17. Megabase and Gigabase Templates: Direct Automated Sequencing off Microbial and Eukaryotic Chromosomal DNA 

S. Kozyavkin, A. Malykh, O. Malykh, Y. Mirokhin, and A. Slesarev 
Fidelity Systems, Inc., 7961 Cessna Avenue, Gaithersburg, MD 20879-4117 
http://www.fidelitysystems.com 
fsi1@fidelitysystems.com 

Combination of the robust dye terminator and ThermoFidelase chemistries has provided solution for the automated sequencing directly from microbial Megabase-long templates. Novel approach streamlines gap closure in the large-scale projects and does not rely on extra subcloning or combinatorial PCR. Redesign of genome sequencing and gene hunting strategies promises to substantially reduce the volume of and eventually completely eliminate shotgun steps in microbial genome projects. 

Our work indicates that Megabase sequencing chemistry provides results with high quality, sensitivity, reproducibility and speed. We will present data on the direct detection of single nucleotide polymorphisms (SNPs) and gross sequence variations between genomic regions from a number of closely and distantly related microorganisms. We will discuss technical and economic feasibility of the new strategy in large-scale projects on comparative genomics. 

The development of sequencing chemistry for Gigabase templates such as human and other complex eukaryotic genomes is one of the most challenging tasks in technology development. Our initial work is focused on the specifics of preparation and handling of Gigabase templates, target selection, achievement of sufficient fluorescent signal strength and quality. We will review basic techniques used in the development of novel chemistry and present our first successful results on the automated sequencing directly from fish and human chromosomal DNA. 

This work is supported in part by DOE grant DE-FG02-98ER82557 and NIH grant 2R44GM55485-02.