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could you explain me how to interpret such situation?

Same tag aligned in several distint places, near different genes... Is the annotation able to distuinguish such 'same' tags, during DGE analysis? Why tags arent unique?

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Is the annotation able to distinguish such 'same' tags, during DGE analysis?

No, you'll end up discarding such probes (assuming you have a reason to actually use microarrays still).

Why aren't tags unique?

Platforms such as HG-U133 date back to 2002 (meaning they were designed a few years before that), when the human reference genome (and the reference transcriptome) were not nearly as "finished" as they are today. Probes that may have made sense given what we knew almost 20 years ago may no longer makes sense now given what we know. Further, microarrays are rather conservative, in that they tend to primarily gain probes over time and not have them removed (lest companies lose customers who are relying on given probes).

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  • $\begingroup$ Thanks, for the answer! Ok, so I have the normalized expression signal from this tag. How can I distuinguish to what gene is this signal corresponds to? For example (very abstract) I have some ambiguous tag which is located in three different genes. So the signal is cumulative value of thesee 3? As a result I am not able to define the expression of these genes separately? $\endgroup$
    – Adamm
    Mar 21, 2019 at 12:35
  • $\begingroup$ As I said, you'll end up excluding this probe most of the time. In modern arrays genes will have multiple probes associated with them (sometimes multiple per transcript). If you really wanted to incorporate this probe, you'd distribute it in some sort of weighted fashion to all genes/transcripts for which it's informative (in a similar to what salmon/kallisto/rsem do for RNA-seq reads). Depending on how elaborate you wanted to get that could be a lot of work, so typically you'd just exclude such probes instead. $\endgroup$
    – Devon Ryan
    Mar 21, 2019 at 12:39
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I always assumed that these tags, such as 239963_at you show, mean that the sequence is not specific of a genome region. This means it cannot be assigned to any gene for DGE.

It could be due to changes on the reference genome or due to a faulty design of the tag sequence. However once in the microarray template they cannot be changed without introducing more problems, that's why doing RNA-seq prevent this.

Sorry I don't have any reference for this (but if someone wants to add this edit the answer).

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