Question: Error using feature counts with DESeq2 : Cannot create DESeqData object
0
gravatar for Cookie-san
27 days ago by
Cookie-san0
Bengaluru, India
Cookie-san0 wrote:

I have a file of read counts with which I want to find the differentially expressed genes from using DeSeq2. The file was generated using feature counts.

           C11  C15 C19 C23 N11 N15 N19 N23

NM_000014   4422    14216   8885    17031   8162    4811    12536   8273
NM_000015   3         0 7   2   0   9   2   6
NM_000016   1063    1192    1608    1345    1118    951 943 1120
NM_000017   164     424 463 507 603 692 494 653
NM_000018   5193    12982   11382   11716   10030   14180   9379    13316
NM_000019   654     1103    1106    1184    743 497 569 844

When I try to create a DeSeq2 object using the DESeqDataSetFromMatrix I get the following error:

DESeq.ds <- DESeqDataSetFromMatrix(countData = readcounts,colData = sample_info,design = ~ condition)
Error in DESeqDataSet(se, design = design, ignoreRank): some values in assay are not integers
Traceback:

1. DESeqDataSetFromMatrix(countData = readcounts, colData = sample_info,  design = ~condition)
2. DESeqDataSet(se, design = design, ignoreRank)
3. stop("some values in assay are not integers")

I checked the entire read counts file, there are no integers in it, so I don't understand why this error keeps occuring. I tried running the sapply(readcounts, class) command as suggested in this thread (which did not give a clear solution) and get the following output:

C11 'numeric'
C15 'numeric'
C19'numeric'
C23 'numeric'
N11 'numeric'
N15 'numeric'
N19 'numeric'
N23 'numeric'

I tried using DESeqDataSet instead, but that requires a RangedSummarizedExperiment object from the function summarizeOverlaps from the Genomic Alignments package. The summarizeOverlaps function does the same job as featurecounts - generate read counts. I don't want to repeat that step.

rna-seq featurecounts deseq2 • 163 views
ADD COMMENTlink modified 8 days ago by Biostar ♦♦ 20 • written 27 days ago by Cookie-san0

How did you obtain the count matrix? Are these raw (=non-normalized) counts? Please show head(readcounts).

You can also try as.integer(readcounts) given that there are indeed integers but they are somewhat misclassified as characters of fators. How did you import the count matrix?

ADD REPLYlink modified 27 days ago • written 27 days ago by ATpoint16k

The file was given to me by my professor as a csv file. Yes these are raw counts. I imported the file using the read.table command. as.integer(readcounts) doesn't work because readcounts is a dataframe.

head(readcounts)
C11 C15 C19 C23 N11 N15 N19 N23
NM_000014   4422    14216   8885    17031   8162    4811    12536   8273
NM_000015   3   0   7   2   0   9   2   6
NM_000016   1063    1192    1608    1345    1118    951 943 1120
NM_000017   164     424 463 507 603 692 494 653
NM_000018   5193    12982   11382   11716   10030   14180   9379    13316
NM_000019   654     1103    1106    1184    743 497 569 844
ADD REPLYlink modified 27 days ago • written 27 days ago by Cookie-san0

Come on, as.integer(as.matrix(readcounts))

ADD REPLYlink written 27 days ago by ATpoint16k

Ah sorry XP New to R.

Edit: Sorry it worked. Should I convert the integer object back to a data frame? Again, sorry if the question is too obvious.

readcounts <- as.integer(as.matrix(readcounts))
head(readcounts)
4422 3 1063 164 5193 654 
DESeq.ds <- DESeqDataSetFromMatrix(countData = readcounts,colData = sample_info,design = ~ condition)
Error in validObject(.Object): invalid class “SummarizedExperiment” object: nb of cols in 'assay' (1) must equal nb of rows in 'colData' (8)
ADD REPLYlink modified 27 days ago • written 27 days ago by Cookie-san0

Ah sorry XP New to R.

No problem, sorry did not intend to sound harsh :)

mode(readcounts) <- "integer" is the last thing I could think of.

ADD REPLYlink modified 27 days ago • written 27 days ago by ATpoint16k

Have you tried importing just the top of the file? Maybe there is one line that got corrupted. Is dim(readcounts) what you expect? You might have a weird whitespace hiding in there somewhere.

ADD REPLYlink written 26 days ago by swbarnes25.5k
tmp <- gsub(" ", "", readcount); mode(tmp) <- "integer"

might be worth a try

ADD REPLYlink written 26 days ago by ATpoint16k

Hey, thank you SO MUCH for asking me to check the dimensions of my data frame. I was indeed missing around 8000 genes. I figured out the error; I was using this code to make my Gene_IDs unique and convert them to row names:

readcounts <- aggregate(readcounts[-1],readcounts[1],mean)
row.names(readcounts) <- readcounts[,1]
readcounts$Gene_ID <- NULL
head(readcounts)

That block of code was somehow chopping off the last 8k of my genes. I also figured that it was adding some non-integer values. So I replaced the code with this instead:

rownames(readcounts) = make.names(readcounts$Gene_ID, unique=TRUE) #To make unique rownames
readcounts$Gene_ID <- NULL
head(readcounts)
                   C11  C15 C19 C23 N11 N15 N19 N23
NR_075077   0   5   0   4   8   2   2   6
NM_001276352    0   5   0   4   8   2   2   6
NM_001276351    0   5   0   4   8   2   2   6
NM_000299   1220    5980    5089    2792    5223    9731    4365    4755
NM_001005337    1220    5980    5089    2792    5223    9731    4365    4755
NM_012102   7436    15741   13205   14024   15995   14659   9167    12504

I found that bit of magic here. I was able to create a DESeqObject now. Thanks swbarnes and ATpoint!

ADD REPLYlink modified 26 days ago • written 26 days ago by Cookie-san0
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