Microarray Multimapping/Cross-hybridizing probesets

Question

[Nathaniel]

I am analysing an Affymetrix Mouse Gene ST 2.0 array (http://www.affymetrix.com/catalog/131476/AFFY/Mouse+Gene+ST+Arrays)

Looking at the annotation file I have realised that some probes map to multiple genes. Should we remove them for the downstream analysis?

Answer 1

[andrew.j.skelton73]

Typically these are different oligonucleotide capture sequences for the same gene at different positions (or they are for Illumina BeadChips, so I'm assuming it's a similar principle). Basically they're for the same gene at different points. You can map the probe IDs to nuIDs and convert the nuID to a nucleotide sequence, if you blast those sequences it should show the target region on the gene. Short answer, is keep them in the experiment.

edit: I read that too quick - Can you explain what you mean by them mapping to multiple genes? How did you determine that happened?

[svlachavas]

Dear Andrew,

I think Nathaniel's post is similar to the general issue of also continuously evolving and changing annotation platforms and annotating generally microarrays through various annotation packages-- there is an example with affycore tools below (via select, etc):

https://www.bioconductor.org/packages/release/bioc/vignettes/affycoretools/inst/doc/RefactoredAffycoretools.pdf

In detail, it is mentioned also in the paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1283542/

One is the probe set redundancy, which is the case you mention above--but also there is the other case:

"Non-specific probes"

A significant increase of cDNA/EST/genome sequence information leads to the possibility a probe thought to be specific for one gene can actually hybridize to transcripts from additional genes or non-coding transcripts. As shown in Table 1, according to the current version of the UniGene database, for most GeneChips 10 - 30% of probe sets contain at least one non-specific probe. Probe alignment to genomic sequences also reveals that 5 - 16% of probe sets contain a probe(s) with more than one genomic sequence hit(s). The difference between the UniGene- and genome-based criteria may largely be due to UniGene clustering or EST sequencing errors.

Best,
Efstathios

[andrew.j.skelton73]

Very comprehensive, thanks!

[Nathaniel]

Exactly, in the microarray annotation file you can see a single probeset matching multiple gene tags.

To avoid any problem, I decided to remove all such probesets from the analysis, but I definitely not know what is the best way to go.

[andrew.j.skelton73]

Ah, I take it these are "cross hybridising probes" - i.e. the capture sequences map to more than one gene, in that case I'd definitely remove them, as they'd most likely confound your results.

Answer 2

[Nathaniel]

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[svlachavas]

Unfortunately(for some reason I didn't get any updates for the new comments and answers) I believe Nathaniel you have probably moved along with this issue---just to pinpoint my opinion on this matter: there isn't a clear option on this specific issue, but you can move similarly to the above vignette in affycoretools and with the most "naive way" keep the first mapping like:

https://www.bioconductor.org/packages/release/bioc/vignettes/affycoretools/inst/doc/RefactoredAffycoretools.pdf

Alternatively, you could consider the above approach i have mentioned, and download and proceed with customCDF arrays--

Hope that helps,

Efstathios