On Thu, Jan 23, 2014 at 6:48 PM, Gordon K Smyth <smyth@wehi.edu.au> wrote:

> My worry is that with this contrast, I'm effectively just testing two
>> groups against each other, and by having 4 groups in the design I will be
>> estimating dispersions that are not appropriate for the test that I'm
>> doing, and hence I will overstate my confidence.
>>
>
> The dispersions remain unchanged regardless of the contrast you test. The
> dispersions have been estimated after removing all differences between the
> four groups, i.e., without bias.
>

But had he more simply coded the samples as belonging to two groups, AB and
CD, then the dispersions could be larger, and the AB vs. CD mean
differences could be less significant than in his four-subgroup design.  I
think that was the intent of Ryan's question.  Is it fair to stratify along
a priori expected subgroupings to minimize variance and then ask
group-level questions?  As you later say, if there is "lots of DE" between
A vs. B and/or C vs. D (read: larger group-wise variance) then the test may
need to be "carefully interpreted".

Thanks for your insight,
-Aaron

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