Hi all,

in relation to my mail from January this year, I followed Simon's advice to
do my analyses in DESeq2 instead of DESeq.

I am working on an RNASeq from c. elegans. I have mapped the data with the
ensembl genome build WBcel215. I have ran tophat2 to map and featureCounts
to counts the reads (both with the defaults parameters).

I have two conditions, control and a knock-out with each three replica. Now
I am trying to find differentially regulated genes between the two
conditions using DESeq2.

This is the script I am using to read my raw count table into DESeq2:

featureCountTable <- read.table("featureCountTable_RawCounts.txt",
sep="\t", quote=F)

colData <- data.frame(row.names=names(featureCountTable), condition =
c(rep("wt",3), rep("cpb3", 3)))

cds <- DESeqDataSetFromMatrix (
    countData =     featureCountTable,
    colData   =     colData,
    design    = ~    condition
    )

fit = DESeq(cds)
res = results(fit)

 But I am getting the same problem with DESeq2 as I have got with DESeq.
When I ran the DESeq command I get a warning:
    Warning messages:
    1: In log(ifelse(y == 0, 1, y/mu)) : NaNs produced
    2: step size truncated due to divergence

So again I have tried to change the fitType.
fit = DESeq(cds, fitType="local")

 Which than came back without any warnings.
The two dispersion plots can be found here
<http://s23.postimg.org/pvopnmtxj/DESesq2_local.png> (local fit) and here
<http://s23.postimg.org/uk4pitj47/DESesq2_parametric.png>
(default/parametric fit). The red line goes through the point-cloud in both
cases (as Simon defined a good fit in the last communication, I wish it
would have bin so easy :-) .
In the local fit type there a more outliers and the right end of the slope
is going up again. I am not sure whether or not this is a good thing or not.

So, my question is - which of the two options is better?
I understand, that in general the parametric (default) option is better,
but here it gives me a warning, so that something in the fit calculations
is not good.

How can I understand theses plots?

Thanks for the help
Assa

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