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Old 11-14-2013, 12:50 AM   #21
areyes
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Hi @thanhhoang,

It does not look like a common error. What is the output of your sessionInfo()?
What is the output of doing:

all( is.na( fData(ecs)$dispBeforeSharing ) )

?

How does the distribution of exon counts look like:

hist( log( rowMeans(counts(ecs)) ) )

?

Alejandro
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Old 11-14-2013, 08:31 PM   #22
nbahlis
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Hello Alejandro and Ryan

I ran DEXSeq on 22 samples (2 conditions: pre and post, biological replicates) as per my post earlier. I did get 50 warnings after running > ecs <- estimateDispersions ( ecs )

> Done
There were 50 or more warnings (use warnings() to see the first 50).
Here's one of the warnings:

Warning messages:
1: In chol.default(XVX + lambda * I, pivot = TRUE) :
the matrix is either rank-deficient or indefinite Error in cat(list(...), file, sep, fill, labels, append) :
argument 2 (type 'S4') cannot be handled by 'cat'


Any advice what I may have done wrong?

thank you
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Last edited by nbahlis; 11-16-2013 at 03:44 AM.
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Old 11-16-2013, 05:10 AM   #23
areyes
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Hi nbahlis,

One question, do you have paired samples? If so, your analysis might benefit from adding the pairing information as an additional covariate, and might also help with the error message.

The reason for the warning is that DEXSeq assumes a mean-variance relation a bit different from the one in your data (e.g. http://www.ncbi.nlm.nih.gov/pmc/arti...195/figure/F2/). I could not read the x-axis labels, but the left part of your plot seems to be a bit strange. Have you tried to filter more strictly on lower counts (e.g. only allowing exon bins with more than 200 counts)? The minCount parameter of estimateDispersions could do this for you.

Alejandro
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Old 11-16-2013, 06:27 AM   #24
nbahlis
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thank you Alejandro

they are paired data /samples. Can you please advise how to add the pairing as a variable. I will repeat the analysis with better (strict) filtering of the counts.
Thank you for your prompt responses, truly appreciate it
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Old 11-18-2013, 02:40 AM   #25
areyes
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Hi @nbahlis,

No prob!

You can find the instructions of how to specify this in the section "Additional technical or experimental variables". Shortly, you have to specify the pairing information in your design matrix when creating your ExonCountSet object and modify your formulas as in the vignette in order to add the pairing information as a covariate. The vignette describes how to do this by specifying the sequencing library type, you should substitute this with your pairing variable!

Alejandro
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Old 11-30-2013, 01:10 PM   #26
nbahlis
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Hi Alejandro

Is there a way to figure out what the bin number exactly represents. In other words what exon or exon-fusion/splice it represents?

thank you
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Old 12-01-2013, 08:12 AM   #27
areyes
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Hi nbahlis,

It is possible to map the exonic bins to a transcript database by overlaping the exonic bins with the coordinates of the genomic transcripts, for example:

Quote:
library(DEXSeq)
library(GenomicFeatures)
library(GenomicRanges)

data("pasillaExons", package="pasilla")
exonBins <- GRanges(
seqnames=fData(pasillaExons)$chr,
ranges=IRanges(
fData(pasillaExons)$start, fData(pasillaExons)$end ),
strand=fData(pasillaExons)$strand)
transcriptDb <- makeTranscriptDbFromGFF("/home/alejandro/Work/Graveley/Reanalisis/Annotations/Drosophila_melanogaster.BDGP5.25.62.mychr_tss.gtf", format="gtf")

exonsByTranscript <- exonsBy(transcriptDb, "tx", use.names=TRUE)

findOverlaps( unlist(exonsByTranscript), exonBins )
This will go back from the exonic bin definition to the original transcripts annotaions!
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Old 12-01-2013, 08:17 AM   #28
nbahlis
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Many thanks!
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