Why can't I replicate DESeq's Log2FC calculation?
1
1
Entering edit mode
8.8 years ago

Hello,

I am trying to figure out how DESeq2 calculates its log2FC measure - we see a strange pattern in the FCs in our data and I'd like to reproduce them by hand from scratch to make sure this pattern does not reflect an error with my pipeline.

Right now, I'm doing this:

deseqOutput<-DESeq(data_collapsedTechnicalReps)
estSizeFactors <- estimateSizeFactors(deseqOutput_DUP)
RLEnormedData <- data.frame(counts(estSizeFactors, normalized=TRUE))
meanOfRLECounts <- data.frame( rowMeans( RLEnormedData[,1:2]) , rowMeans( RLEnormedData[,3:5])  ) # here, condition 1 = cols 1 and 2, condition 2=cols 3,4,5

colnames(meanOfRLECounts)<-c('Condition1','Condition2')

meanOfRLECounts$log2FC <- log2(meanOfRLECounts$Condition1/meanOfRLECounts$Condition2)

Here, condition 2 is the wild type condition - i.e. the samples that I indicate when I use relevel().

Can anyone spot what I'm doing wrong? What data does DESeq2 use to generate its log2FC estimates?

Thank you!

deseq2 rnaseq RNA-Seq deseq • 4.0k views
ADD COMMENT
6
Entering edit mode
8.8 years ago

Take a look at this thread and the DESeq2 manuscript

They are using a moderated (shrinkage) estimate.

ADD COMMENT

Login before adding your answer.

Traffic: 1966 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6