A microchannel electrophoresis DNA sequencing system
In order to increase the DNA sequencing throughput of the Joint Genome Institute, we have developed a microchannel electrophoresis system. The critical new and unique elements of this system include 1) a process for the production of arrays of 96 and 384 microchannels on bonded glass substrates up to 14 x 58 cm and 2) new sieving media for high resolution and high speed separations. With custom fabrication apparatus, microchannels are etched in a borosilicate substrate, and then fusion bonded to a top substrate 1.1 mm thick that has access holes formed in it. SEM examination shows a typical microchannel to be 40 micrometers deep x 180 micrometers wide by 46 cm long. This technology offers significant advantages over discrete capillaries or conventional slab-gel approaches. High throughput DNA sequencing with over 550 base pairs resolution has been achieved in roughly half the time of conventional sequencers. In February 1999, we begin a pre-production evaluation protocol for the microchannel and for three glass capillary electrophoresis systems (two from industry and one developed by Lawrence Berkeley National Laboratory for the Joint Genome Institute). In order to utilize these instruments for DNA production sequencing, we have been evaluating and implementing software to convert raw electropherograms into called DNA bases with an associated probability of error. Our original intent was to utilize the DNA base calling software known as Plan and Phred developed by the University of Washington. This software has been outstanding for our slab gel electrophoresis systems currently in the production facility. In our tests and evaluations of this software applied to microchannel data, we observed that the electropherograms are of a different statistical and underlying signal structure compared to slab gels. Even with substantial modifications to the software, base calling performance was not satisfactory for the microchannel data. In this paper, we will present o The microchannel DNA sequencing system and show the advantages compared to current slab gel and capillary systems. o The signal processing modules needed including correction of multiple wavelength channels, signal averaging, non-uniform sampling, variable DNA mobility, and peak shape and spreading effects. o A comparison of the DNA base signatures in the raw data of microchannels vs. slab gels including some simple modeling results. This will be propagated through the base calling software to show the impact on DNA sequencing.
- Research Organization:
- Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
- Sponsoring Organization:
- USDOE Office of Defense Programs (DP)
- DOE Contract Number:
- W-7405-Eng-48
- OSTI ID:
- 7986
- Report Number(s):
- UCRL-JC-133132; KP1103010; ON: DE00007986
- Resource Relation:
- Conference: 33rd Annual Conference on Information Sciences and Systems, Baltimore, MD, March 17-19, 1999
- Country of Publication:
- United States
- Language:
- English
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