On Thu, Nov 20, 2008 at 3:48 PM, Cei Abreu-Goodger <cei@ebi.ac.uk> wrote:

> Hi Nick, and others,
>
> Apologies for not making my question more clear, but I guess there have
> been some interesting answers anyway. I was in fact thinking of expression
> arrays. And my main interest was from the standpoint of probe annotation.
>
> It now does seem pretty clear that there are many regions in the genome
> that encode transcripts on both strands. If a probe is designed to such a
> region, the expression microarrays will be measuring both transcripts, and
> you will essentially have a "perfectly" cross-hybridizing probe.
>

Not really.  It depends on the protocol being used.  For illumina, you will
end up with a product that goes on the array that is strand-specific.  That
is not true of all array platforms.


>
> Now, annotation-wise, what should we do? Ignore such probes? At least flag
> them up? The problem is, many bioconductor annotation packages only allow a
> single gene to be assigned to each probe. So, in many cases you many be led
> to believe that your experiment has measured differential expression for a
> particular gene (with its set of GO terms, KEGG pathways, etc) when in fact
> the changing gene was the one on the other strand.


I don't think this comes up very often, but it is always possible that for
any given gene there is another explanation for differential expression as
observed.  That is why for a given gene, it is important to validate using a
different technology.  Globally (as in sets of genes), it hopefully won't be
too much a factor.


>
>
> These "problems" tend to show up on the list occasionally, for example when
> people find out that different databases (Ensembl/Biomart, NCBI, the
> manufacturer or a bioC annotation package) lists different genes for the
> same probe. Obviously not all, but many of these differences have been due
> to overlapping transcripts. In fact, Ensembl recently patched their probe
> mapping pipeline to be "strand-aware". If you think that this would affect a
> tiny portion of probes, think again: the Affymetrix probes affected on the
> human and mouse genomes was around 10%:
>
> http://osdir.com/ml/science.biology.ensembl.devel/2008-06/msg00052.html
>
> Also, from talking to some of the NuID/Illumina mapping people it seems
> that they simply don't consider the strand of the probe. But they do
> calculate a "uniqueness" score to avoid probes that map to multiple genes.
>
> In the end, I would ideally prefer "cross-hybridizing" probes (of whatever
> sort) to be annotated in a way that they could be identified. But I have no
> idea of how much a nightmare that would be for the developers of the current
> annotation packages...
>

There is no attempt to map probes in bioconductor annotation packages (at
least those maintained by the core).  The annotation from which the
annotation packages are derived come directly from the manufacturers,
generally.  Herve Pages just posted some code to the list that will allow
you to align your own probes to the genome or, more probably, to a
transcript database of your choice.  Then, you can use your own definitions
for probes.  I used to do this on a large scale for all arrays that we used,
but I have backed away because the answers that one gets are very similar
for the vast majority of probes.

Sean



>
> Nick Henriquez wrote:
>
>> Dear Cei, Steve,
>>
>> There are two versions of the correct answer depending on whether we are
>> talking about an expression or CGH/SNP type array;
>>
>> If we are using an EXPRESSION array
>>
>> 1) It does not matter on which strand the gene resides.
>> 2) It a not matter of bad probe design. It is either a negative control or
>> a
>> misnomer derived from genome annotation.
>>
>> For ANY probe to hybridise it has to be the RC of cDNA and therefore the
>> DNA
>> homologue of the original RNA sequence. (I'll let you work that one out
>> for
>> yourself).
>>
>> If the probe WAS encoded on "the opposite strand" your labelled target
>> would
>> not hybridise as it would be the reverse complement of the actual
>> sequence.
>> The annotation "opposite strand" stems from the convention that we call
>> one
>> strand the "coding strand" and the other strand the non-coding or
>> "opposite"
>> strand. By definition then a gene cannot be encoded by the "opposite"
>> strand.
>> However, what often happens when sequencing genomes is that we find
>> several
>> genes encoded on one strand (which we will then call the coding strand)
>> and
>> then somewhat later also one or more genes on the "opposite" strand. This
>> annotation is (wrongly in my opinion) retained when genomes are assembled
>> and thus part of the annotation of the probes.
>>
>> So an opposite strand probe is at best a kind of negative control, at
>> worst
>> a misnomer annotation retained when the genome was assembled. Mostly we
>> now
>> try to use terms like + and - but even that has the drawback that we
>> generally associate + with coding and - with noncoding. As we all know
>> BOTH
>> strand encode functional RNAs of various kinds including those coding for
>> proteins.....
>>
>> If we are talking about DNA targets, e.g. a SNP array
>>
>> 1) It does not matter on which strand a gene resides, any overlap is a
>> matter of coincidence- "genes" are rare events on the genome.
>> 2) It is not a matter of bad probe design. Usually it simply does not
>> matter
>> and this is a sequence that was used historically without knowledge of the
>> gene (often discovered later). Sometimes the sequence on the coding strand
>> may have a problem with background or sequence similarity. To get around
>> this one can try to use the RC (i.e. "opposite strand" sequence) which is
>> often different enough. Of course if more than 2 similar sequences exist
>> the
>> problem remains as we can use this trick only once.
>>
>> Hope this helps,
>>
>> Nick
>>
>> N.V. Henriquez, Senior Research Associate
>> Dept. Of Neurodegenerative Diseases
>> Institute of Neurology, UCL, Queen Square House rm 124
>> Queen Square
>> London WC1N 3BG
>>
>>
>>
>>
>> Message: 8
>> Date: Wed, 19 Nov 2008 10:45:52 -0500
>> From: Steve Lianoglou <mailinglist.honeypot@gmail.com>
>> Subject: Re: [BioC] Does the strand of a microarray probe matter?
>> To: Cei Abreu-Goodger <cei@ebi.ac.uk>
>> Cc: Bioconductor Newsgroup <bioconductor@stat.math.ethz.ch>
>> Message-ID: <7710F044-03D5-4572-8EE4-2DB96F4C348C@gmail.com>
>> Content-Type: text/plain; charset=US-ASCII; format=flowed; delsp=yes
>>
>> Hi Cei,
>>
>> On Nov 19, 2008, at 3:51 AM, Cei Abreu-Goodger wrote:
>>
>>  Hello all,
>>>
>>> Related issues have arisen before, where the probe of a particular  array
>>> platform was annotated to a gene on the opposite strand. But I  was just
>>> asked if this even matters, or should it simply be  considered a case of bad
>>> probe design.
>>>
>>> Does the protocol for different manufacturer's arrays always produce
>>>  amplified product of both strands for the transcript to be measured?  I
>>> could imagine that protocols that amplify based on poly-A tails  would tend
>>> to produce an anti-sense biased amplification product  (older Affy arrays?),
>>> whereas those based on random priming could  produce products of both
>>> strands (and so the actual strand that is  on the array becomes
>>> meaningless).
>>>
>>> Does someone know what is the case in particular for Illumina
>>>  Beadarrays?
>>>
>>
>>
>> I've never worked on the bench-side of a microarray experiment, but  for
>> gene expression arrays I was under the impression that most  protocols:
>>
>> (i) extract the the RNA from cell lysate using their poly-A tails as
>>  targets
>> (ii) reverse transcribe to cDNA and amplify the cDNA w/ random primers.
>> (iii) hybridize amplified cDNA to the array
>>
>> If that's the case, I don't think that the strand of the probe should  be
>> an issue.
>>
>> I'd be interested, of course, to hear other people's thoughts on this,
>>  too (while this info should be easily available from the  manufacturer's
>> site, or the Methods section of many papers, let's see  if the lazy-web can
>> help :-).
>>
>> -steve
>>
>> --
>> Steve Lianoglou
>> Graduate Student: Physiology, Biophysics and Systems Biology
>> Weill Medical College of Cornell University
>>
>> http://cbio.mskcc.org/~lianos <http://cbio.mskcc.org/%7Elianos>
>>
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>
>
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