[BioC] RNAseq expression analysis using DESeq: technical replicates, paired samples
Michael Muratet
mmuratet at hudsonalpha.org
Mon Nov 7 23:44:49 CET 2011
On Nov 7, 2011, at 4:11 PM, Simon Anders wrote:
> Dear Michael
>
> On 2011-11-07 22:56, Michael Muratet wrote:
>> I would like to verify that 18 months later, adding counts for
>> technical
>> replicates is still the best approach for combining technical
>> replicates.
>>
>> We are constrained to single biological replicates, but we're
>> interested
>> in using the technical replicates if we can. I recall that limma had
>> ways to set up the model matrix.
>
> Your question sounds as if you consider the issue as a technical
> limitation of the available methods. Unfortunately, it is not, and I
> am afraid, 18 months are not enough time for fundamental principles
> of statistics to change.
Hi Simon
>
> If you want to know whether a difference between control and
> treatment may be attributed to the treatment, you need to verify
> that this difference is large compared to what you expect from
> random biological variation, and without biological replicates you
> cannot know the extent of biological variation.
No, I really was just checking for updates in how to arrange the
input. I understand the limitations of no biological replicates.
However, as noted in section 6 of the vignette, '...such experiments
are often undertaken...' and we hope to confirm what we find by other
means.
I thought I had seen documentation somewhere on how to incorporate
technical replicates into deseq, but I could not put my hands back on
it, so I thought I'd ask. I have actually processed the original data
once and got what results we could get. I don't expect the tech
replicates to change the answer.
Cheers
Mike
>>
> Of course, you can perform the analysis on the level of technical
> replicates, as you can with microarrays in limma. Then, you will get
> lots of results, but any call of significant differences may only be
> interpreted to mean that your two biological samples differ in this
> gene. You may not infer that the difference is more likely to be the
> result of your treatment than just due to chance differences between
> the samples.
>
> Best regards
> Simon
Michael Muratet, Ph.D.
Senior Scientist
HudsonAlpha Institute for Biotechnology
mmuratet at hudsonalpha.org
(256) 327-0473 (p)
(256) 327-0966 (f)
Room 4005
601 Genome Way
Huntsville, Alabama 35806
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