Hi,


My experiment consist of control and diseased samples; looking like this:

colData<-DataFrame(condition=factor(c("A","A","B","B","C","C","D","D","E","E","F","F","H","H","I","I")),
type=factor(c("plasma","plasma","plasma","plasma","plasma","plasma","plasma","plasma","tissue","tissue","tissue","tissue","tissue","tissue","tissue","tissue")),
diseased=factor(c("no","no","no","no","yes","yes","yes","yes","no","no","no","no","yes","yes","yes","yes")))

To search for differential expression I compare
AvsB, AvsC, BvsD and BvsD
then
EvsF, EvsH, FvsI and HvsI,

So I use the results(dds,contrast=c("condition","H","I")) option in DESeq2.

And then, these are the results I do not get;
look at this example:

geneid comparison log2foldchange padj countA1 countA2 countC1 countC2
(counts are the normalized values)
xxx AC 2.40588491829641 0.281844992240883 0 0 0 0
yyy AB 3.98976903585264 0.0455960023534269 0 0 0 0
or .. a log2fold change has been found between A and C of 2.4 and almost 4
in AvsB, while all of these samples are.

I noticed that in all cases where the counts and the normalized counts of
the two conditions that I tested were all zero I do get a fold change, even
up to (2log) 5. Often I see a padj of "NA". So I was satisfied with that,
as pseudocounts could result in some kind of fold change. Leaving those
with padj "NA" out would be good then.
But as the example above, I sometimes get a padj, which is even significant
in the AB example above. This propably has something to do with all the
other values (A1, A2, B1, B2,....,I1,I2);
yyy  0    0    0    0    0    0    0    11    4897697    4167458
4337956    4139125    3731919    9711412    4043504    3796036  (i.e.
massively present in tissue samples but not in blood, because of the
discrepancies between blood and tissue I am not comparing them directly)

I would like to understand why there is a significance here between A and
B??
Or should I 'just' split my data in tissue and plasma?? But then still. I
will find the AC example above, as B and D have counts in that example,
however small numbers (E-I are also small numbers)


Hanneke





> sessionInfo()
R version 3.0.2 (2013-09-25)
Platform: x86_64-pc-linux-gnu (64-bit)

locale:
 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C
 [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8
 [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8
 [7] LC_PAPER=en_US.UTF-8       LC_NAME=C
 [9] LC_ADDRESS=C               LC_TELEPHONE=C
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C

attached base packages:
[1] parallel  stats     graphics  grDevices utils     datasets  methods
[8] base

other attached packages:
[1] DESeq2_1.2.2            RcppArmadillo_0.3.910.0 Rcpp_0.10.4
[4] GenomicRanges_1.14.3    XVector_0.2.0           IRanges_1.20.4
[7] BiocGenerics_0.8.0

loaded via a namespace (and not attached):
 [1] annotate_1.40.0      AnnotationDbi_1.24.0 Biobase_2.22.0
 [4] DBI_0.2-7            genefilter_1.44.0    grid_3.0.2
 [7] lattice_0.20-23      locfit_1.5-9.1       RColorBrewer_1.0-5
[10] RSQLite_0.11.4       splines_3.0.2        stats4_3.0.2
[13] survival_2.37-4      XML_3.98-1.1         xtable_1.7-1

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