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Issues of EST in whole genome gene discovery

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Issues of EST in whole genome gene discovery

Postby cyberpostdoc » Mon Jun 27, 2005 4:24 am

EST is short for expressed sequence tags, where partially sequenced cDNA fragments are used to represent gene expression. It has the power of gene discovery on a genomic level, However, there are limitations of this technology. Of course the advantage of EST is that it provides direct evidence of the expression of a gene. But there are several drawbacks I could think of:

1. mRNA abundance limitation: it is estimated that genes have an average express ion level below 5% of the mRNA population is less likely to be detected by EST technology. Therefore, genes that are expressed at extreme low level won't be able to be detected by EST, so the 27K in this sense should be an lower estimate (if we only focus on this matter). Now, one of the other technique is more sensitive, it is called SAGE, for serial analysis of gene expression, where tags at 3' end of genes are collected and sequenced to represent mRNA expression.

2. development stage limitation: some of the genes in our genome may only express at certain development stage, therefore, if EST libraries cannot cover this development stage, both for technical and ethical reasons, there is no way these gene can be detected.

3. diseased stage limitation: on the same line of #2, but from an opposite point of view, some mRNA might be a result of diseased stage, not neccissary be a normally expressed gene. Or aberrent expression that are not constitute normal behavior of human genome, hence, enriched EST libraries from cancer cell lines (for example) will likely to result in over estimation of human transcriptome.

4. sequencing error: this could post extreme difficulties for bioinformatics to
further map these ESTs back to genome to identify genes. Especially from paralog genes.

Finally, besides gene discovery, EST can be used to study post transcriptional control on a genomic level, which have been shown very successful, including studies in alternative splicing and alternative polyadenylation. Pioneer of these include Michael Zhang at CSHL, Chris Burge at MIT, and Chris Lee at UC.

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Re: Issues of EST in whole genome gene discovery

Postby Gabrielwer » Wed Sep 07, 2011 9:50 am

Except for what the top guy has mentioned, some more limitations are listed below:

1.tissue-specific limition: some genes express only in specific tissue.
So, if EST libraries is not the tissue-specific one, it is impossible for these genes to be detected.

2.cell-type limition:some genes express only in specific cell type. So,the same thing will happen as issuse 1.

3.signal-specific limitation: It is a more strict requirement. Some gene express only induced by specific signal esspecially in neural system.
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Postby JackBean » Wed Sep 07, 2011 11:17 am

I think that's exactly what he wrote :roll:
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Re: Issues of EST in whole genome gene discovery

Postby merv » Sun Oct 02, 2011 7:46 pm

cyberpostdoc wrote:EST is short for expressed sequence tags, where partially sequenced cDNA fragments are used to represent gene expression. It has the power of gene discovery on a genomic level,


i think the term 'transcriptome' is used in place of genome in this case - you are looking at cDNA not genomic, thus not a genomic level.

However, there are limitations of this technology. Of course the advantage of EST is that it provides direct evidence of the expression of a gene.


I understand what you are saying, but most people are only interested in whether you get an active protein or not - you say expression of a gene - but really what matters is the protein - so direct evidence of expression of a gene is not that - also, as an EST represents the cDNA, which could be a spliced variant which is the cells own way of making (potentially) a dominant negative or constitutively active form of the protein.

But there are several drawbacks I could think of:


see previous comment

1. mRNA abundance limitation: it is estimated that genes have an average express ion level below 5% of the mRNA population is less likely to be detected by EST technology. Therefore, genes that are expressed at extreme low level won't be able to be detected by EST, so the 27K in this sense should be an lower estimate (if we only focus on this matter). Now, one of the other technique is more sensitive, it is called SAGE, for serial analysis of gene expression, where tags at 3' end of genes are collected and sequenced to represent mRNA expression.


what is the 27K you are referring to here please?

secondly, in my opinion, these days, it would be more efficient to clone cDNA for the tissue of interest and sequence the lot using high throughput DNA sequencing, unless you want to do it piecemeal employing many people to do what a library-sequence through a high-throughput sequencing lab would achieve. It would also have the advantage of providing a picture as to which spliced variants occur. High throughput normalisation (subtractive hybridisation would eliminate 90% of the known housekeeping as well (or DNA chip screening, but you do miss the advantage of the splice variants).


2. development stage limitation: some of the genes in our genome may only express at certain development stage, therefore, if EST libraries cannot cover this development stage, both for technical and ethical reasons, there is no way these gene can be detected.

regardless of my personal technical abilities or ethics, I don't consider either of these to be limitations- the issue of expression level of hypothesised highly powerful proteins required at vanishingly small quantaties will probably never be solved, I suggest, withoutfuture technologies unless we perhaps evolvie containing future technologies (e.g. genes for breathing fire without a fireguard would cause spontaneous combustion - a dragons argument for Gof.).


3. diseased stage limitation: on the same line of #2, but from an opposite point of view, some mRNA might be a result of diseased stage, not neccissary be a normally expressed gene. Or aberrent expression that are not constitute normal behavior of human genome, hence, enriched EST libraries from cancer cell lines (for example) will likely to result in over estimation of human transcriptome.

if you consider mRNA editing to be a real phenomena, then the human transcriptome (i see you are familair with the word) is actually infinite, in each of us, but see the dragon argument.

4. sequencing error: this could post extreme difficulties for bioinformatics to
further map these ESTs back to genome to identify genes. Especially from paralog genes.

I agree - a real problem with high throughput work without bench confirmation. But protein action is what matters, no protein then there is unlikely to be a gene effect, although it might have evolutionary implications, regardless of EST detection. if the EST is there but the protein is undetectable tthen you either need more cells but if this is unfeasible, then you need would need a more sensitive assay, eg. a gene silencing experiment with an assay that you can detect.

Finally, besides gene discovery, EST can be used to study post transcriptional control on a genomic level, which have been shown very successful, including studies in alternative splicing and alternative polyadenylation. Pioneer of these include Michael Zhang at CSHL, Chris Burge at MIT, and Chris Lee at UC.


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Last edited by JackBean on Mon Oct 17, 2011 8:40 am, edited 2 times in total.
Reason: Fixed quote fail, please be careful in the future
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Re: Issues of EST in whole genome gene discovery

Postby JackBean » Mon Oct 17, 2011 8:40 am

merv wrote:
However, there are limitations of this technology. Of course the advantage of EST is that it provides direct evidence of the expression of a gene.


I understand what you are saying, but most people are only interested in whether you get an active protein or not - you say expression of a gene - but really what matters is the protein - so direct evidence of expression of a gene is not that - also, as an EST represents the cDNA, which could be a spliced variant which is the cells own way of making (potentially) a dominant negative or constitutively active form of the protein.

Not true. Many people are looking on the transcriptome level, that's why we have transcriptomics. Of course, knowing exact level (or even activities!) of proteins would be better, but it's also much harder to determine ;)

merv wrote:
secondly, in my opinion, these days, it would be more efficient to clone cDNA for the tissue of interest and sequence the lot using high throughput DNA sequencing, unless you want to do it piecemeal employing many people to do what a library-sequence through a high-throughput sequencing lab would achieve. It would also have the advantage of providing a picture as to which spliced variants occur. High throughput normalisation (subtractive hybridisation would eliminate 90% of the known housekeeping as well (or DNA chip screening, but you do miss the advantage of the splice variants).


2. development stage limitation: some of the genes in our genome may only express at certain development stage, therefore, if EST libraries cannot cover this development stage, both for technical and ethical reasons, there is no way these gene can be detected.

Just for curiosity, what ethical reasons are you refering to?
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Re: Issues of EST in whole genome gene discovery

Postby merv » Tue Oct 18, 2011 10:37 pm

JackBean wrote:
merv wrote:
However, there are limitations of this technology. Of course the advantage of EST is that it provides direct evidence of the expression of a gene.


I understand what you are saying, but most people are only interested in whether you get an active protein or not - you say expression of a gene - but really what matters is the protein - so direct evidence of expression of a gene is not that - also, as an EST represents the cDNA, which could be a spliced variant which is the cells own way of making (potentially) a dominant negative or constitutively active form of the protein.

Not true. Many people are looking on the transcriptome level, that's why we have transcriptomics. Of course, knowing exact level (or even activities!) of proteins would be better, but it's also much harder to determine ;)


'many' is a vague term, and there are many people and many discoveries and many that lead no-where and some of the many for good reason! ; and just because one can think up a new word like transcriptomics doesnt mean one is clever, with all due respect. What matters to me, and many biochemists, is the evidence of function, which you well know cannot occur without protein (with the rare exception of ribozymes). Determining this may be harder, but to use an analogy, just because you have a car manual doesnt mean you have a car, and yes I add then that if you can only see the bookshelf then determining if someone has a car is harder to determine in the molecular biology world than if they have a manual on the bookshelf!! You don't need to tell me that the transcriptome is useful information, but one needs sufficient evidence to present a functional case.

JackBean wrote:
merv wrote:
secondly, in my opinion, these days, it would be more efficient to clone cDNA for the tissue of interest and sequence the lot using high throughput DNA sequencing, unless you want to do it piecemeal employing many people to do what a library-sequence through a high-throughput sequencing lab would achieve. It would also have the advantage of providing a picture as to which spliced variants occur. High throughput normalisation (subtractive hybridisation would eliminate 90% of the known housekeeping as well (or DNA chip screening, but you do miss the advantage of the splice variants).


2. development stage limitation: some of the genes in our genome may only express at certain development stage, therefore, if EST libraries cannot cover this development stage, both for technical and ethical reasons, there is no way these gene can be detected.


Just for curiosity, what ethical reasons are you refering to?


Well, with all due respect, what are your ethics? If you have none, there is no point in my trying to tell you what these are. Most countries have ethical guidelines, I can't see how you can have overlooked this.
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Re: Issues of EST in whole genome gene discovery

Postby JackBean » Tue Oct 25, 2011 3:17 pm

merv wrote:'many' is a vague term, and there are many people and many discoveries and many that lead no-where and some of the many for good reason! ; and just because one can think up a new word like transcriptomics doesnt mean one is clever, with all due respect. What matters to me, and many biochemists, is the evidence of function, which you well know cannot occur without protein (with the rare exception of ribozymes). Determining this may be harder, but to use an analogy, just because you have a car manual doesnt mean you have a car, and yes I add then that if you can only see the bookshelf then determining if someone has a car is harder to determine in the molecular biology world than if they have a manual on the bookshelf!! You don't need to tell me that the transcriptome is useful information, but one needs sufficient evidence to present a functional case.

Transcriptomics has several advantages over work with proteins. It is easier and more reliable than proteomics and it shows which which paralogues are specifically expressed in contrary to activity measurement. Even detection of proteins by means of proteomics doesn't have to tell you the amount of the active proteins.
I'm not saying that doing proteomics is bad or useless thing, just explaining, why is it easier and sometimes better to do transcriptomics.

merv wrote:Well, with all due respect, what are your ethics? If you have none, there is no point in my trying to tell you what these are. Most countries have ethical guidelines, I can't see how you can have overlooked this.

I asked you to which ethical reasons are you referring to with respect to transcriptomics. But I see, you rpefer to answer with a question and not to provide real answer.
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