Hi Bob,
Have you tried an different software program that might have a different
basecalling algorithm? One possibility is to analyze the results with
phred. If you do not have phred readily available, just send me some
chromatogram files and I would be happy to give it a try.
ABI should take note that many labs would love to use flourescent
sequencing to detect methylayed C's. Nice trick regarding the hidden menu
for 377s. Did you find it by randomly banging the keyboard in fustration?
- George
At 03:19 PM 4/6/01 -0400, Robert Lyons wrote:
>ABRFers,
>
>Perhaps someone has a suggestion about how to handle some
>problematic samples in our DNA sequencing lab.
>
>We occasionally receive samples with highly skewed nucleotide
>compositions. Two situations produce such samples: organisms
>with bizarre base compositions; and a procedure used to detect
>the presence (or absence) of 5-Me-C using dimethylsulfate (?)
>degradation of unmethylated bases. Both of these situations
>produce highly skewed nucleotide compositions.
>
>ABI's Sequence Analysis software has a problem with such samples.
>It assumes that all nucleotides are present at "roughly" similar
>numbers, and tries to normalize the color intensities before base-
>calling. If in fact there are very few G's or A's, the software
>just amplifies the background chatter up to the level of the
>"real" peaks. The sequence is trashed.
>
>On a 377, there's a trick that allows one to circumvent this
>problem. A "hidden" menu can be called up in the SABasecaller*
>that allows the operator to override the assumed nucleotide
>composition. It has some bugs (mis-labeled nucleotides, strange
>numbers), but we have been able to fiddle with the settings and
>get these skewed-composition samples to sequence quite well.
>
>Here's the problem - there's no such "hidden" menu on the 3700.
>According to ABI, there's no way to adjust the expected nucleo-
>tide compositional balance. I would *greatly* prefer to run
>these on the 3700, but see no alternative but to fire up one of
>our old gel-based machines.
>
>Anyone have ideas on how we might run these on a 3700?
>
>Bob Lyons
>University of Michigan
>
>
>P.S. -
>The hidden menu is obtained by: double-click BasecallerPPC, then
>hold down the option key while pulling the 'Edit' menu to 'Settings'.
>*** Make sure you restore the changes to default values before going
>on to normal samples! Click 'Default' in the Settings menu. ***
>
________________________________________________________________
George Grills
Director, DNA Sequencing, Genotyping and GeneChip Microarray Facilities
Director, DNA Sequencing Group, Einstein Genome Center
Albert Einstein College of Medicine
713 Ullmann Building
1300 Morris Park Avenue
Bronx, New York 10461-1602
Tel: (718) 430-2657
Fax: (718) 430-8778
E-mail: grills@aecom.yu.edu
DNA Sequencing: http://leper1.ca.aecom.yu.edu/dnacore
Genome Center: http://sequence.aecom.yu.edu/chr12
________________________________________________________________
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