently have a link to that study, but I can probably get it in a few days when my co-worker who initially found it is available.


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