[BioC] Analysis of Affy-data from amplified RNA
ririzarr at jhsph.edu
Wed Jan 16 05:37:19 CET 2008
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Samuel Wuest wrote:
> Dear Rafa,
> Hope you are fine… I have become a big fan of you when I came across the
> analysis of Affy-Chips, so thanks for the nice toolboxes you have
> created so far.
> Although I am not a statistican but a geneticist, I have been chewing
> for quite a while on the problem of analyzing chips from amplified RNA,
> and couldn't come up with a solution for making an accurate
> Present-Absent decision matrix so far…
> I am trying to modify the Affymetrix MAS5 present-absent calls
> algorithm, adjusting for probe-position bias in the data from amplified
> samples. I have tried the normal MAS5 and the half-price method, but
> both of these methods do not cope too well with the amplification bias…
> Up to date, I have the basic ideas and the tools for their
> implementation/checking the new algorithm, but would need some help on
> certain issues (details, if interested, see below). I think that your
> group has the best expertise on the field: do you know anyone that I
> could contact for a small collaboration?
> Thanks for any help on this, best regards.
> Wuest Samuel
> Smurfit Institute of Genetics
> Trinity College Dublin
> Dublin 2, Ireland
> Phone: +353-1-896 2444
> Mobile: + 353-85-735 5821
> Email: wuests at tcd.ie <mailto:wuests at tcd.ie>
> Details: (supporting illustration are available if necessary: quality
> control images, 3' positional bias, sensitivity index density, ROC
> curves of the MAS5 algorithm on my data, etc).
> I am interested in the sexual reproduction of plants, and thus have
> isolated single cells of reproductive importance in Arabidopsis, such as
> the egg cell. The samples needed two round of RNA amplification, and
> there was a slight problem with RNA degradation, so it took me quite a
> while to get data of suitable quality…
> My goal is the creation of a modified MAS5-calls algorithm that takes
> into account both probe sensitivity and amplification bias:
> for this, I want to make use of probe-sensitivity indices modelled
> across multiple chips, all from samples that have been amplified. This
> allows to estimate both, the probe sensitivity and the amplification
> bias at the same time.
> The sensitivity-index can be taken as a measure of how reliable a signal
> from a certain probe actually is.
> So I would change the one-sample Wilcox implemented in the mas5calls
> into a two sample Wilcox, comparing the measured signals against a
> null-distribution (consisting of 11 zero-values, one for each probe).
> For each single probe, an assigned null-value is used for comparison,
> and this null-value is chosen according to the modelled
> probe-sensitivity ( e.g. based on quantile information of the respective
> sensitivity). The new algorithm is assessed using ROC curves that make
> use of the knowledge that is available so far for the different cell
> types (true positives) and of a negative probes (where reannotation has
> led to probes with no targets).
> I would need help on the following points: a) how to create a
> null-distribution from which to choose null-values and b) general
> feedback on the method (e.g. is there any major statistical pitfalls in
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