|Genome Sequencing Section
DOE Human Genome Program Contractor-Grantee Workshop
11. LANL Finishing Team Accomplishments in FY98
J. Buckingham, L. Goodwin, C. Munk,
L. Saunders, S. Thompson, S. Ueng, D. Ricke, and M. Mundt
In response to the Joint Genome Institute's goal of finishing 20 MB of high quality sequence, the Finishing Team was formed at Los Alamos National Laboratory's Center for Human Genome Studies. The task for this diverse group of biologists, computer scientists and mathematicians was to design an efficient process to quickly close clone projects and bring the sequence quality up to a high standard. The tools to do this job were largely untested and disorganized, and many new protocols and strategies had to be formulated to address problems, even as the "conditions of contest" changed over the past year. Timely feedback to reduce unnecessary work was also an important factor to our success.
Initially, LANL's sequencing capacity was directed at the double-ended plasmid SASE approach, using TAQ and later TAQ-fs. Two major improvements were switches to BigDye terminator reactions for production and ET dye primer chemistry on ABI 373's for finishing. The boost in quality from these two process changes was quite evident using both our own base caller and Phred. Phred, Phrap, and Consed had not previously been used at LANL, so several technicians went through UNIX training to become specialists at interacting with these programs. Auxiliary Java programs were also designed to analyze Phrap assembly structure and to suggest finishing reactions consisting of dye primer redos and primer walks. A paper trail system was converted to an automatic submission system for our oligo synthesizers. Following the lead of the production crew, we now use halfTERM (Genpak,Ltd.) with BigDye for our primer walks. In addition, we are adding DMSO to the formulation to improve the reactions. We have also used shatter libraries successfully and transposons not so successfully to close final difficult gaps that exist due to, for example, high GC content. Part of our automation schemes included the use of Hydras and multichannel pipettors for setting up finishing reactions.
We are now streamlining our approach to efficiently address the issues involved with "draft" sequencing. We have defined a prefinishing step based on our "Strand Gap" report that will also help evaluate cost functions to feed back to our production team to determine level of shotgun required. Research plans include trying halfTERM with dye primer reactions and working with the Mermade oligo synthesizer that should be delivered in the next few months. We are also investigating the potential benefits of programming robots to select templates for reaction set ups and weighing these against potential disadvantages such as reduced quality. We will present relevant statistics to demonstrate the quality of our finishing reactions and their utility in alignments to our final consensus. This work contributed to the completion of 2.8Mb of sequence in FY98. Supported by US DOE under contract W-7405-ENG-36.
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