This collaborative project is aimed at the development of a """"""""Microbead INtegrated DNA Sequencer"""""""" (MINDS) that efficiently integrates all of the major steps in DNA sequencing, from library construction to final sequence output exploiting low-cost microfluidic devices. The automated MINDS system will combine three fundamental steps: 1) library construction, amplification, and selection using microbead colony technologies 2) nanoliter cycle sequencing sample preparation and purification, and 3) microfabricated capillary array electrophoresis (uCAE)-based separation of DNA sequencing fragments. The library construction and amplification process will input sheared, sized DNA fragments and construct an emulsion PCR amplified library of template on beads, with each bead representing a single DNA fragment. Single beads will then be processed in a 25 nL cycle sequencing reactor to produce fluorescently labeled sequencing fragments that are efficiently captured concentrated and purified using on-chip affinity capture. The fragments are then separated and sized on a proven microfabricated uCAE sequencer. ? ? This proposal will combine the efforts of Microchip Biotechnologies Inc. (MBI) with subcontracts to three collaborating academic institutions. MBI will develop a prototype microchip-based DNA sample preparation nanoscale thermal cycling module and an advanced rotary scanner with a prototype uCAE sequencing system using conventional external chemistries. These will then be integrated to produce a MINDS microchip with arrays of 25 nL cycle sequencing sample preparation, affinity purification, and uCAE sequencing. When this has been accomplished, by 30 months, MBI will further integrate the microbead-based library technology being developed by the Mathies laboratory to create 400 channel MINDS System prototypes ready for beta-testing. These developments will build upon novel methods and strategies developed in tandem by the academic collaborators, in particular the uCAE separation system and bead-based microfluidic """"""""cloning"""""""" methods. A subcontract to the Mathies lab at U.C. Berkeley will support the development of new microtechnologies for the amplification and selection of clones, and the integration of these methods and processes with prototype microfabricated sequencing systems. In collaboration with Mathies, the Barron lab at Northwestern will develop and test novel DNA separation matrices that are easily loaded into and replaced from chip microchannels, and that provide rapid, high-resolution separations with at least a 700-base read. A subcontract to the Ju lab at the Columbia Genome Center will support the development of new methods for genomic clone production as well as for beta-testing the integrated sequencing systems produced by MBI. The Columbia group will also work with the Berkeley group toward improving methods for clone production and selection, and for on-chip sample clean-up. The project goal is to place a beta version of the fully integrated, prototype Sanger sequencing system at Columbia Genome Center and to demonstrate its capability to perform genomic sequencing and resequencing at 100-fold lower cost and a throughput of about 7 million bases/day/machine by producing over 1.5 gigabase of shotgun sequence. ? ? The MINDS system will greatly reduce the cost of shotgun sequencing and resequencing, by exploiting the ability of well established uCAE devices to analyze sub-nanoliter volumes through preparation of samples in volumes more closely matched to the analytical requirements, reducing cycle sequencing reagent consumption by 100-fold. Library construction will be automated in the bead-based format, with amplification and selection performed at full scale in a single bulk reaction, again reducing reagent consumption and cost. A novel polymeric separation matrix designed for microchips already shows good performance and, along with microfluidic volume reductions, will minimize matrix expense. With these combined innovations, the MINDS system will drive CAE instrumentation close to the ultimate performance possible for four-color Sanger fluorescent DNA sequencing in an ultra-high-throughput implementation for genome centers. Future work will explore the development of lower-throughput versions appropriate for core and individual laboratories. ? ?
