SEQanswers

Go Back   SEQanswers > Applications Forums > RNA Sequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
Velvet tests using ILM DH10B data winsettz De novo discovery 9 03-24-2013 08:03 AM
burden tests fadista General 0 12-21-2012 05:36 AM
RNA-seq expression measurements and tests Rainbird Bioinformatics 3 12-10-2012 02:16 AM
a new user tests if posting is successful wellswu Bioinformatics 0 10-19-2012 06:14 AM
why cuffdiff cds-tests empty? joseph Bioinformatics 2 02-17-2011 10:33 AM

Reply
 
Thread Tools
Old 01-21-2013, 01:51 AM   #1
vaibhav_jain
Junior Member
 
Location: INDIA

Join Date: Jan 2011
Posts: 6
Default cuffdiff not performing splicing tests and promoter tests

Hi all,

I am using cufflinks package to look for differentially spliced exons. The pipeline I followed is tophat -> cufflinks -> cuffmerge/cuffcompare(tried with both) -> cuffdiff.
Result on command line is like this :-

Performed 23758 isoform-level transcription difference tests
Performed 20313 tss-level transcription difference tests
Performed 18306 gene-level transcription difference tests
Performed 17903 CDS-level transcription difference tests
Performed 0 splicing tests
Performed 0 promoter preference tests
Performing 0 relative CDS output tests
Writing isoform-level FPKM tracking
Writing TSS group-level FPKM tracking
Writing gene-level FPKM tracking
Writing CDS-level FPKM tracking
Writing isoform-level count tracking
Writing TSS group-level count tracking
Writing gene-level count tracking
Writing CDS-level count tracking
Writing isoform-level read group tracking
Writing TSS group-level read group tracking
Writing gene-level read group tracking
Writing CDS-level read group tracking
Writing read group info
Writing run info

Command I used to run cuffdiff is :-
./cuffdiff --no-update-check -o day0 -p 2 -N -b /home/Vaibhav/Alt_transcriptome_data/ws230/ws230.fa -u merged_asm/merged.gtf /home/Vaibhav/Alt_transcriptome_data/new/again_X_day0/accepted_hits.bam /home/Vaibhav/Alt_transcriptome_data/new/again_Y_day0/accepted_hits.bam

I have no replicates of the sample. Now I am getting some differential results for gene, cds and isoform but getting nothing for splicing and promoter something I am interested in. I also tried lowering c option till 2, but got the same result.Can someone please suggest me what should I do so that cuffdiff performs splicing tests.
Really in need of the differential spliced exons for my data.

Thanks a lot
vaibhav_jain is offline   Reply With Quote
Old 03-04-2013, 01:05 AM   #2
krespim
Member
 
Location: Dresden

Join Date: Jul 2012
Posts: 49
Default

hi vaibhav_jain,

did you solve the problem? I having similar issues and can't for the life of me figure out what is going on.
krespim is offline   Reply With Quote
Old 03-18-2013, 06:31 AM   #3
leifive
Member
 
Location: Nanjing, China

Join Date: Mar 2013
Posts: 10
Default

Hi, vaibhav_jain and krespim:
Have you solved the problem? This has puzzled me almost one week. Why cuffdiff performed 0 splicing test?
leifive is offline   Reply With Quote
Old 03-18-2013, 01:30 PM   #4
krespim
Member
 
Location: Dresden

Join Date: Jul 2012
Posts: 49
Default

Quote:
Originally Posted by leifive View Post
Hi, vaibhav_jain and krespim:
Have you solved the problem? This has puzzled me almost one week. Why cuffdiff performed 0 splicing test?

Not really a solution, but when I load the results in cuffdiff I do get DE isoforms. Do try that. Maybe it is just a bug in the reporting.
krespim is offline   Reply With Quote
Old 03-18-2013, 05:17 PM   #5
leifive
Member
 
Location: Nanjing, China

Join Date: Mar 2013
Posts: 10
Default

Quote:
Originally Posted by krespim View Post
Not really a solution, but when I load the results in cuffdiff I do get DE isoforms. Do try that. Maybe it is just a bug in the reporting.
Thanks for your reply, krespim. But I don't think this is just a bug in the reporting, because there is no "OK" status in my splicing.diff file from Cuffdiff, but NOTEST or LOWDATA instead. And I have tried both combined.gtf and merged.gtf as input of cuffdiff, results are almost the same.
leifive is offline   Reply With Quote
Old 03-20-2013, 07:28 AM   #6
krespim
Member
 
Location: Dresden

Join Date: Jul 2012
Posts: 49
Default

Quote:
Originally Posted by leifive View Post
Thanks for your reply, krespim. But I don't think this is just a bug in the reporting, because there is no "OK" status in my splicing.diff file from Cuffdiff, but NOTEST or LOWDATA instead. And I have tried both combined.gtf and merged.gtf as input of cuffdiff, results are almost the same.

Well, I can't help much further but I can tell you what I end up doing. As I am working with species that have relative well annotated genomes/transcriptomes (Homo sapiens and Mus musculus), I forego the initial cufflinks step and go straight to cufdiff after mapping using the gtf provided in iGenomes as a reference. Here are my settings:

# cuffdiff v2.0.2

$cuffdiff -L "GFP,KD" -o $cuff_output -p 8 -u $gtfFile $gfp_a,$gfp_b $srsf1_a,$srsf1_b

And that seems to work.
krespim is offline   Reply With Quote
Old 03-27-2013, 05:44 PM   #7
leifive
Member
 
Location: Nanjing, China

Join Date: Mar 2013
Posts: 10
Talking

Quote:
Originally Posted by krespim View Post
Well, I can't help much further but I can tell you what I end up doing. As I am working with species that have relative well annotated genomes/transcriptomes (Homo sapiens and Mus musculus), I forego the initial cufflinks step and go straight to cufdiff after mapping using the gtf provided in iGenomes as a reference. Here are my settings:

# cuffdiff v2.0.2

$cuffdiff -L "GFP,KD" -o $cuff_output -p 8 -u $gtfFile $gfp_a,$gfp_b $srsf1_a,$srsf1_b

And that seems to work.
Thanks krespim. I have solved the problem by going back to Cufflinks 1.3.0, and I'm not sure whether there is a bug in the lastest version of Cufflinks or something incompatible in my reference file. However, it's done now.
leifive is offline   Reply With Quote
Old 08-27-2014, 05:25 AM   #8
krespim
Member
 
Location: Dresden

Join Date: Jul 2012
Posts: 49
Default Update

Quote:
Originally Posted by leifive View Post
Thanks krespim. I have solved the problem by going back to Cufflinks 1.3.0, and I'm not sure whether there is a bug in the lastest version of Cufflinks or something incompatible in my reference file. However, it's done now.
I went back to my notes - because I had to run cuffdiff again - and the problem seems to occur when the annotation gft is not a cufflinks output or from iGenomes (not 100% sure about iGenomes though). The reason is that to perform the splicing tests the gtf needs to have some extra annotated features.

Solution: run cuffcompare in the orginal gtf.
cuffcompare -s hg19.fasta -CG -r EnsemblGene-67.gtf EnsemblGene-67.gtf

This will create a cuff* ready annotation file.
krespim is offline   Reply With Quote
Reply

Tags
bioinformatics, ngs

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 10:57 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO