singletons in dexseq_count.py

[Jetse]

Hello everyone,

I am using the DEXseq library in R. To get the read counts per exon, I used the script dexseq_count.py. When executing this script I get many warnings:

/usr/local/lib64/python2.7/site-packages/HTSeq/__init__.py:592: UserWarning: Read 1350_690_366_F3 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
  "which could not be found. (Is the SAM file properly sorted?)" )

The used data is paired end dat in color-space format. The BAM file is created with Tophat. When using samtools flagstat I receive this output:

9158190 properly paired (6.38%)
89796971 singletons (62.59%)

This data is converted to sam after sorting the bam file with samtools sort. The conversion to SAM is done by samtools view -o <outFile> <inFile>.

When ignoring the warnings of dexseq_count.py, I can load the data in R. But when using the function estimateDispersions, I get the error:

Dispersion estimation. (Progress report: one dot per 100 genes)
Error in FUN(c("ENSG00000000003", "ENSG00000000419", "ENSG00000000457",  : 
  Underdetermined model; cannot estimate dispersions. Maybe replicates have not been properly specified.
In addition: Warning messages:
1: In .local(object, ...) :
  Exons with less than 10 counts will not be tested. For more details please see the manual page of 'estimateDispersions', parameter 'minCount'
2: In .local(object, ...) :
  Genes with more than 70 testable exons will be omitted from the analysis. For more details please see the manual page of 'estimateDispersions', parameter 'maxExon'.

This is my R code:

samples <- data.frame(condition,type,row.names=condition)
pairedGenes <- read.HTSeqCounts(countfiles = c(paired,single), design = samples, flattenedfile = annotationfile)
pairedExons <- estimateSizeFactors(pairedGenes)
pairedExons <- estimateDispersions(pairedExons)

How can I solve this error?
Is this error occur because of the warnings of dexseq_count.py?
And does the error of dexseq_count.py occur because of the singletons from tophat?

[areyes]

Dear Jetse,

As the error in DEXSeq indicates, it occurs because you do not have biological replicates.

Alejandro