We are about to embark on a large scale cosmid (genome) sequencing
venture and I would like to survey people to get their opionions on a
couple of alternative approaches. We are planning to shotgun clone
cosmid and sequence using an ABI 373A (and hopefully a 377 later).
1. subcloning - is it better to use M13 (ssDNA - better read lengths?)
or a ds plasmid (e.g. pBluescript - better for joining contigs, 2
sequences per template).
2. screening subclones. Does it make sense (in terms of money & effort)
to screen subclones (by restriction digestion and hybridization) to
eliminate no-insert clones and clones containing cosmid vector DNA or
just go ahead and sequence everyting and sort it out later? What about
3. sequencing reaction - dye primer vs dye terminator? When should you
use TaqFS vs Thermosequenase?
4. what sort of read lengths are people routinely getting (from 373A,
with & without stretch and 377)?
Thanks to everyone in anticipation of your response.
=======================================================================Peter J. Myler phone: (206) 284-8846x332
Seattle Biomedical Research Institute FAX: (206) 284-0313
4 Nickerson Street e-mail: MYLERPJ at U.WASHINGTON.EDU
Seattle, WA 98109-1651