Extension of Read Length--need new ideas

Steve Steve
Wed Jun 11 19:04:53 EST 1997


In article <5n98e4$627 at net.bio.net>, caribou at u.washington.edu wrote:

> 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?

Have you tried using the Semi-adaptive base caller. This may help improve
the reads.

> 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? 
 
Length of read is related to the WTR distance. When we install an
instrument with 48 cm Plates we must get a minimun of 650 bases with 98.5%
accuracy of the long read standard. There also must be less than 2% N's as
well. When we install with 36 cm plates this drops to 450 bases with the
same accuracy. What you are getting is the normal parameters for your
configuration. The 48 cm plates will give you longer reads. You need to be
careful with you DNA preps for 48 cm runs. Clean template is extremely
important to get good sequence on the 48cm plates.
 
> 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?

Not sure how this will affect the length of read.
 
> 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.)

Clean DNA preps are very important to getting good sequences. The cleaner
the DNA the longer read you will get up to a point. When I worked in the
sequencing Core Facility at Caltech before I joined ABD we got read
lengths of 600 bases routinely when we prepped the DNA on the Autogen
Miniprep machine. People that did sloppy preps were lucky to get 400
bases. The people who took their time and made clean preps got longer
reads. We reccomend the Quiagen miniprep systems for sequencing on the
377. The promega kits are not reccomended because the results vary from
kit to kit.

Steve Marsh Field Service Engineer So. Calif
marshsr at ccmail.apldbio.com



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