SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
Cufflinks, differentially expressed genes statsteam Bioinformatics 5 11-15-2013 11:28 AM
still too many differentially expressed genes. thejustpark Bioinformatics 2 11-03-2013 01:54 PM
What to do after finding differentially expressed genes? sazz Bioinformatics 1 07-15-2013 10:39 AM
very different numbers of differentially expressed genes by DESeq bliu1 RNA Sequencing 2 08-22-2012 08:41 AM
DESeq and EdgeR: too many differentially expressed genes!?!? cutcopy11 Bioinformatics 5 12-08-2011 12:14 AM

Reply
 
Thread Tools
Old 02-24-2014, 07:44 AM   #1
Parharn
Member
 
Location: Sweden

Join Date: Jul 2013
Posts: 84
Default Differentially expressed genes

Hi,
I am doing RNA-seq analysis using Tuxedo pipeline. I wonder when do you name it DIFFERENTIALY expressed genes? Does it have to be significant genes out of cuffdiff analysis or any sort of fold changes between two conditions.
I am asking it because my two conditions show very few significant changes in gene expression. And that is between G2 and S phase cells I just get 16 significant differentialy expressed genes.
Let me know if you need further information.

thanks.
Parharn is offline   Reply With Quote
Old 02-24-2014, 01:02 PM   #2
Bukowski
Senior Member
 
Location: Aberdeen, Scotland

Join Date: Jan 2010
Posts: 388
Default

Significance in cuffdiff is when the q-value (FDR adjusted p-value) is less than the p-value itself and it is not dependent on the magnitude of the fold change.

(Source, the cuffdiff manual : http://cufflinks.cbcb.umd.edu/manual.html#gene_exp_diff )

It's impossible to tell why you have few significant genes without more information about your experimental set up. Too few reads per sample, or too few replicates would be possible explanations.

Last edited by Bukowski; 02-24-2014 at 01:04 PM. Reason: added link
Bukowski is offline   Reply With Quote
Old 02-24-2014, 01:16 PM   #3
Parharn
Member
 
Location: Sweden

Join Date: Jul 2013
Posts: 84
Default

Yes, I understood how Cuffdiff defines significant, but my question is if differentially expressed genes are specifically those that are expressed significantly?

Second, since we were establishing RNA-seq for the first time in our lab, we did only one replicate of each condition. We just wanted to go all the way to the end before doing actual experiment. I recently read that number of replicates have great impact on the accuracy of data analysis. My data has between 10 to 20 million reads per sample. I guess having one replicate is making the problem. What do you think?

How many replicates do you suggest with how many reads in each?
Please let me know what other information will make it more clear.

Thanks.
Parharn is offline   Reply With Quote
Old 02-25-2014, 11:27 PM   #4
TiborNagy
Senior Member
 
Location: Budapest

Join Date: Mar 2010
Posts: 329
Default

Great article about the replicates/read numbers
http://www.ncbi.nlm.nih.gov/pubmed/24319002
TiborNagy is offline   Reply With Quote
Old 02-26-2014, 08:20 PM   #5
Jeremy
Senior Member
 
Location: Pathum Thani, Thailand

Join Date: Nov 2009
Posts: 190
Default

Most qPCR papers use a minimum fold change of 2 and statistical significance before calling anything differentially expressed. Since RNA-seq is basically high throughput qPCR it would make sense to use the same thresholds.
Jeremy is offline   Reply With Quote
Old 02-27-2014, 02:03 AM   #6
reema
Member
 
Location: Scotland

Join Date: Feb 2014
Posts: 27
Default

How about using logFold change?
reema is offline   Reply With Quote
Old 02-27-2014, 07:04 AM   #7
Parharn
Member
 
Location: Sweden

Join Date: Jul 2013
Posts: 84
Default

Thanks people.
Parharn is offline   Reply With Quote
Old 02-27-2014, 12:07 PM   #8
Krish_143
Member
 
Location: Sweden

Join Date: Jan 2012
Posts: 45
Default

When we have no replicates.. then i suggest u to try Gfold.
Gfold > 0 || gfold < 0 means it Upregulated or down regulated.

For more information check.
http://compbio.tongji.edu.cn/~fengjx/GFOLD/gfold.html
__________________
Krishna
Krish_143 is offline   Reply With Quote
Old 02-27-2014, 02:45 PM   #9
Parharn
Member
 
Location: Sweden

Join Date: Jul 2013
Posts: 84
Default

Thanks a lot Krish. Looks very interesting, how can I get the software? Apt-get I guess? I am new to Linux environment.
Parharn is offline   Reply With Quote
Old 02-27-2014, 10:30 PM   #10
Krish_143
Member
 
Location: Sweden

Join Date: Jan 2012
Posts: 45
Default

You can download at
https://bitbucket.org/feeldead/gfold/downloads

For downlading to linux :

wget https://bitbucket.org/feeldead/gfold....V1.1.1.tar.gz

Check Readme and installation files in gfold.
__________________
Krishna

Last edited by Krish_143; 02-27-2014 at 10:35 PM.
Krish_143 is offline   Reply With Quote
Old 02-27-2014, 10:56 PM   #11
Parharn
Member
 
Location: Sweden

Join Date: Jul 2013
Posts: 84
Default

Thanks a million Krish. I will give it a try.
Parharn is offline   Reply With Quote
Old 03-04-2014, 02:16 AM   #12
Parharn
Member
 
Location: Sweden

Join Date: Jul 2013
Posts: 84
Default

This Gfold was a game changing program. Thanks for suggesting it Krish.
Parharn is offline   Reply With Quote
Reply

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 10:00 AM.


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