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Shi, Tao
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@shi-tao-199
Last seen 9.6 years ago
hi Michael,
I have a simple RNAseq experiment: a cell line treated with a compound
at various doses and time. ?Here are the htseq counts for one of gene
below along with the treatment conditions and sizeFactor estimated by
DESeq2.
> tmp
? count sizeFactor time dose
1 11396 1.0069598 8 0
2 12306 1.0913848 8 0
3 11761 1.0611031 8 0.1
4 12177 1.0764237 8 0.1
5 12017 1.1675793 8 1
6 10667 1.0375095 8 1
7 11423 1.0693406 8 10
8 11529 1.1097800 8 10
9 7845 0.9187615 24 0
10 8600 0.9620073 24 0
11 3200 0.9502428 24 0.1
12 2964 0.8491865 24 0.1
13 1079 0.8893242 24 1
14 1391 1.0884646 24 1
15 1261 0.9130893 24 10
16 1451 1.0182247 24 10
The design matrix I used is : ~time + dose + time:dose
Here are the coefficient estimates for this particular gene:
baseMean 7.266687e+03
baseVar 1.854839e+07
allZero 0.000000e+00
dispGeneEst 1.000000e-08
dispFit 6.694323e-04
dispersion 4.994657e-04
dispIter 1.000000e+01
dispOutlier 0.000000e+00
dispMAP 4.994657e-04
Intercept 1.238851e+01
time8 1.007756e+00
time24 -9.953678e-01
dose0 8.800724e-01
dose0.1 2.014315e-01
dose1 -5.854584e-01
dose10 -4.836570e-01
time8.dose0 -8.127168e-01
time24.dose0 8.201522e-01
time8.dose0.1 -1.467029e-01
time24.dose0.1 1.484047e-01
time8.dose1 5.176693e-01
time24.dose1 -5.226157e-01
time8.dose10 4.502645e-01
time24.dose10 -4.543508e-01
And some of the contrasts I tested:
for time24.dose10 vs time24.dose0
baseMean log2FoldChange lfcSE stat pvalue padj
7266.687 -1.274503 0.02810044 -45.35527 0 0
for time8.dose10 vs time8.dose0
baseMean log2FoldChange lfcSE stat pvalue padj
7266.687 1.262981 0.02804676 45.03128 0 0?
The thing that puzzles me a lot is that, for the log2FC of the 2nd
contrast, I can't see how it can be ~1.26 given the sizefactor
corrected mean counts for the gene for these two conditions are
11296.41 and 10535.41, respectively. To me, the log2FC should be
negative or close to 0.
Attach is the tmp matrix you saw above. ?I know you can't replicate
all the estimates using the tmp matrix alone without other genes in
the dataset, but that's all I can provide for confidentiality.
?Hopefully, you can figure out where the problem is.
I'm using DESeq2 v1.4.5, R3.1.0, under Win 7.
Thank you very much!
Tao