Entering edit mode
Dear Nick,
If you go back to the post from 2010 that you give the URL for, you
will
see that I was giving very briefly the same advice about checking
Poisson
variability that Ryan has explained at greater detail.
You don't give any information about read lengths, sequence depths or
alignment methods. I would be surprised if MiSeq and HiSeq would
generate
perfect Poisson replicates of one another, especially if the read
lengths
from the two platform are different or the alignment and counting
software
has been varied. So you may well end up back at the blocking idea.
Best wishes
Gordon
---------------------------------------------
Professor Gordon K Smyth,
Bioinformatics Division,
Walter and Eliza Hall Institute of Medical Research,
1G Royal Parade, Parkville, Vic 3052, Australia.
http://www.statsci.org/smyth
On Sun, 31 Aug 2014, Ryan wrote:
> Thanks to the underlying theory behind dispersion estimation, you
can
> easily test whether your "technical replicates" really do represent
> technical replicates. Specifically, read counts in technical
replicates
> should follow a Poisson distribution, which is a special case of the
> negative binomial with zero dispersion. So, simply fit a model using
edgeR
> or DESeq2 with a separate coefficient for each group of technical
> replicates. Thus all the experimental variation will be absorbed
into the
> model coefficients and the only thing left will be the technical
> variability of of the replicates. For true technical replicates, the
> dispersion should be zero for all genes. So if you estimate
dispersions
> using this model, and plotBCV/plotDispEsts shows the dispersion very
near
> to zero, then you can be confident that you really have technical
> replicates. If the dispersion is nonzero, then there is some
additional
> source of unaccounted-for variation.
>
> I have used this method on a pilot dataset with several technical
> replicates for each condition. edgeR said the dispersion was
something like
> 10^-3 or less for all genes except for the very low-expressed genes.
>
> -Ryan
>
> On 8/28/14, 9:23 AM, Nick N wrote:
>> Hi,
>>
>> I have a study where a fraction of the samples have been replicated
on 2
>> Illumina platforms (HiSeq and Miseq). These are technical
replicates - the
>> library preparation is the same using the same biological
replicates -
>> it's only the sequencing which is different.
>>
>> My hunch was that I shall introduce the platform as as an
additional
>> (blocking) factor in the analysis. Than I stumbled upon this post:
>>
>> https://stat.ethz.ch/pipermail/bioconductor/2010-April/033099.html
>>
>> It recommends pooling the replicates. The post seems to apply to a
>> different case ("pure" technical replicates, i.e. no differences in
the
>> sequencing platform used) so I probably shall ignore it. But I
still feel
>> a bit uncertain of the best way to treat the technical replicates.
Can
>> you, please, advise me on this?
>>
>> many thanks!
>> Nick
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