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Old 12-13-2012, 01:09 AM   #1
Location: The netherlands

Join Date: Nov 2012
Posts: 38
Default singletons in

Hello everyone,

I am using the DEXseq library in R. To get the read counts per exon, I used the script When executing this script I get many warnings:
/usr/local/lib64/python2.7/site-packages/HTSeq/ 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, 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
And does the error of occur because of the singletons from tophat?
Jetse is offline   Reply With Quote
Old 12-14-2012, 01:05 AM   #2
Senior Member
Location: Heidelberg

Join Date: Aug 2010
Posts: 165

Dear Jetse,

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

areyes is offline   Reply With Quote

dexseq,, singletons, tophat

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