Efficient low redundancy large-scale DNA sequencing at EMBL

Abstract

An efficient low redundancy DNA sequencing strategy should allow high accuracy determination of the consensus sequence on both strands of a DNA fragment from a minimal number of sequencing reactions with minimal overlap. At EMBL we developed a directed strategy for cosmid-scale sequencing based on primer walking, whereas most other sequencing projects of this scale rely on the random 'shotgun' strategy. In our strategy, highly accurate raw data are obtained from automated double-stranded Sanger dideoxy sequencing with inexpensive walking primers (8 to 10 $ per primer), T7 DNA polymerase and internal labelling by fluorescein-15- dATP on A.L.F. DNA sequencers (Pharmacia Biotech). The use of 60-cm long glass plates enables reading length of up to 1000 bases. Comparing various random and directed sequencing strategies in the course of the European Community yeast genome sequencing project on cosmids from chromosomes IX, XI and XV, primer walking was found to be the strategy resulting in the lowest possible redundancy of 2.6 to 2.8. Future development of the sequencing strategy is based on the new EMBL 2-dye sequencing device for simultaneous sequencing on both strands, and implementation of an initial limited random sequencing phase to reduce the number of walking primers required by a factor of 3, while still maintaining a low redundancy of approx. 3.