I would like to address this enquiry to experienced practioners of automated
fluorescent M13 shotgun sequencing:
Essentially, we are interested in developing a new approach to assembling
sequencing templates from cosmid clones, in order to reduce the redundancy
associated with this type of sequencing effort. For comparative purposes, we
are interested in the efficiency of the shotgun approach that appears to be
still very much in vogue. It would be greatly appreciated if anyone was able
to point us in the right direction with regard to answers for the following
points:
1. Number of reads of each base deemed acceptable for accurate assembly of
a complete sequence?
2. Average cosmid coverage of a shotgun programme both before and after
finishing (degree of redundancy)?
3. Proportion of a cosmid sequence which must be acquired by "special means";
eg. primer walking?
(Are a given number of M13 clones sequenced, and then primer walking
commences?)
4. Average ambiguity content of each gel read?
(Taq FS/Thermosequenase, dye terminator chemistry)
Is manual editing performed with/without the assistance of overlapping
sequence data?
5. Average "accuracy" of each unedited/edited gel read; eg. the identity of a
given single read to the equivalent portion of the final assembled sequence?
Many thanks in advance for any help that anyone is able to provide.
Mike Starkey
UK Human Genome Mapping Project Resource Centre
Hinxton Hall
Hinxton
Cambridge. CB10 1SB
Tel: 01223 494572
Fax: 01223 494512
E-mail: (mstarkey at hgmp.mrc.ac.uk)
