Hi all,
in relation to a mail from January this yearin the r-help community, 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.
Apparently this time both fitTypes are almost similar (at least to my inexperienced eyes.)
I add both dispersion Plots. 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
default/parametric fit
local fit
P.S.
I also tried to post it on the bioC help site, but got no responses, so I try it here.
When did you post this to the Bioconductor list? I can't seem to find it. The DESeq2 authors are usually very good at replying in a timely manner, so I'm surprised they didn't get back to you.
About a week ago I sent the mail to bioconductor@r-project.org. Is it still the correct address?
It seems that my question was also not posted in the daily Bioconductor digest, so I can't really say why is that.
Can it be that it has a problem with images?
Perhaps it was the images, since it didn't go through. Give it another try.
In the dispersion plot, it looks like the slightly sigmoidal slope with an up-tick at the end is just not fit easily with the parametric equation. My guess is that Michael or Simon will advise you to go with the local fit, but of course it's always more definitive to get that answer from them.