DNA Sequencing Difficult Template Study
George S. Grills
grills at aecom.yu.edu
Sat Jan 30 16:00:03 EST 1999
of the ABRF DNA Sequencing Committee
Difficult Template Study
Dear Fellow DNA sequencers:
During the last few years there have been numerous improvements in DNA
sequencing chemistry and hardware, including the introduction of several new
DNA sequencing instruments. To evaluate the effects of these improvements, the
ABRF DNA Sequencing Research Committee 1999 Survey has a two part approach.
The first part, called the Standard Template Study, involves the continuation
of our "Never Ending Story" study with the standard template, pGEM.
Instructions on how to anonymously participate in this part of the study are
posted at http://mbcf.dfci.harvard.edu/dsrc.html. The second part, called the
Difficult Template Study, involves sequencing two challenging templates.
The Difficult Template Study will open for sequencing data submissions on
February 1, 1999. We are mailing to you two difficult templates, "Lunatic" and
"Sasha," and the M13(-24) reverse primer. Please electronically submit the
chromatogram (electropherogram) files of the results of sequencing these
templates with this primer and fill out a sample information web form for each
submitted sample. Instructions on how to anonymously submit your analysis files
and fill in the forms can be found at http://brcweb.bio.cornell.edu/dsrc99.
The primer is included for dye terminator chemistries. Participants using dye
primer chemistries can use their own primer based on the primer sequence
included on the web site.
If you wish to participate in this study and you have not received the
difficult templates by February 5, 1999, please go to the web site for
instructions on how to request the templates and primer.
We hope to obtain sufficient data that will allow us to answer the following
· Has advances in sequencing technology improved the quality of sequence
· How do dye chemistries and enzymes from different sources compare with
· How does data quality compare between different automated sequencer
manufacturers and models?
The first goal of the Difficult Template Study is to determine if current
improvements in sequencing technologies allow more accurate sequencing of an
already characterized difficult GC rich template, Lunatic. In our 1997 study
using Lunatic, the average number of errors over 600 bases for all machines was
65 and the average number of bases with a confidence level >20 was 270. In
contrast, in our 1998 study using pGEM, the average number of errors over 600
bases was 13 and the average number of bases with a confidence level >20 was
518. By asking labs to sequence Lunatic again, we hope to draw conclusions
about the effects of advances in DNA sequencing technology on sequencing
difficult templates. The second goal of this study is to provide a challenge
and a chance to exercise your problem solving abilities when faced with Sasha,
a very GC rich template.
Please keep in mind that this is a survey study, not a contest. A typical run
or a poor run can provide as valuable information as your best run. We hope to
receive results from the oldest machines to the newest, from beginners to
experts. This is an opportunity for self-evaluation and to share your
successful techniques with others.
Data received by Feb. 20, 1999 will be presented in a preliminary report at the
ABRF '99 meeting in March of this year. A full report will be presented on a
web poster later in the year on the ABRF home web site at http://www.abrf.org
under Research Committees/DNA Sequence.
We hope that you have fun with these challenging templates. Your participation
will help us define the "art" of DNA sequencing in 1999.
The ABRF DNA Sequencing Research Committee
George Grills, Albert Einstein College of Medicine, Bronx, NY (chair)
Pamela Scott Adams, Trudeau Institute, Saranac Lake, NY
Duane Bartley, John Hopkins University, Baltimore, MD (ad hoc)
Mary Kay Dolejsi, Fred Hutchinson Cancer Research Center, Seattle, WA
Susan Hardin, University of Houston, Houston, TX
Doug McMinimy, The Jackson Laboratory, Bar Harbor, ME
Paul Morrison, Dana-Farber Cancer Institute, Boston, MA (ad hoc)
Theodore Thannhauser, Cornell University, Ithaca, NY
More information about the Autoseq