Question: Cuffdiff gene name
0
gravatar for caromanty3000
2.9 years ago by
United States
caromanty30000 wrote:

Hi!

I am analyzing RNA-seq using tophat-cufflinks-cuffdiff, but at the end, in the cuffdiff table I don't get the gene name. I don't know if I am skipping a important step. Could you let me know if I have to add additional information in one of the steps.

I appreciate your collaboration.

Carolina

rna-seq cufflinks • 963 views
ADD COMMENTlink modified 2.9 years ago • written 2.9 years ago by caromanty30000
1
gravatar for Jennifer Hillman Jackson
2.9 years ago by
United States
Jennifer Hillman Jackson25k wrote:

Hello -

This prior question covers the basics for including this type of annotation in output files from this tool package. Cufflinks output files

Key point: apply the right annotation reference dataset

Detailed help that covers the full spectrum of practical usage guidance for this tool suite is in the Galaxy support wiki here https://wiki.galaxyproject.org/Support#Tools_on_the_Main_server:_RNA-seq

(Also see the "best of" help links at the top of that same wiki - many Galaxy and community resources cover RNA-seq analysis, ranging from general usage/analysis examples all the way to troubleshooting results)

Broader help/context: There are also many other posts on this forum that cover the same topic in different levels of detail. Try searching by some keywords: RNA-seq, any of these tool names, reference annotation, gene annotation, etc. Putting those terms into a google can also yield useful results (that may point back to this forum or others that cover the topic). Or try to Galaxy user search tool. Just keep in mind that the title/subject of a question or email is only about 50% related to the root issue - the content/keywords in the replies are what to focus on - which can clutter search results. 

This is a very common question for users. The solution is not always the same, but give the initial advice a try then dig deeper into the others are needed. If you get completely stuck and are working on http://usegalaxy.org (or can replicate your issue there), send an email to galaxy-bugs@lists.galaxyproject.org for personalized, confidential advice based on your exact data/jobs. Include a shared history link with the target datasets left undeleted, plus any other info that you think will help with context/goals.

Best, Jen, Galaxy team

 

 

 

ADD COMMENTlink written 2.9 years ago by Jennifer Hillman Jackson25k
0
gravatar for caromanty3000
2.9 years ago by
United States
caromanty30000 wrote:

Hi!

Thank you for your answer.

I try to add the reference annotation, but the cufflinks is an empty file. I am not sure to understand how to import the reference annotation.

Thank you again,

Carolina 

ADD COMMENTlink written 2.9 years ago by caromanty30000
  1. Upload the reference annotation dataset into your history. It should be a dataset in GTF or GFF3 format.
  2. Add in the database metadata attribute if needed on the Edit Attributes: Datatype form (reached through the upper pencil icon of the dataset's box). 
  3. Double check that the chromosome identifiers in the reference annotation are an exact match for those in the target reference genome (the same one used for Tophat). This is critical - if there is a mismatch, the annotation will not be linked to the results. Convert the BAM Tophat output to SAM format, if needed, for the inspection. Support#Reference_genomes
  4. Select the annotation dataset for Cuffdiff on the tool form under the option Transcripts (first option on the form)

Reference annotation can be the original GTF/GFF3 dataset from an external source (for just differential expression of known transcripts) or can be a merged annotation that includes novel transcripts discovered during the Cufflinks job (for differential expression with discovery). For the latter use, merge Cufflinks result GTF datasets with the external reference GTF/GFF3 datasets using Cuffmerge, then use that result as the reference annotation dataset input to Cuffdiff. More protocol detail is in the manual and in several Galaxy RNA-seq tutorials/training examples.

Hopefully this helps!

ADD REPLYlink modified 2.9 years ago • written 2.9 years ago by Jennifer Hillman Jackson25k
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