DESeq2 error in data.frame (multiple treatments and multiple replicates)

[KYR]

I have a text file, containing read counts per gene for each treatments and control, with the following column

[Gene Symbol] [C1] [C2] [C3] [A1] [A2] [A3] [B1] [B2] [B3]

• C is Control
• A is Treatment 1
• B is Treatment 2

-> Each of C, A an B have 3 replicates

When I do data.frame it generates an error

library( "DESeq2" )
library("Biobase")
mydata = read.table("matrix.txt", header=TRUE)
col1 <- mydata[,1]

## Error message
ExpDesign = data.frame(row.names=col1, condition=c("C", "C", "C", "A", "A", "A", "B", "B", "B")
Error in data.frame(row.names = col1, condition = c("C", "C", "C", "A",  : 
  row names supplied are of the wrong length

## The following is what I would next if I didn't have any error message

countdata <- assay( mydata )
head( countdata )
coldata <- colData( mydata )
rownames( coldata ) <- coldata$run
colnames( countdata ) <- coldata$run
head( coldata[ , c("C", "C", "C", "A", "A", "A", "B", "B", "B") ] )

Eventually the goal is to have a heatmap with each replicates in the control, treatment A and B.

I think the problem comes from the fact that I should subset my data, though I have no clue how to do to that. Any suggestions on where is the error message coming from and how to subset data? (If I should ever subset that..)

[Michael Love]

This is your countData, which has as many rows as genes:

mydata = read.table("matrix.txt", header=TRUE)
col1 <- mydata[,1]

It looks like this will be the colData (sample information table).

ExpDesign = data.frame(row.names=col1, condition=c("C", "C", "C", "A", "A", "A", "B", "B", "B")

...which has as many rows as samples.

So the error comes when you try to name the rows of your colData using the gene names in col1.

You will also get an error later when you try to run

assay( mydata )

because mydata is a data.frame. assay() is a function for getting a matrix from SummarizedExperiment objects. You can just use

as.matrix( mydata )

in order to supply a matrix to DESeqDataSet.

[rookie_genomics]

Hi,

I followed your advice and tried to import as a matrix. But when I try to set up col.data I still get an error

This is my code

deseq2_analysis2 <- read_excel("deseq2_analysis2.xlsx")
> View(deseq2_analysis2)
> analysis3 <- as.matrix(deseq2_analysis2)
> (condition <- factor(c(rep("group1", 4), rep("group2", 4), rep("group3", 4), rep("group4", 4))))
[1] group1 group1 group1 group1 group2 group2 group2 group2 group3 group3 group3 group3 group4
[14] group4 group4 group4
Levels: group1 group2 group3 group4
> (coldata <- data.frame(row.names=colnames(analysis3), condition))
Error in data.frame(row.names = colnames(analysis3), condition) :
row names supplied are of the wrong length

This is my result for head command

head(deseq2_analysis2)
# A tibble: 6 x 17
gene Sample1_group1 Sample2_group1 Sample3_group1 Sample4_group1 Sample1_group2 Sample2_group2
<chr> <dbl> <dbl> <dbl> <dbl> <dbl> <dbl>
1 YAL0~ 0 0 0 0 2 0
2 YAL0~ 0 0 0 0 0 0
3 YAL0~ 243 242 109 130 271 233
4 YAL0~ 16 7 52 30 23 10
5 YAL0~ 23 21 21 33 11 28
6 YAL0~ 38 42 76 88 47 40
# ... with 10 more variables: Sample3_group2 <dbl>, Sample4_group2 <dbl>, Sample1_group3 <dbl>,
# Sample2_group3 <dbl>, Sample3_group3 <dbl>, Sample4_group3 <dbl>, Sample1_group4 <dbl>,
# Sample2_group4 <dbl>, Sample3_group4 <dbl>, Sample4_group4 <dbl>

What am I doing wrong here?

[rookie_genomics]

Originally Posted by Michael Love

This is your countData, which has as many rows as genes:

mydata = read.table("matrix.txt", header=TRUE)
col1 <- mydata[,1]

It looks like this will be the colData (sample information table).

ExpDesign = data.frame(row.names=col1, condition=c("C", "C", "C", "A", "A", "A", "B", "B", "B")

...which has as many rows as samples.

So the error comes when you try to name the rows of your colData using the gene names in col1.

You will also get an error later when you try to run

assay( mydata )

because mydata is a data.frame. assay() is a function for getting a matrix from SummarizedExperiment objects. You can just use

as.matrix( mydata )

in order to supply a matrix to DESeqDataSet.

Hi,

I followed your advice and tried to import as a matrix. But when I try to set up col.data I still get an error

This is my code

deseq2_analysis2 <- read_excel("deseq2_analysis2.xlsx")
> View(deseq2_analysis2)
> analysis3 <- as.matrix(deseq2_analysis2)
> (condition <- factor(c(rep("group1", 4), rep("group2", 4), rep("group3", 4), rep("group4", 4))))
[1] group1 group1 group1 group1 group2 group2 group2 group2 group3 group3 group3 group3 group4
[14] group4 group4 group4
Levels: group1 group2 group3 group4
> (coldata <- data.frame(row.names=colnames(analysis3), condition))
Error in data.frame(row.names = colnames(analysis3), condition) :
row names supplied are of the wrong length

This is my result for head command

head(deseq2_analysis2)
# A tibble: 6 x 17
gene Sample1_group1 Sample2_group1 Sample3_group1 Sample4_group1 Sample1_group2 Sample2_group2
<chr> <dbl> <dbl> <dbl> <dbl> <dbl> <dbl>
1 YAL0~ 0 0 0 0 2 0
2 YAL0~ 0 0 0 0 0 0
3 YAL0~ 243 242 109 130 271 233
4 YAL0~ 16 7 52 30 23 10
5 YAL0~ 23 21 21 33 11 28
6 YAL0~ 38 42 76 88 47 40
# ... with 10 more variables: Sample3_group2 <dbl>, Sample4_group2 <dbl>, Sample1_group3 <dbl>,
# Sample2_group3 <dbl>, Sample3_group3 <dbl>, Sample4_group3 <dbl>, Sample1_group4 <dbl>,
# Sample2_group4 <dbl>, Sample3_group4 <dbl>, Sample4_group4 <dbl>

What am I doing wrong here?