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Question: voomWithQualityWeights: question on min. number of replicates per group + design to use
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gravatar for Guido Hooiveld
18 days ago by
Guido Hooiveld2.2k
Wageningen University, Wageningen, the Netherlands
Guido Hooiveld2.2k wrote:

I have an RNA-seq data set (8 treatments, 2 replicates) that I would like to process by voom transformation for analysis in limma. I also would like to include sample-specific quality weights, so I know using the function voomWithQualityWeights() is the way to go.

However, since I have only 2 replicates per group/treatment, I wonder whether it would be best to provide as argument for design  the actual design matrix of the experiment, or rather (still) the default (intercept only) design matrix.

 

The reason I am asking is based on this statement in the limma user guide (for the related function arrayWeights()) [section 14.4 "When to Use Array Weights", page 68]:

For example, in a two-group comparison with just 2 replicates in each group, the array weights should be estimated with the default (intercept) design matrix, otherwise each array is compared only to its partner rather than to the other 3 arrays.

 

Does this also apply for other designs with only 2 replicates per group? I noticed that when providing the design matrix of the experiment the sample-specific weights for each duplo sample are identical, whereas when providing the intercept design matrix the sample-weights are (indeed) different for all samples.

 

What would would be the best approach? Any insights on this would be appreciated!

 

> design
   Grp1 Grp2 Grp3 Grp4 Grp5 Grp6 Grp7 Grp8
1     0    0    0    0    1    0    0    0
2     0    0    0    0    1    0    0    0
3     0    0    0    0    0    0    1    0
4     0    0    0    0    0    0    1    0
5     0    0    0    0    0    0    0    1
6     0    0    0    0    0    0    0    1
7     0    1    0    0    0    0    0    0
8     0    1    0    0    0    0    0    0
9     0    0    0    1    0    0    0    0
10    0    0    0    1    0    0    0    0
11    1    0    0    0    0    0    0    0
12    1    0    0    0    0    0    0    0
13    0    0    1    0    0    0    0    0
14    0    0    1    0    0    0    0    0
15    0    0    0    0    0    1    0    0
16    0    0    0    0    0    1    0    0
attr(,"assign")
[1] 1 1 1 1 1 1 1 1
attr(,"contrasts")
attr(,"contrasts")$Treatment
[1] "contr.treatment"

>

 

 

ADD COMMENTlink modified 16 days ago by Gordon Smyth32k • written 18 days ago by Guido Hooiveld2.2k
3
gravatar for Matthew Ritchie
17 days ago by
Australia
Matthew Ritchie690 wrote:

Yes, this advice also applies when using voomWithQualityWeights on RNA-seq data.

Using the actual design matrix will work best if you have 3 or more replicates per group with this model.

If you have fewer than this, an intercept model is the best you can do.

ADD COMMENTlink written 17 days ago by Matthew Ritchie690
1
gravatar for Gordon Smyth
16 days ago by
Gordon Smyth32k
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
Gordon Smyth32k wrote:

Just to refine Matt's answer: It isn't wrong to apply voomWithQualityWeights() or arrayWeights() with just n=2 in each group, but the result will be to apply a weight to each group rather than to each individual sample. It's easy to understand why it has to be like this -- when you have just two samples in a group, it isn't possible to tell which of them is the outlier (if any)!

ADD COMMENTlink written 16 days ago by Gordon Smyth32k
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