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Offlinechibiabos
Cosmic Pond Scum
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Registered: 03/16/17
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Re: FYI: DNA-Based Taxonomic ID Available to Customs and LE [Re: Alan Rockefeller]
    #24723265 - 10/19/17 09:06 PM (6 years, 3 months ago)

Why would anybody skip that step?  Seems like a pretty big fuckup, with respect to quality control.  :shrug:


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OfflineAlan RockefellerM
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Re: FYI: DNA-Based Taxonomic ID Available to Customs and LE [Re: chibiabos]
    #24723274 - 10/19/17 09:09 PM (6 years, 3 months ago)

Quote:

chibiabos said:
Why would anybody skip that step?  Seems like a pretty big fuckup, with respect to quality control.  :shrug:




Because if you are sequencing 96 reactions at a time and doing your own PCR cleanup, it can cost as little as $2 for Sanger sequencing.    For that kind of money, it does not make sense to waste time with electrophoresis because you are only saving $2 per PCR that does not work.  Also sometimes there is enough amplified DNA there to get a good sequence, but not enough to show up on the gel.    A lot of professionals just sequence everything, and if the sequence does not come back clean they run the PCR again.

For many researchers, the time spent with electrophoresis does not justify saving a couple dollars of their bosses money.


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Offlinechibiabos
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Registered: 03/16/17
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Re: FYI: DNA-Based Taxonomic ID Available to Customs and LE [Re: Alan Rockefeller]
    #24723301 - 10/19/17 09:19 PM (6 years, 3 months ago)

Granted, but if you skip that step in the interest of saving money then you still need some way to verify that you were actually studying one sequence instead of several of them.  You can always compare it to other sequences in order to justify a guess that it is some particular region of a genome, but unless the way in which you actually end up solving the sequence of your PCR products allows some way to tell how many, distinct, sequences you are solving (without knowing beforehand) then you're making assumptions about your PCR products which you can't really justify.  That might be fine in industry, but it's not science.


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OfflineAlan RockefellerM
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Re: FYI: DNA-Based Taxonomic ID Available to Customs and LE [Re: chibiabos]
    #24723327 - 10/19/17 09:36 PM (6 years, 3 months ago)

Quote:

chibiabos said:
Granted, but if you skip that step in the interest of saving money then you still need some way to verify that you were actually studying one sequence instead of several of them.




If you have multiple templates being amplified by the PCR reaction, when you get the chromatogram back it is garbage because you have lots of overlapping peaks.  The base caller finds N's all throughout the sequence, it isn't anything like a clean sequence you are supposed to get back.

About 1% of my sequences come back this way, either because there was a fungus eating the mushroom or a pipetting error where I put two PCR products into one tube.


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InvisibleStonehenge
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Registered: 06/20/04
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Re: FYI: DNA-Based Taxonomic ID Available to Customs and LE [Re: chibiabos]
    #24725501 - 10/20/17 06:50 PM (6 years, 3 months ago)

Chibiabos, you seem very knowledgable in many areas. May I ask what kind of work you do?


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