Hello everyone,

I have a specific problem with Deseq2. I tried to google and find similar error codes in bio-conductor web page and other sites. But couldn't find a similar error code, so i am sending this e-mail to you guys so that you could suggest me a solution.

 I am trying to analyze my data and i am trying to compare the expression of small RNAs between two stages. Here is the code and the error that i get.

> temp=read.csv("results.csv",header=TRUE, sep=",")
> head(temp)
        X.miRNA Sample.1 Sample.2 Sample.3 Sample.4 Sample.5 Sample.6 Sample.7 Sample.8
1 aae-bantam-5p       0       0       0       0       0       0       0       0
2 aae-bantam-3p       0       3       0       0       0       1       0       0
3     aae-let-7    6732     759      11      12      14      10      22      59
4     aae-miR-1      72      11       3       0       2       0      24    1360
5    aae-miR-10    1136     246      24      19       8      25    1095   70971
6   aae-miR-100    4181     300      24      19       9      32      38   31840
> rownames(temp) <- make.names(temp[,1], unique=TRUE)
> head(temp)
                    X.miRNA Sample.1 Sample.2 Sample.3 Sample.4 Sample.5 Sample.6
aae.bantam.5p aae-bantam-5p       0       0       0       0       0       0
aae.bantam.3p aae-bantam-3p       0       3       0       0       0       1
aae.let.7         aae-let-7    6732     759      11      12      14      10
aae.miR.1         aae-miR-1      72      11       3       0       2       0
aae.miR.10       aae-miR-10    1136     246      24      19       8      25
aae.miR.100     aae-miR-100    4181     300      24      19       9      32
              Sample.7 Sample.8
aae.bantam.5p       0       0
aae.bantam.3p       0       0
aae.let.7          22      59
aae.miR.1          24    1360
aae.miR.10       1095   70971
aae.miR.100        38   31840
> countdata <- temp [,-c(1)]
> head(countdata)
              Sample.1 Sample.2 Sample.3 Sample.4 Sample.5 Sample.6 Sample.7 Sample.8
aae.bantam.5p       0       0       0       0       0       0       0       0
aae.bantam.3p       0       3       0       0       0       1       0       0
aae.let.7        6732     759      11      12      14      10      22      59
aae.miR.1          72      11       3       0       2       0      24    1360
aae.miR.10       1136     246      24      19       8      25    1095   70971
aae.miR.100      4181     300      24      19       9      32      38   31840
> condition <- factor(c('Stage1', ' Stage 2', ' Stage3', ' Stage 4', ' Stage 5', ' Stage 6', ' Stage 7', ' Stage 8'))
> coldata <- data.frame(row.names = colnames(countdata), condition)
> coldata
                      condition
Sample.1           Stage1
Sample.2           Stage2
Sample.3           Stage3
Sample.4           Stage4
Sample.5           Stage5
Sample.6           Stage6
Sample.7           Stage7
Sample.8           Stage8
> ddsprep <- DESeqDataSetFromMatrix(countData = countdata, colData = coldata, design =~condition)
> colData(ddsprep)$condition<-factor(colData(ddsprep)$condition,levels=c("Stage1"," Stage2"))
> dds <- DESeq(ddsprep)
Error in designAndArgChecker(object, betaPrior) : 
  variables in the design formula cannot have NA values

ddsprep$condition

condition

<factor>
Stage1
Stage2
NA
NA
NA
NA
NA

NA

As you can see, there are NAs and obviously that is the reason why the error is showing up. Then i tried to subset my samples according to the instructions given in the manual to eradicate the error above using the command and i get a similar error.

> dds <- ddsprep[ , ddsprep$condition == " Stage1"," Stage 2"]
Error: subscript contains NAs

It seems that NAs isn't accepted when you finally run the Deseq2, but i don't know how to remove NAs (i tried is.na(), but i get the error, is.na() applied to non-(list or vector) of type 'S4') and i don't know how to subset the samples for my data analysis. Could you please suggest a solution? I just wanted to compare Stage 1 and Stage 2. I am a biologist with very little knowledge on R, so this might appear as a stupid question, but i really appreciate your time and patience.

p.s: stage 1 to 8 doesn’t have replicates. The main purpose of this experiment is to do a exploratory and hypothesis generating analysis

Thanks for your help in advance

You can use the following:

dds <- DESeqDataSetFromMatrix(countData = countdata, colData = coldata, design =~condition)

dds <- dds[, dds$condition %in% c("Stage1","Stage2") ]

dds <- DESeq(dds)