[BioC] edgeR: mixing technical replicates from Illumina HiSeq and MiSeq
rct at thompsonclan.org
Fri Aug 29 10:42:50 CEST 2014
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.
On 8/28/14, 9:23 AM, Nick N wrote:
> 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:
> 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!
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