Re: [DIYbio] Cambrian Genomics

I wonder what folks in the Caruther's lab are doing... seems like you
can only squeeze so much out of this control loop the way it's setup
now, before you simply have to go back to the basics and recajigger
the whole system.

http://chem.colorado.edu/index.php?option=com_content&view=article&id=246&Itemid=185

On Thu, Apr 25, 2013 at 10:14 PM, Anselm Levskaya <levskaya@gmail.com> wrote:
> Where to begin...
>
> The cost of synthesis is a sum of the cost of the instruments (or its
> depreciation over time), the cost of the people running it, the fixed
> overhead costs of the company doing it, the cost of shipping, and lastly the
> cost of the reagents. Among other things. "Cost" is not a simple thing.
>
> The cost of phosphoramidites is really low (at a minimum) given that they're
> produced in china. The container the phosphoramidites come in can be more
> expensive than the contents. CpG isn't that expensive either... I mean
> let's take it to a ridiculous limit-point: an oligo synthesis run costs a
> few hundred dollars in reagents, maybe a thousand. You're working with a
> ~mole of material. Divide that by avogadro's number. 1e3/6e23 = about a
> tenth of an attocent per monomer.
>
> If you had a magic daemon that coud assemble single-molecules, your reagent
> cost for a 100mer would be about 10 attocents. (We don't know how to make
> magic daemons yet, but I suspect that physics does allow for a
> mesoscopically-coupled polymerase that could act as a decently accurate
> 'molecular printer' if you coupled it to some single-molecule sequencing
> feedback.) Why does a bulk reagent limit the ultimate cost? If you're
> efficient it doesn't have to.
>
> Reagents are expensive when you waste them. For traditional gene synthesis,
> the cost of oligos does dominate. Even the lowest-scale bulk synthesis
> reactions make about 1000x more material than is needed for a synthesis
> reaction. Microarray synthesis scales usage way down (though the amount of
> dna on a microarray spot is actually too low to use directly for reliable
> assembly, so some amount of post-processing is required) However,
> microarrays jumble everything together and make the error-correction problem
> much more severe.
>
> With regards to enzymes/reagents don't think that the price you pay for them
> is going to be the price an industrial purchaser is going to pay- most
> endusers of anything in biology are paying large margins. What you're
> paying for as an enduser isn't the stuff in the tube, it's the quality
> control that guarantees the stuff in the tube will do what you hope it will,
> as well as the convenience of that little tube shipped to you the moment you
> need it.
>
> Don't get fixated on a single aspect of the cost structure. Ingots of
> ultrapure silicon aren't cheap, but their cost is only distantly related to
> the price of a cpu. If you have a relatively fixed amount of cost for
> people and machines and reagents, the only way you get improved cost per
> unit is by making a shitload more units by using more efficient processes.
> That's the idea.
>
>>Using lasers seems cool but how much time does it save over taking a few
>> minutes to run something over an HPLC column?
> Have you ever run an HPLC column? How do you propose to do it for a million
> different sequences? You can't just run the whole batch: 1) there's not
> anywhere near enough material, and 2) HPLC retention times are highly
> sequence-dependent, not just length dependent, so it's not a batch
> purification strategy.
>
> The paper you reference used a poor sequencing strategy that made exactly
> the same kinds of base-calling errors as the synthesis errors is was
> supposed to detect. If you don't match your filter to your noise source,
> you're not going to get good rejection ratios.
>
> Quick reference for error sources in DNA:
> chemical synthesis errors : ~1 in 100
> thermal depurination errors : 1 in 10^5 (or worse, depending on time spent
> above 60C)
> polymerase copy errors: 1 in 10^6 (for modern, phusion-like enzymes)
> in-vivo bacterial copy errors: 1 in 10^8-10^9 per replication.
>
> Even if you magic away all the synthesis errors, you still have to worry
> about thermal depurination and large assembly errors. It's all about
> building quality control into the whole system. And that is why cheap
> sequencing is a big deal: it was a necessary precedent for cheap synthesis.
> Of course, the devil is in the details of how you leverage it.
>
> -A
>
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--
-Nathan

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