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  • singletons in dexseq_count.py

    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:
    Code:
    /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:
    Code:
    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:
    Code:
    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:
    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?

  • #2
    Dear Jetse,

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

    Alejandro

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