Help with Cuffdiff

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Help with Cuffdiff

Maria Hoffman
Hello,

I am new to cuff diff and just got my data output back and it doesn't look like anything is statistically significant. There are three treatment groups with two biological replicates each group. I am not sure if I made an error somewhere along the line, need to adjust the parameters, or if there really could be no change. The samples are from sheep and I have been using the OvisAries3.0 reference I downloaded from UCSC.

Thanks
Maria


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Re: Help with Cuffdiff

Jen Hillman-Jackson
Hi Maria,

More details are needed to help. Would you like to share a history with me?

You might also find the tutorials and other resources for RNA-seq analysis we have helpful. Many are linked from here:
https://wiki.galaxyproject.org/Support#Tools_on_the_Main_server:_RNA-seq

I've also added the latest sheep build to the set of genomes to be indexed for the RNA-seq mapping tools (bowtie & bowtie2). I'll try to include them in the upcoming snapshot if at all possible. But if not this one (is nearing completion), will be added to the next.

Please review the help, then send me a share link if the suggested protocols are not addressing your questions (direct).

Jen
Galaxy team

On 2/6/14 9:09 AM, Maria Hoffman wrote:
Hello,

I am new to cuff diff and just got my data output back and it doesn't look like anything is statistically significant. There are three treatment groups with two biological replicates each group. I am not sure if I made an error somewhere along the line, need to adjust the parameters, or if there really could be no change. The samples are from sheep and I have been using the OvisAries3.0 reference I downloaded from UCSC.

Thanks
Maria



___________________________________________________________
The Galaxy User list should be used for the discussion of
Galaxy analysis and other features on the public server
at usegalaxy.org.  Please keep all replies on the list by
using "reply all" in your mail client.  For discussion of
local Galaxy instances and the Galaxy source code, please
use the Galaxy Development list:

  http://lists.bx.psu.edu/listinfo/galaxy-dev

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please use the interface at:

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-- 
Jennifer Hillman-Jackson
http://galaxyproject.org

___________________________________________________________
The Galaxy User list should be used for the discussion of
Galaxy analysis and other features on the public server
at usegalaxy.org.  Please keep all replies on the list by
using "reply all" in your mail client.  For discussion of
local Galaxy instances and the Galaxy source code, please
use the Galaxy Development list:

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Re: Help with Cuffdiff

Jen Hillman-Jackson
Hi Maria,

I didn't notice any obvious problematic usage, format, or content issues
with the Tuxedo pipeline execution in your history. Your protocol is
right on track. This leaves data and parameter inputs to consider.

I did notice that you are mainly using defaults and omitting the use of
reference annotation that Cuffdiff uses to generate the full compliment
of statistics.

The "NOTEST" result indicates that the coverage is too shallow. You
could follow the advice here, by adjusting "-c" to be lower. This is
"Min Alignment Count:" and is set to "10" in your runs.
http://cufflinks.cbcb.umd.edu/faq.html#notest

Adding in a reference annotation file could also potentially help.
Aligned sequences may be falsely fragmenting without a reference
transcript to help bind them together. But, this is just a guess - I
didn't examine any assembly regions. This is however something that you
could do. The UCSC Table Browser is one source for a GTF file.

Experimenting with other parameters as you are doing also is worth it.
The manual and such cover these in detail, and there is always the tool
author's google group for detailed questions/advice.

Good luck with your project,

Jen
Galaxy team

On 2/6/14 12:38 PM, Maria Hoffman wrote:

> Hello,
>
> Thank you for your help. I have found that wiki page very helpful and
> actually us it very often (I was using it this AM too before I emailed
> you). In looking at the wiki again, nothing is really standing out to
> me ( my chromosome notation matches up etc). I am going to keep
> looking etc but I did send you my history too. I did try running
> another cuffdiff playing with the dispersion estimation method too out
> of curiosity.
>
> Thank you so much for your help! This is my first real data set doing
> this and we have abstracts due soon, so the pressure is on!
>
> Thanks!
> Maria

--
Jennifer Hillman-Jackson
http://galaxyproject.org

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Jennifer Hillman-Jackson
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