Automated fluorescent M13 shotgun DNA sequencing

M. Starkey mstarkey at hgmp.mrc.ac.uk
Wed Sep 18 09:05:10 EST 1996


 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)





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