32. Sheath-Flow Capillary Array DNA Sequencer Development at JGI/LBNL 

Jian Jin, William F. Kolbe, Yunian Lou, Earl W. Cornell, Alex Cheung, and Joseph M. Jaklevic 
Ernest Orlando Lawrence Berkeley National Laboratory, University of California, Engineering Science Department, 1 Cyclotron Road, Berkeley, CA 94720 
Jian_Jin@lbl.gov 

We have developed a 96-channel capillary electrophoresis system capable of production-level sequencing at increased rates and, more importantly, with improved automation. The system is based on an adaptation of the best available technology developed by several laboratories. In particular, we employ the sheath-flow excitation/detection geometry and a DNA sequencing protocol using linear polyacrylamide as sieving media. In addition, we have developed an effective base-calling software platform using a combination of algorithms. The sequencing system is fully integrated and includes a fixture for off-line capillary-array coating, gel-replacement and sample loading. The four-color sequencing instrument employs a cooled-CCD camera for data acquisition. Our custom base-calling software has been fully integrated with existing assembly algorithms including calibrated Phred scores and Phrap-based assembly. Currently we achieve a total run time of less than two hours, separating 750 bases per channel, with an average read-length (defined as having a Phred score > 20) of 350-400 bases/trace. The turn-around time between runs is less than 5 minutes. Over the past 9 months we have conducted a full evaluation of the system using sequencing templates taken directly from JGI production runs. During this time we generated more than 6 Mb of raw sequence data for system performance evaluation and protocol development. Those results have demonstrated that our system produces sequencing data with a quality comparable to commercial slab-gel systems currently employed in production sequencing. Detailed comparison data will be presented and future plans and discussed. 

This work was supported by the Director, Office of Energy Research, Human Genome Program, of the U.S. Department of Energy under Contract N0.DE-AC03-76SF00098