Sorry this is a bit long but here goes:
Bruce Roe's comments on the above yesterday, and especially the problems of
getting longer sequences from the ABI sequencer got me thinking.
Much of our sequencing is "into the unknown"" rather than routine and
so the rate limiting factor is not the speed of gel reading but the speed
with which we can get the next oligo made. Hence it is vital that we
maximise read length from any reaction set. We have extensively played
with mannual sequencing to optimise this. Sequenase sequencing using both
manganease and extension mix in the reaction labels well and evenly from
approx 10 to 2000 plus bases from the primer and Hydrolink Long Ranger gels
maintain good tight bands (albeit closely spaced) up to at least 750 bases
from primer (best read to date).
When I was looking to select an automated sequencer, read length was a
significant factor. Because I knew what was possible from manual
sequencing, I asked the ABI guys about the flexibility of electrophoresis
on the 373A and was basically told that, since the bands must pass the
detection zone at a fixed rate (12+-2 laser scans per band), you did
electrophoresis how they told you to do it or it wouldn't work and that was
it. Ie. They were not interested in getting longer reads. Since, if current
adverts are to be believed, both LKB and LiCor are way ahead of ABI on this
score, this seemed an extreemly short sighted attitude. All power,
therefore, to Bruce and his collegues who are trying to improve matters but
I do wonder if there might be an "easier" way.
Point 1: I know that reactions can be made good for a couple of thousand
Point 2: the standard ABI base calling algorithm works (or you wouldn't
get any sequence at all)
Point 3: surely then, we should be looking not to make the algorithms even
more complicated (and thus vunerable) than they are at the moment but to
improve the electrophoresis conditions so as to be able to better space the
bands that are far from the primer.
LKB make extensive use of Hydolink Long Ranger gels in the ALF which
presumably must help acheive their long reads. The problem with
these gels is that you usually run them more slowly than standard gels
(hence the scans/band problem). Has anyone out there in 373A land tried any
of the following to increase read length:
Long Ranger gels run slow or fast (by altering electrical supply and/or
buffer concentration or gradient)
Composite gels with standard acrylamide in the detection zone (so bands
pass through it at the correct rate) and Long Ranger above (to keep the
bands tight), or gradients of the 2 forms of acrylamide.
or anything else on the electrophoresis front
My 2 cents worth,
David A. Johnston
Dept of Zoology, The Natural History Museum, Cromwell Road,
South Kensington, London SW7 5DB.
(tel 071 9389297, fax 071 9388754, email daj at nhm.ic.ac.uk)