SEQanswers

Go Back   SEQanswers > Applications Forums > RNA Sequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
explain unimodal GC-content bias shuteo Illumina/Solexa 2 08-29-2012 01:43 PM
RNA-Seq: Summarizing and correcting the GC content bias in high-throughput sequencing Newsbot! Literature Watch 0 02-11-2012 03:00 AM
RNA-Seq: GC-Content Normalization for RNA-Seq Data. Newsbot! Literature Watch 0 12-20-2011 03:00 AM
RNA-Seq: Improving RNA-Seq expression estimates by correcting for fragment bias. Newsbot! Literature Watch 0 03-18-2011 03:00 AM
RNA-Seq: Length Bias Correction for RNA-seq Data in Gene Set Analyses. Newsbot! Literature Watch 0 01-22-2011 03:02 AM

Reply
 
Thread Tools
Old 02-27-2013, 03:45 PM   #1
federete
Junior Member
 
Location: Spain

Join Date: Feb 2013
Posts: 2
Default RNA-seq and GC content bias

Dear all,

I am analyzing a dataset of RNA-seq (from Illumina Genome Analyser IIx), and have found some interesting expression pattern trends related to the GC content of genes. After reading the literature, I have noticed that RNA-seq has some biases related to the GC content. Hence, I am not sure of the validity of my inferences.

So, my question, or questions are:
-How can I determine if this is a biologically meaningful trend or rather so a bias of the methodology?
-To what extent does the GC content biases the results of RNA-seq experiments? (the "trends" that I observe are strong)
-I am working with published RPKM values, and was wondering if I need to recalculate all these values considering GC content biases or if I could just test how much bias is in the data.

Any clue with any of these questions is highly appreciated!
Federico
federete is offline   Reply With Quote
Old 02-28-2013, 02:07 AM   #2
dpryan
Devon Ryan
 
Location: Freiburg, Germany

Join Date: Jul 2011
Posts: 3,480
Default

Have a look at the conditional quantile normalization paper from Hansen et al. (http://biostatistics.oxfordjournals..../13/2/204.full). I expect that has much of what you're looking for.
dpryan is offline   Reply With Quote
Old 03-01-2013, 02:02 AM   #3
federete
Junior Member
 
Location: Spain

Join Date: Feb 2013
Posts: 2
Default

Many thanks, dpryan

I am not sure whether I could guess from the paper the extent of the influence of the GC content, but will do some tests with my data to see what can I see

Fede
federete is offline   Reply With Quote
Old 03-01-2013, 07:24 AM   #4
amcloon
Member
 
Location: Germany

Join Date: Sep 2012
Posts: 15
Default

This was also a concern of mine since I work with a GC-rich species, and it is clear from Aird et al, 2011 (Genome Biology 12:R18) that PCR conditions during library prep can non-uniformly amplify transcripts with different GC contents. If you are really seeing systematic biases, I would figure out what you did/had done during library construction (polymerase, buffers, etc.), to know if you can anticipate that you had biased amplification.

I am not sure this will help your analysis any, but at least if your preparation didn't take this into account, you can write off some of the variability that way.

Anna
amcloon is offline   Reply With Quote
Reply

Tags
gc content bias, illumina ga iix, rpkm

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 11:17 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO