Dear Michael and DESeq2 users,

I started using DESeq2 three weeks ago. Even I was having some normal
problems for a newby person my experience was really positive and I was and
I am detecting more DEG than with DESeq(1).

However suddenly I am encountering a new problem that wasn't coming up
previously:

> resSig <- res[ res$padj < .1, ]
Error in normalizeSingleBracketSubscript(i, x, byrow = TRUE, exact = FALSE)
:
  subscript contains NAs

For me this is pretty weird because I've been using the exactly same
RScript and datasets for the last 3 weeks and I wasn't having this error
before.

My RScript is:

#Count matrix input
Cele_MRvsER_old = read.csv (file.choose(), header=TRUE, row.names=1)
CeleDesign <- data.frame(
  row.names = colnames(Cele_MRvsER_old),
  condition = factor(c("MR", "MR", "ER", "ER", "ER", "ER")))
dds <- DESeqDataSetFromMatrix(countData = Cele_MRvsER_old,
                              colData = CeleDesign,
                              design = ~ condition)
dds

#Est size factor = normalize for library size
dds<- estimateSizeFactors(dds)
ddsLocal <- estimateDispersions(dds, fitType="local")
ddsLocal <- nbinomWaldTest(ddsLocal)
#plot dispersion
plotDispEsts(ddsLocal)


#Differential expression analysis
resultsNames(ddsLocal)
res <- results(ddsLocal, name= "condition_MR_vs_ER")
res <- res[order(res$padj),]
head(res)
plotMA(ddsLocal)
sum(res$padj < .1, na.rm=TRUE)


#filter for upregulated and downregulated genes
resSig <- res[ res$padj < .1, ]
head(resSig)
write.table(as.data.frame(resSig [ order(resSig$padj), ]),
file="BRvsMR_old_MostSigGenes.txt")
write.csv(as.data.frame(resSig[ order( resSig$log2FoldChange,
-resSig$baseMean ), ]),
            file="BRvsMR_old_DownRegulated.csv")
write.csv(as.data.frame(resSig[ order( -resSig$log2FoldChange,
-resSig$baseMean ),]),
          file="BRvsMR_old_UpRegulated.csv")

#up-regulated
head(resSig[order(resSig$log2FoldChange,-resSig$baseMean),])
#down-regulated
head(resSig[order(-resSig$log2FoldChange,-resSig$baseMean),])

#list most sig genes
head( resSig[ order(resSig$padj), ] )

And my sessionInfo():

> sessionInfo()
R version 3.0.2 (2013-09-25)
Platform: x86_64-apple-darwin10.8.0 (64-bit)

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

attached base packages:
[1] parallel  stats     graphics  grDevices utils     datasets  methods
base

other attached packages:
[1] DESeq2_1.2.0            RcppArmadillo_0.3.920.1 Rcpp_0.10.5
GenomicRanges_1.14.1
[5] XVector_0.2.0           IRanges_1.20.0          BiocGenerics_0.8.0

loaded via a namespace (and not attached):
 [1] annotate_1.40.0      AnnotationDbi_1.24.0 Biobase_2.22.0
DBI_0.2-7
 [5] genefilter_1.44.0    grid_3.0.2           lattice_0.20-24
 locfit_1.5-9.1
 [9] RColorBrewer_1.0-5   RSQLite_0.11.4       splines_3.0.2
 stats4_3.0.2
[13] survival_2.37-4      tools_3.0.2          XML_3.95-0.2
xtable_1.7-1* *


Any idea about why am I having this error now but not in the past and about
how to fix it?

Thank you!!!

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