PCR template preparation for capillary DNA sequencing

Fluorescence-based capillary DNA sequencing has facilitated the early compl etion of several several complex sequencing projects. While capillary syste ms offer great benefits in terms of ease of use and automation, we find tha t they are sufficiently different from slab gel separation methodologies, d emanding re-examination of the protocols used to generate and use DNA seque ncing templates. We have recently initiated a large-scale Human Open Readin g Frame(TM) EST project involving 30 laboratories feeding 11 MegaBace(TM) 1 000 capillary sequencers. The group has already produced more than 300 000 valid sequences. The most successful template preparation protocol we have found is described here. We have found that a crucial step is the standardi zation of the quantity and quality of the templates, which have been achiev ed by overnight bacterial culture followed by PCR using limiting amounts of primers. Using this protocol, there is no need for post-PCR purification, and the final preparation cost is US $0.09/template. After sequencing 10 84 8 templates using this protocol, 78% of the reads were accepted (after disc arding vectors without inserts and inserts smaller than 100 nucleotides), a nd 85% of the total number of bases had Phred scores of 15 or above.