Help With Cuffdiff

Question: Help With Cuffdiff

4.8 years ago by

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

• 1.2k views

ADD COMMENT • link •

modified 4.8 years ago by Jennifer Hillman Jackson ♦ 25k • written 4.8 years ago by Maria Hoffman • 30

4.8 years ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

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

ADD COMMENT • link written 4.8 years ago by Jennifer Hillman Jackson ♦ 25k

4.8 years ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

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

ADD COMMENT • link written 4.8 years ago by Jennifer Hillman Jackson ♦ 25k

Please log in to add an answer.

Similar posts • Search »