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Limma - RNA-Seq DE genes - Quantile normalized log transformed RPKM data

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Avinash S ▴ 40

@avinash-s-4979

Last seen 10.4 years ago

Hello All, I'm trying to understand the method of differential expression analysis described in : * * *Stem cell transcriptome profiling via massive-scale mRNA sequencing* *Nicole Cloonan et al* *NATURE METhODS | VOL.5 NO.7 | JULY 2008 | 613* http://www.nature.com/nmeth/journal/v5/n7/abs/nmeth.1223.html *Section: Statistical Analysis * To calculate differential expression of SQRL tag data we analyzed the normalized gene signals (tags per Refseq transcript, length- normalized) for each library using the Limma package in R. After Quantile normalization, we used Limma to fit a linear model to the log2-transformed data using an empirical Bayes method32 to moderate standard errors. Differentially expressed genes were defined as those with a B statistic > zero. I have quantile normalized log2-transformed RPKM data and I wanted to find DE genes based on B statistic and log2foldchange. *Sample Rawdata:* GeneModel CON1 CON2 TR1 TR2 1s00200.1 2.723945276 3.775939811 3.623211245 3.717795434 1s00210.1 4.999354495 3.738129253 3.268468778 3.822220668 1s00220.1 1.450861588 1.265013193 0.942706046 0.744551693 I'm using the following R-script: library(limma) raw.data <- read.delim("INPUT-QuantileNormalizedLog2Transformed-RPKM-Data.txt") attach(raw.data) names(raw.data) d <- raw.data[, 2:5] rownames(d) <- raw.data[, 1] Group <- factor(c("CON","CON","MYC","MYC"), levels=c("CON","MYC")) design <- model.matrix(~0+Group) colnames(design) <- c("CON","MYC") fit <- lmFit(d, design) fit <- eBayes(fit) *Warning message:* *Zero sample variances detected, have been offset* topTable(fit,coef=2,number=1000,genelist=fit$genes) - THIS LISTS THE GENES ALL POSITIVE LOGFC VALUES - NON -NEGATIVE. R version 2.14.0 (2011-10-31) Platform: i386-apple-darwin9.8.0/i386 (32-bit) locale: [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] limma_3.10.0 Can someone please let me know what I'm doing wrong here. Is it in the array design or input data? Thank you, Avinash [[alternative HTML version deleted]]

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ADD COMMENT • link updated 7.6 years ago by Gordon Smyth 52k • written 13.1 years ago by Avinash S ▴ 40

score 0 · Answer 1 · 2017-06-06

Dear Avinash,

Briefly, you have used

design <- model.matrix(~0+Group)

when you needed

design <- model.matrix(~Group)

However, I do not recommend a limma analysis of RPKM values, because it does not respect the mean-variance relationship inherent in the count data. Nicole Cloonan's excellent Nature Methods paper was written four years ago, and our understanding of the statistical analysis RNA-Seq data has moved on considerably since then. Please see the last case study in the limma User's Guide, which analyses the Nigerian HapMap data, for how I recommend limma be used to analyse RNA-Seq data.

Best wishes
Gordon

PS. This answer was originally posted 4 December 2011: Limma - RNA-Seq DE genes - Quantile normalized log transformed RPKM data