Question: Cuff diff output vs genotype discrepancy
gravatar for Hermione
3.9 years ago by
Hermione0 wrote:

Hi there,

I am using tuxedo pipeline to analyse differential gene expression between my samples. I have 3 biological replicates from each group and 4 groups. Two groups are wild type whereas the other two groups are from knock out cells. However I am seeing this bizarre output where I am getting hits for the silenced gene in the knock out groups.

These are 100bp paired end reads from Illumina and I have done Trimmomatic to  remove adaptor sequences, TopHat,  Cufflink, Cuffmerge of all the  Cuff link output files and Cuff diff.

I am clueless on what to do, could someone please help me? Any comments/suggestions welcome.




rna-seq cufflinks galaxy • 906 views
ADD COMMENTlink modified 3.9 years ago by Jennifer Hillman Jackson25k • written 3.9 years ago by Hermione0
gravatar for Jennifer Hillman Jackson
3.9 years ago by
United States
Jennifer Hillman Jackson25k wrote:


Did you run Tophat and/or Cufflinks with a reference annotation dataset? Try running without.

Modified: I should add in that cross-over hits are possible regardless of inputs/settings, as much depends on the genome source/finished state and what the knock-out is. For example, plants with a high ploidy count can be problematic in this type of analysis. But if I were analysing, I would check to see if the knock-out is included in a gene family resulting from a duplication event(s) or is perhaps contains a mix of unique/variable regions (IG, Zinc Finger, etc). Visualization of the NGS hits along with a good reference annotation dataset may be a big help in sorting out the results. Abundance statistics, maybe not so much if coverage is low to begin with, as a high quality sequenced duplicated region can be just as greedy as any other region (e.g. will capture hits just fine). Where the hits are can reveal a great deal.

Most of this is a guess from the given information, but perhaps helps.

Best, Jen, Galaxy team

ADD COMMENTlink modified 3.9 years ago • written 3.9 years ago by Jennifer Hillman Jackson25k

Hi Jen,

Thanks for the reply.

Just a brief description of the data set for you.

These cells are from wild type and KO mice for a particular gene. I have confirmed their genotype with PCR primers. here's the link to the visualization of the region of interest for this gene in two of the biological replicates from each group. 

Please have a look and let me know your comments/suggestions.

I am re-running the cufflinks without the reference annotation.

Thanks again

ADD REPLYlink modified 3.9 years ago • written 3.9 years ago by Hermione0
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