Illumina BeadChip Replicate Probes

Question

[andrew.j.skelton73]

Hi,

I'm dealing with Illumina HT12 v4 BeadChip data, I understand that there are replicate probes on the arrays, but these replicate probes have a different nuID and hence a different probe sequence attached to the bead. Has anyone seen any literature or knows off the top of their head, if these replicate probes have different sequences for different transcript variants or just a different binding site of the parent transcript?

Ultimately I'm trying to gauge what the effect is if a given gene X has one probe that's differentially expressed and has 2 others that are not differentially expressed. Biologically and statistically.

Any input is appreciated!

Thanks,

Answer 1

[Ying W]

I think you are getting some things confused here. On Illumina arrays, most genes only have one probe associated with it. There are multiple replicates of a single probe (will share the same probe sequence). Occasionally, you will get two probes per gene (is this what you mean by replicate?). With Affy probe designs, you will get multiple probes per gene (probeset) but each probe might only be represented once (or very few times). Illumina's probe design (one probe, multiple copies) has been shown to be more accurate. If you want to learn more about this topic, I would have a look at the beadarray and lumi bioconductor manuals. I believe lumi was the one that introduced the concept of nuIDs but I have personally never found a use for them.

For your question, you will need to subset down to the few genes with multiple probes for that gene, typically if a gene has more than one probe it will be due to things like known alternative exon usage and alternative start sites. You can probably find all the info you need in this annotation set: http://www.bioconductor.org/packages/release/data/annotation/html/illuminaHumanv4.db.html

[andrew.j.skelton73]

Thanks for the response! Yeah, I think I got a little bit confused. Thanks for the suggestion on reading the beadArray package information (I've already read the lumi package information), it provided some key bits on information I was missing. So essentially, (specific to Illumina bead arrays), for each nuID (unique probe sequence), there are many replicate beads. In most cases there is one probe per gene, but in some cases there are more than one probe.

I guess this brings me on to a followup question.... If there are more than one probe for a given gene, is this to fill space on the array? Target potential transcript variants? Or are transcript variants not really accounted for because it's a gene level assay? I looked at a couple of genes in my data with more than one probe and they appeared to target the same exon but in slightly different locations.

[Ying W]

I think the original intention was to look for alternative splicing but I am not sure. Also some of the probes may be less specific, the annotation resource I linked to also has some probe quality metrics.