[BioC] Log2 fold change single replicate RNA seq

Marc Carlson mcarlson at fhcrc.org
Wed Jul 13 18:39:34 CEST 2011

Hi John,

You have not really told us enough to help you but I will try to guess 
about what you need anyways.  To just get started, the thing you most 
need is to count how many of your aligned reads in the two samples are 
landing on each gene of interest.  You probably want to look at the 
Shortread and GenomicFeatures packages to get your alignments read in 
and some annotations to compare them to, and then you will want to use 
countOverlaps() from the GenomicRanges package to count how many reads 
land in each annotation element for each sample.  Then you will at least 
have counts that you can play with. (and compare to the quantifications 
from your microarray experiment).  Once you have that you can at least 
start to think about fold changes.

Hope this helps,


On 07/12/2011 12:18 PM, john herbert wrote:
> I have microarray data, which is 2 colour agilent human of 3 technical
> replicates.
> Green dye case and Red dye control. I have analysed in Limma,
> normalising within arrays and between arrays using aQuantile
> normalisation.
> I also have some Next gen RNAseq data that has been mapped to the
> Refseq transcriptome and I have these raw counts.
> However there are no replicates; only one case and one control.
> I want to plot how the Log2 Fold change is correlated between the two
> data sets as they are looking at similar samples.
> The microarray data is easy as Limma reports log2 fold change but NGS
> on the other hand does not.
> What would be the best package/approach to generating a log2 fold
> change of the next gen counts?
> I am thinking they should be quantile normalised as the microarray data is????
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