8. Process Description of a 5 Mb / Year Finish Sequencing Operation Using 100% Plasmid Double End Sequencing 

David C. Bruce, Leslie A. Chasteen, Donna L. Robinson, Myrona D. Jones, Jennifer Bryant, Nancy C. Brown, Beverly Parson-Quintana, Darrell O. Ricke, Mark O. Mundt, P. Scott White, Norman Doggett, and Larry L. Deaven 
Human Genome Center, Los Alamos National Laboratory, Mail Stop M888, Los Alamos, NM 87545 
bruce@telomere.lanl.gov 

Beginning in Oct. 1997, the Center for Human Genome Studies (CHGS) at Los Alamos National Laboratory (LANL), as part of the Joint Genome Institute (JGI, http://www.jgi.doe.gov/), committed to first year finished sequencing of 2.84 MB of human sequence. A steady state finish rate of 5 MB / year, by the end of the first year is projected. An exhaustive description of the JGI Sequence quality standards and sequencing targets is available at http://www.jgi.doe.gov/Docs/JGI_Seq_Quality.html. Prior to Oct 1997, the CHGS had submitted 224 KB of finish sequence to GenBank using only Applied Biosystems software, minimal sample tracking, and little automation. As of Oct 4, 1998, the CHGS had finished 3.2 MB total and 2.8 MB unique sequence and reached a finish sequence output of 0.6 MB / month using phred/phrap/consed finishing tools, select automation, sample tracking system in conjunction with a fully redesigned process. The sequencing strategy is 6X plasmid end sequencing using dye terminator chemistry in production, quality gap closure using alternate chemistry / gel conditions in pre-finish and final gap closure using a combination of primer walking, transposon bombing and small insert libraries in finish. Implementation of this aggressive ramp will be presented including; sequencing strategy design, process analysis, personnel reorganization, automation, informatics, and quality / cost control with emphasis on the production phase of sequencing.