Hi, I'm trying to run DEseq2. As a test I'm using RNAseq data from 8 samples.  My countdata, coldata, and rowdata objects look (to me) formatted as they should, the dimensions/lengths match, count data is correct, etc. But when I run DESeqDataSetFromMatrix() I get this error:

> ddsFull <- DESeqDataSetFromMatrix(countData = countdata,
+    colData = coldata, rowData = rowdata, design = ~ type + sex)
Error in seq_len(length(idx) - 1) : 
  argument must be coercible to non-negative integer
In addition: Warning message:
In DESeqDataSet(se, design = design, ignoreRank) :
  58 duplicate rownames were renamed by adding numbers

Here is the (detailed) step-by-step. First I generate my SE object (works without problems):

> ex3 <- summarizeOverlaps(features=grl, reads=bamLst, ignore.strand=T, singleEnd=T)
> class(ex3)
[1] "RangedSummarizedExperiment"
attr(,"package")
[1] "SummarizedExperiment"

Then I create the countdata, coldata, rowdata objects (without problems):

> countdata <- assay(ex3)
> coldata <- colData(ex3)
> rowdata <- rowRanges(ex3)
> class(coldata)
[1] "DataFrame"
attr(,"package")
[1] "S4Vectors"
> class(rowdata)
[1] "GRangesList"
attr(,"package")
[1] "GenomicRanges"
> class(countdata)
[1] "matrix"
> length(rowdata)
[1] 24943
> dim(coldata)
[1] 8   6
> dim(countdata)
[1] 24943   8

> head(countdata)
         OM_003  OM_005  OM_014  OM_023
A1BG        259      69     116      69
NAT2          6      11       0       0
ADA        1785     396     964     441
CDH2        119      52      35      45 ...

> head(rowdata)
GRangesList object of length 6:
$A1BG 
GRanges object with 15 ranges and 2 metadata columns:
       seqnames               ranges strand |   exon_id   exon_name
          <Rle>            <IRanges>  <Rle> | <integer> <character>
   [1]    chr19 [58346806, 58347029]      - |    264625        <NA>
   [2]    chr19 [58347353, 58347640]      - |    264626        <NA> ...

> head(coldata)
DataFrame with 6 rows and 6 columns
            type      sex   status    height   weight     tech
           <factor> <factor> <factor> <numeric> <numeric> <factor>
OM_003       AA        F     yes      15.9     36.67        2
OM_005       AA        M     no       10.5     83.35        1
OM_014       BB        F     yes      14.3     31.22        7 ...

And then the error:

> ddsFull <- DESeqDataSetFromMatrix(countData = countdata,
+    colData = coldata, rowData = rowdata, design = ~ type + sex)
Error in seq_len(length(idx) - 1) : 
  argument must be coercible to non-negative integer
In addition: Warning message:
In DESeqDataSet(se, design = design, ignoreRank) :
  58 duplicate rownames were renamed by adding numbers

The traceback():

7: eval(expr, envir, enclos)
6: eval(quote(list(...)), env)
5: eval(quote(list(...)), env)
4: standardGeneric("paste")
3: paste(rnms[idx[-1]], c(seq_len(length(idx) - 1)), sep = ".")
2: DESeqDataSet(se, design = design, ignoreRank)
1: DESeqDataSetFromMatrix(countData = countdata, colData = coldata, 
       rowData = rowdata, design = ~type + sex

Any thoughts?? Count data is correct (zeros and positive integers, no "negative" counts), colData is correctly formatted, rowData seems correct as well. I am not sure what paste(rnms[idx[-1]], c(seq_len(length(idx)-1), sep=".") means, but it seems like maybe that is where the error is generating??

Figured it out!

The problem: I had replaced the original rownames in ex3 (the SE object) from entrezID numbers (which make zero sense to me) to gene symbols (which at least make some sense). I didn't think changing the rownames would matter so long as they matched rowdata, but it seems that was the big problem.

Solution: Anyway, I re-generated the SE object (took a while!) without replacing rownames and now DESeqDataSetFromMatrix worked without a problem. I was able to run the DESeq2 pipeline to results. Then I can add the gene symbols/names to the results data frame.

hi,

As a thought, the manual (summarizedexperiment 1.2.2) specifies rowData accepts a DataFrame and rowRanges a GRangesList (as you supply). If you  try with 'rowRanges = rowdata' instead of 'rowData=rowdata' do you see the same error?

tom