Creating a data.frame for DESeq2

[KHubbard]

Hey All,

I am a novice R user and am having a difficult time getting a data.frame set up for DESeq2. I have done the following:

datafile <- "E:/Longitudinal Paper/DESeq/0 to 1.txt"

> datafile

[1] "E:/Longitudinal Paper/DESeq/0 to 1.txt"

> countTable = read.delim(datafile, header=TRUE, row.names=1)

> head(countTable)

C1 C2 C3 D1 D2 D3 D4
COX1 244969 257554 243872 235001 237881 178634 202627
Hsp90ab1 185595 200135 228766 143571 147350 99427 116190
CYTB 149810 151470 148100 129141 140353 86705 107290
Eef2 142818 151153 163043 140762 134884 94678 99470
Col4a1 126366 130972 227447 126424 122709 83403 77633
ND1 116379 117694 111396 91746 99802 62038 76769

> ExpDesign = data.frame(row.names=colnames(countTable),
+ condition=c("C1", "C2", "C3", "D1", "D2", "D3", "D4"),
+ libType = c("paired-end","paired-end","paired-end","paired-end","paired-end","paired-end","paired-end"))

> ExpDesign

condition libType
C1 C1 paired-end
C2 C2 paired-end
C3 C3 paired-end
D1 D1 paired-end
D2 D2 paired-end
D3 D3 paired-end
D4 D4 paired-end

I then try to do the following and get the subsequent errors:

> data("countTable")
Warning message:
In data("countTable") : data set ‘countTable’ not found

> countData <- counts(countTable)
Error in (function (classes, fdef, mtable) :
unable to find an inherited method for function ‘counts’ for signature ‘"data.frame"’

> colData <- pData(countTable)[,c("condition", "type")]
Error: could not find function "pData"

What steps am I missing in between? Thanks for any help!

[dpryan]

data("countTable") doesn't mean what you think. It tries to load an R dataset called "countTable", not convert your data.frame into a format for DESeq2. In the vignette, that works, since it's just loading a specific dataset that comes with an R package. Instead, just use:

cds <-DESeqDataSetFromMatrix(countData = countTable, colData=ExpDesign$condition, design=~ExpDesign$condition)

BTW, your experimental design is really just ExpDesign$condition, since they're all paired-end (so you needn't include it). Also, are your groups really unreplicated?

[KHubbard]

Thanks for the reply!

In answer to your question, all the "C's" are replicates of one another as are the "D's".

[dpryan]

Ah, good. Then you want instead:

condition=c("C", "C", "C", "D", "D", "D", "D")