I meet some problem for using Deseq2.

Question

[codezy]

Dear all,

I am new in this area and trying to learn how to do the differential analysis now. I have got some bam file from the mapping tool STAR. Now I want to use the DESeq2 to continue the analysis, but DESeq2 ask a matrix as input. Therefore, I use htseq-count to convert the reads bam file into a txt file by the code

 htseq-count -f bam file genome.gtf >file.txt

But the outcome of it is like this when I use read.table in R:

> head(df)
                      V1    V2.x    V2.y
1 __alignment_not_unique 2812420 2666754
2            __ambiguous   14080   15004
3           __no_feature 8863085 8241851
4          __not_aligned       0       0
5        __too_low_aQual       0       0
6     ENSG00000000003.14       0       0

I have try the code

  condition <-factor("V1","V2.x","V2.y")
df2 <- data.frame(df,condition = condition)
dds <- DESeqDataSetFromMatrix(df2, DataFrame(condition), ~ condition)

But it doesn't work, and give me the error

Error in DESeqDataSetFromMatrix(df2, DataFrame(condition), ~condition) : 
ncol(countData) == nrow(colData) is not TRUE

could anyone help me with this? How can I use this htseq outcome for DESeq2? Thank you

[Vitis]

Reading the tutorial of DESeq2 would be a good start.

http://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#htseq-count-input

For the error, you didn't import the read count file generated from htseq-count, thus the "cannot open the connection" error.

[codezy]

I have solved the problem with these codessampleFiles <- grep("count",list.files("F:/"),value=TRUE) sampleCondition <- sub("(.*count).*","\\1",sampleFiles) sampleTable <- data.frame(sampleName = sampleFiles, fileName = sampleFiles, condition = sampleCondition)

[swbarnes2]

You realize that DESeq2 doesn't really work if you have one sample-one condition concordance, right?

Go through the tutorial. Just because your command lines are being parsed doesn't mean the software is going to give you the answer you want.

Answer 1

[swbarnes2]

The best advice anyone can give you is to walk through the DESeq2 tutorial. Look at the example data files, and google things about R to learn how to make your data look like the data in the tutorial.

Answer 2

[leaodel]

Hi, codezy. You should really dive into DESeq2's tutorial, it's an essential resource for learning. With that being said, when you have files from HTSeq you should use another function to import your samples. Try this:

Create a variable with the file's path:

dir <- "~/pathtoyourfiles/"

Add the file's name as a column on the table you'll use to feed the sampleTable argument.

myTable$fileName <- grep("count", list.files(dir), value = T)

Finally, import your samples:

ddsHTseq <- DESeqDataSetFromHTSeqCount(sampleTable = myTable, directory = dir, design = ~control+treatment)

You can also find more details about this function with:

?DESeqDataSetFromHTSeqCount()

Answer 3

[codezy]

Thank you for all your answer. I have solved the problem by these codes:

sampleFiles <- grep("count",list.files("F:/"),value=TRUE) 
sampleCondition <- sub("(.*count).*","\\1",sampleFiles) 
sampleTable <- data.frame(sampleName = sampleFiles, fileName = sampleFiles, condition = sampleCondition)