[BioC] Limma analysis of focused arrays vs. whole genome arrays
smyth at wehi.edu.au
Wed Jun 8 12:23:24 CEST 2005
>Date: Tue, 7 Jun 2005 09:33:51 -0400
>From: Mike Schaffer <mschaff at bu.edu>
>Subject: [BioC] Limma analysis of focused arrays vs. whole genome
>To: bioconductor at stat.math.ethz.ch
>The lab I work with has used "whole genome" human arrays (~18,000
>genes) for a couple years and I have helped with the analysis using
>Limma. Now, due to costs, they are now considering switching from
>whole genome arrays to focused arrays with ~400 genes of interest
>(selected from the whole-genome array results).
>The obvious analysis problems with a focused array where most genes are
>1. LOESS normalization assumes most genes are not changing. If most of
>the genes are expected to change, there is no basis to recenter the
>data around zero. The response from the lab was that they would be
>willing to include 100-150 genes that are not expected to change.
>2. The B-statistic in Limma requires a parameter indicating a certain
>fraction of genes are changing. The corresponding moderated
>t-statistic uses the data from all genes to moderate the standard error
>in the t calculation. Both of these could change dramatically if most
>of the genes on the array are changing.
>My questions are:
>1. Are my concerns valid and are there ways around around them? Are
>there other analysis pitfalls with this scenario?
>2. Can Limma handle situations where most of an array is expected to
>change? What modifications, if any, need to be made to the Limma
>analysis to account for this?
To quote from the Limma User's Guide (page 15):
"In such a situation, the best strategy is to include on the arrays a
series of non-differentially
expressed control spots, such as a titration series of whole-library-pool
spots, and to use the
up-weighting method discussed below. In the absence of the such control
of boutique arrays requires specialist advice."
>3. Alternatively, is there a more appropriate statistical package to
>use in this case?
I don't know of any other available methods. In my opinion, you have to put
down control spots, "house-keeping" genes if that is all you can get, but
preferably constructed spots as described above.
More information about the Bioconductor