I just came across an interesting paper in the new issue of Genome Research
9:588-595, 1999. http://www.genome.org Titled: Improved DNA Sequencing
Accuracy and Detection of Heterozygous Alleles Using Manganese Citrate and
Different Fluorescent Dye Terminators by C. Korch and H. Drabkin.
It concluded that when manganese was added to dRhodamine it produced
results superior to BigDye in terms of base calling accuracy and peak
height uniformity (even superior peak height uniformity than obtained with
dye primer chemistry!).
Our Core Facility has chosen BigDye (in spite of the cost) over dRhod
primarily because of the increased sensitivity. I'm sure that many of you
will agree that weak or no signal strength is perhaps the most commom
reason for poor sequencing results. (I envy those genome sequencing centers
working with those perfectly uniform quality and quantity templates!)
We've always been concerned that a lower sensitivity chemistry will knock
our poor quantifiers below a detectable level.
Let's assume that the addition of manganese to dRhod produces sequence
profiles superior to BigDye in read length, peak uniformity and
heterozygote determination (the paper reports the addition had minimal
effect on BigDye). However, BigDye can read those otherwise marginal
signals and those increasingly larger molecules. It can also be diluted to
reduce costs.
Say you're a core facility that receives all varieties of templates with
very divergent qualities and quantities. You are also a fairly high through
put facility with a large and impatient waiting queue. Mixing and matching
chemistry with template is impractical. What chemistry would you choose as
your standard?
I would very much enjoy a discussion of the pros and cons of each. Thanks.
Best regards,
Skip Vaught
DNA Sequencing Service
University of Arizona
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Skip Vaught- jev@u.arizona.edu
Brian Coullahan- coullaha@u.arizona.edu
Mark Schwartz- schwartm@u.arizona.edu
Felisa Blackmer- blackmer@u.arizona.edu