77. High Throughput Fingerprinting and Contig Assembly to Supply Sequence Ready Templates to the JGI-PSF 

Linda Meincke, Robert Sutherland, Connie Campbell, Joe Fawcett, Phil Jewett, Lynn Clark, Cliff Han, Larry Deaven, and Norman Doggett 
Joint Genome Institute, Center for Human Genome Studies, Los Alamos National Laboratory, Los Alamos, NM 87545 
meincke@telomere.lanl.gov 

LANL's mapping goal for FY99 is to produce 48 Mb of sequence ready maps (40% of the JGI production goal). Our primary target is the q arm of chromosome 16 for which approximately 5000 BAC clones have been identified. A probe-content map is first constructed with the use of overgo probes (see abstract by Cliff Han for details). Gaps in this map are closed by PCR-screening with BAC-end STSs of a single-tier pooled BAC library (see abstract by David Torney for details). Clones are selected for fingerprinting based on map order and DNA is prepared in 96 well deepwell dishes. This DNA is restricted with EcoR1, also in a 96 well format, and is run on twelve fingerprinting gels. Gels are stained with ethidium bromide and images are captured on the Biorad Fluor-S MultiImager system and scored using the Bio Image Advanced Quantifier software. Fragment data is generated and organized into Excel spreadsheets and processed into a Sybase mapping database. Input files for contig assembly are generated from the Sybase database and passed to GRAM, a program that provides graphical representation and utilizes algorithms to assist in contig assembly. Minimal tiling sets of clones are then selected from the GRAM contigs and these are fingerprinted with two additional enzymes. Overlap among the tiling set is confirmed with the additional fragment data using GRAM. These confirmed clones are then released for sequencing.  

Supported by the US DOE, OBER under contract W-7405-ENG-36.