[pmiguel]

It occurs to me that you probably centrifuge and aspirate the Ethanol. That will not work for the throughputs we are using.

Yes, the CleanSeq at $ 20,000 per liter (appoximately) is far more expensive than the big dye per reaction.

Actually we just do inverted spins to remove the ethanol. However we do use a 6K RPM plate centrifuge for the precipitation spin. The protocol is nothing special. The only changes that were critical were reducing the sodium acetate concentration 1000x (probably no different than adding only ethanol to the precipitation) and using somewhat less than 2 volumes of ethanol. The former aids electrokinetic injection of product and the latter helps with signal droop.

But I admit this may not scale well. To tell you the truth, Sanger sequencing is so obviously trailing edge at this point, I just can't find the motivation to continue to tinker with it. At this point I just "put out fires" but otherwise leave the technique as is.

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Phillip

[pmiguel]

I am using 0.05 microliters big dye per well in 384 well plate.

Ah, up-thread you wrote:

I am using 2 microliter reactions for big dye in a 384 well plate

So I presumed you meant 2ul of Big Dye (1/4th) reactions. But you must have meant 2ul total volume reactions and 1/160th reactions. I don't know what you are paying in CleanSeq, but you say it is more than the 4 cents you pay in Big Dye. I am going to guess that it is at least 6 cents. So you still have polymer, capillary and service contract costs. Even with preferential government pricing on your instrument service contracts I guess you are looking at near $20K/year per sequencer. Call it $50/ day or another $0.04/reaction.

Well, to spare everyone the rest of the blow-by-blow I think it is safe to say that you are looking at costs of at least $0.20/reaction. So, let's call this $0.20 per 1E-03 bases or $200/megabase of raw sequence. This puts you at least 1 order of magnitude above the cost of a GS-FLX or Ion Torrent data set of the same size. And that means an Illumina data set of the same size would run you 2 orders of magnitude less.

Which is not to say that Sanger sequence will not get you the most bang for your particular buck. But given the level of optimization you have gone to for Sanger, my guess is that you would have little difficulty re-tooling for a Next Generation protocol that would drop your costs 10x, if not more. I hasten to add that I am not saying it would be easy, but for you I doubt it would be hard. Although change is frequently painful, you might as well get used to the current sequencing paradigms, if you have a choice.


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Phillip