[Automated-sequencing] Re: variation in libraries was: Autoseq
Digest, Vol 4, Issue 5 - Email found in subject
Phillip San Miguel
pmiguel at purdue.edu
Wed Sep 21 10:38:02 EST 2005
Fulton, Ciaran wrote:
> Thanks Phillip for the response...we have noticed a difference in the
> size of pellets with regards the QPix, that was something noticed from
> the very first time we used it and the other issues you mentioned we
> have also encountered and are looking at addressing. The QPix aside,
> we're finding the pellets the same size when we pick manually but the
> yields don't necessariy tally with the LOR result, we can get a good
> read length with next to nothing in the well, whereas wells where the
> yeild looks respectable fail. Admittedly we don't have a plate reader
> and the yield quantifications are on a respresentative number of random
> wells but in most of the cases there is no correlation. What is the
> range of yield that you work with Phillip?
> The fact that everything in our facility is so well standardised this
> seemed to happen with the start of the 'new' cDNA library with the
> obvious difference being the host strain and method of transformation.
Yes Ciaran, on the whole I do not find DNA concentration to be a good
indication of read length--with the following caveat. Obviously if you
have no DNA (or almost none) then the resulting sequencing reaction can
produce such a low amount of product that the sequencer will not detect
the products against background noise. I would have to check what the
minimum molar amount is that works for us.
What is the nature of "failure" that results in lower read lengths? Can
you examine the traces? Generally failure will fall into 3 general
(1) No (or almost no) signal. No DNA in the reaction, or the reaction
failed for some other reason (contamination with something that poisons
the polymerase, etc.), or the capillary in which the sequence was run is
blocked, or there was a lot of salt competing with your sequence
products for electrokinetic injection.
(2) Mixed sequences. If a sequence primer can anneal in 2 places in your
DNA preps (and this includes having 2 plasmids mixed together) then you
can get mixed sequence.
(3) Broad peaks. I've only ever seen this on capillary instruments--but
it is not uncommon. The first peak will be delayed and all the peaks are
broad. ABI's more modern basecallers (eg, KB basecallers) seems to be
able to compensate for this. But even if compensated for perfectly, the
reads will be shorter--not as many bases have time to roll past the
detector window. And the compensation is not perfect: runs of bases will
often be called where only one is really there. The phenomenon is
generally correlated with certain host strains--although if there is
enough glop in a DNA prep, I think it can happen in any host strain.
However, the strains you mention perform very well in our hands. If,
however, you are using the strains that Invitrogen normally includes
with it's cloning kits--those give us enough trouble that we toss them
and buy DH10B electrocompetant cells.
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