Extension of Read Length--need new ideas

caribou at u.washington.edu caribou at u.washington.edu
Fri Jun 6 09:56:04 EST 1997


Hi again folks,

My next question is to determine methods for extending usable read
length on the 377 DNA sequencer.  My facility only does dye terminator
samples and we generally run them on a 36 cm plate overnight for 7 hours
at a 2X run paramater.  I get approxamately 700-750 bases called, of
which I would bet my life on only 500 bp being accurate. Sometimes that
figure drops to only 400 reliably called bp.

1)  I have tried using Long Ranger solution but the computer basecalling
program (ABI100) does a lousy job, and the base spacing is all off.  Is
there a better way of using Long Ranger?  Do a 3.5 hr 4X run?  Other
suggestions?

2)  I have considered getting a 48 cm set of plates, but am avoiding the
extra cost, since we already invested in two sets of 36 cm plates.  Is
it really the plate length that is the limiting factor for read length? 
That is, if I ran the same sample on the two gels, what would I get?  

3)  Does anyone see improvement by changing the cycling conditions?  Has
anyone done any tinkering with number of cycles, temperatures and
extension lengths to determine if a better read length is obtainable?

4)  Other ideas?  What else have you folks tried?  Which published
referrences should I read to determine what the companies claim?  (Of
course, companies will only declare their best results, which ones are
most reliable?--I took my car to the gas station to get a recommendation
on who fixes Subarus nearby.  The fellow paused uncertainly and said,
"of course, WE see Subarus all the time", but he wasn't very
believable.)

Hope this sparks some discussion and exchange of ideas.

Sincerely, 
Grace
(caribou at u.washington.edu)
HHMI University of Washington
Seattle, WA USA



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