I got error when i run DESeq None of your conditions is replicated. Use method='blind' to estimate across conditions, or 'pooled-CR', if you have crossed factors

Question

[PK]

Hi I have to identify differently expressed genes using DESeq. I have completed up to HTseq and I have HTseq output files also. I have two conditions with two replicates (arabinose Rep1,2 ,glucose Rep1,2) . When i run DESeq i got error like this ( None of your conditions is replicated. Use method='blind' to estimate across conditions, or 'pooled-CR', if you have crossed factors)

My input file is:

    arabinose_1 arabinose_2 glucose_1   glucose_2
NCU00003    831 566 247 371
NCU00004    1164    964 734 1047
NCU00005    466 489 241 270
NCU00006    2527    2517    1828    1821
NCU00007    887 911 417 336

My R version 3.3.2 (2016-10-31)

This is my code: (I just copied and pasted what i get)

> library(DESeq2)

> countsTable <- read.delim ('SRP002628_readCounts.txt',header=TRUE)

> rownames(countsTable) <- countsTable$Gene_ID 

> countsTable <- countsTable[,-1] 

> head(countsTable)

arabinose_1 arabinose_2 glucose_1 glucose_2

1         831         566       247       371

> conditions <- factor(c("arabinose_1","arabinose_2"," glucose_1","glucose_2"))

> countDataSet <- newCountDataSet(countsTable, conditions)

> countDataSet <- estimateSizeFactors(countDataSet)

> sizeFactors(countDataSet)

arabinose_1 arabinose_2   glucose_1   glucose_2 
  1.1572973   1.0995991   0.8849006   0.9018904 

> head(counts(countDataSet))

  arabinose_1 arabinose_2 glucose_1 glucose_2
1         831         566       247       371
2        1164         964       734      1047

> head(counts(countDataSet,normalized=TRUE))

 arabinose_1 arabinose_2 glucose_1 glucose_2

1    718.0523    514.7330  279.1274  411.3582

> countDataSet <-estimateDispersions(countDataSet)

**Error in .local(object, ...) : 
  None of your conditions is replicated. Use method='blind' to estimate across conditions, or 'pooled-CR', if you have crossed factors.**

please help me. I literally confused. I know it is a basic one. But i am not able to solve.

[GenoMax]

Please edit and provide a more informative title. Copying and pasting the error message in the title provides no hints/useful information.

Answer 1

[ddiez]

From the newCountDataSet help about the condition argument:

A factor of experimental conditions (or treatments, or tissue types, or phenotypes, or the like). The length of the factor has to be equal to the number of columns of the countData matrix, assigning a condition to each sample.

And this is how it is used in the example:

cds <- newCountDataSet(countsTable, c( "A", "A", "A", "B", "B" ) )

So, I believe you want in your code:

conditions <- factor(c("arabinose", "arabinose", "glucose", "glucose"))

[PK]

Thanks for your answer. It works. one more thing i have to calculate RPKM values for DESeq. How do i calculate?

[WouterDeCoster]

i have to calculate RPKM values for DESeq

No, you definitely don't have to do that. Input to DESeq2 should be raw read counts.