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Old 03-16-2014, 10:07 AM   #12
Brian Bushnell
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Location: Walnut Creek, CA

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Quote:
Originally Posted by rskr View Post
Well, never do genome mapping because you'll spend more time studying pseudo genes. Now, what are you going to do?
Those are not too hard to identify, as they lack introns and typically have a lot of SNPs with regards to genes. Anyway, pseudogenes also interfere with DNA mapping (in human, for example, many are not in HG19); should DNA mapping be done to the transcriptome as well, to avoid interference?

Quote:
Anyway it doesn't make sense to me that you would get isoforms via genome mapping that you wouldn't get via transcriptome mapping
Often the genome is fairly good, but transcriptomes of complex organisms are probably all incomplete. You can't expect a complete transcriptome from organisms with many life stages or tissue types when some isoforms and genes may only be expressed at certain times.

Quote:
furthermore why would you be looking for different isoforms when you are quantifying relative expression? Is this one of the things where you are just answering the question you want to answer?
Some isoforms are tissue- or condition-specific, and if a gene changes from 99% isoform A to 99% isoform B, that could be very important. Assuming that all the isoforms of a gene are functionally identical would mean there is no reason for alternative splicing to even exist.

Mapping to a transcriptome, you'll be somewhat limited to answering questions that have already been answered, or at least asked. It's like searching for minerals only using a map of known mineral deposits; you'll never discover anything truly novel.

Also, mapping to a genome is more objective and repeatable. Mapping to a transcriptome is very subjective, as there are a huge number of ways to design one. Add a single gene, or a single transcript, and the mappings of all reads may be affected. So, how do you choose which transcripts and isoforms to include? All of them? Just the longest for each gene? Just a full concatenation of all exons per gene? Just the ones that were known prior to date XYZ, or also the two new ones your lab found that you think are relevant? You'll get different results based on this purely subjective decision, possibly allowing results to be tweaked as desired.

Last edited by Brian Bushnell; 03-16-2014 at 10:11 AM.
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