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ABI 3700 and Phred/Phrap

Bruce Roe BROE at aardvark.ucs.ou.edu
Sat Jun 5 20:36:31 EST 1999


Philip San Miguel writes: 
=> Date: Thu, 03 Jun 1999 11:47:48 -0500
=> From: Phillip San Miguel <pmiguel at purdue.edu>
=> Subject: ABI 3700 and Phred/Phrap
=> To: nobody at net.bio.net
=>     Any problems using Phred/Phrap with the ABI3700 chromat files?  I
=> tried Phred out on some DNA standard sequences (*.ab1 files) from the
=> 3700 and the histogram of high quality bases gave very low results.
=> (Less than 200 bases for all 96 capillaries--even though most of the
=> data was pretty good.)
=>     Does anyone have any advice?
=> Phillip San Miguel
=> Purdue Genomics Facillity

Hi all,
	I've been quiet for a while with all the grant writing, etc.

Phillip,
        Data out of the Whitead and WashU indicates that lowering the
capillary chamber temperature from 50 to 40 degC while keeping all the other
loading and running conditions the same should improve the spacing and help
"trick" Phred into making better base calls.  We have done this as well
and the base calls by Phred are much improved.  There is some debate if 
38 degC is better than 40degC but for us, 40 degC works so much better than
the ABI recommended 50degC, I'm not about to worry about the 2 degC
difference.  There also has been a mention of removing the fabric surrounding
the capillaries, but that only seems to have a major effect at 50 degC and
has little or no effect at 38-40 degC.  You also may want to increase the run
time by a half hour if you have the data out that far because the samples
will travel slower at 38-40 degC than they do at 50 degC. ABI also should be
releasing a new low-T mobility file for the 3700 which I was told would be
available on their ftp site some time soon (maybe by the end of this month?)
        Finally, four of the genome centers (WashU, Whithead, Hood and Sanger) 
supposidly were sending Phil Green data so he could re-train Phred for the
3700.  This should result in a more 3700 compatable Phred by the end of the
summer or sooner depending on how quickly Phil Green gets the job done.
I hope this comes out soon but, will not believe it till I see it since
re-training Phred is non-trivial.
Cheers,
--bruce
*************************************************************************
Bruce A. Roe, Ph.D  George Lynn Cross Research Professor
                    Department of Chemistry and Biochemistry
                    University of Oklahoma, Norman, OK 73019-0370, U.S.A.
Phone: (405) 325-4912 or 7610;  FAX: (405) 325-7762;  e-mail: broe at ou.edu
********************** http://www.genome.ou.edu/ ************************




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