Full Text HG-95-001 IMPROVED ELECTROPHORETIC DNA SEQUENCING TECHNOLOGY NIH GUIDE, Volume 23, Number 42, December 2, 1994 RFA: HG-95-001 P.T. 34 Keywords: Nucleic Acid Sequencing Human Genome National Center for Human Genome Research Letter of Intent Receipt Date: February 1, 1995 Application Receipt Date: March 17, 1995 PURPOSE The National Center for Human Genome Research (NCHGR) invites applications for research projects to develop novel automated sequencing technology suitable for large-scale genomic sequencing through the reduction in scale and increased parallelization of existing approaches utilizing Sanger sequencing reactions coupled with electrophoretic separation of fragments. The Request For Applications (RFA) encompasses front end sample preparation, separation, detection, and data acquisition and handling. Technologies solicited include a spectrum of approaches ranging from capillary and ultrathin gel electrophoresis to microfabricated and microelectro mechanical systems (MEMS) that could yield reductions in scale and increased throughput. HEALTHY PEOPLE 2000 The Public Health Service (PHS) is committed to achieving the health promotion and disease prevention objectives of "Healthy People 2000," a PHS-led national activity for setting priority areas. This RFA, Improved Electrophoretic DNA Sequencing Technology, is related to several priority areas, including cancer, heart disease and stroke, diabetes and chronic disability conditions, maternal and infant health, and others. Potential applicants may obtain a copy of "Healthy People 2000" (Full Report: Stock No. 017-001-00474-0) or "Healthy People 2000" (Summary Report: Stock No. 017-001-00473-1) through the Superintendent of Documents, Government Printing Office, Washington, DC 20402-9325 (telephone 202-783-3238). ELIGIBILITY REQUIREMENTS Applications may be submitted by domestic for-profit and non-profit organizations, public and private, such as universities, colleges, hospitals, laboratories, units of State and local governments, and eligible agencies of the Federal government. Collaborations between scientists in academia and industry are encouraged, as are applications from minority individuals and women. Applications from foreign institutions will not be accepted. However, subcontracts to foreign institutions are allowable, with sufficient justification. Applications are particularly encouraged from scientists, such as engineers and physicists, and institutions, such as for-profit institutions and biotechnology companies, that have not traditionally requested research support from the NCHGR. Applicants whose expertise is primarily nonbiological are especially encouraged to interact closely with biologists during the development of the research plan. MECHANISM OF SUPPORT Support for this program will be through the National Institutes of Health (NIH) individual research project grant (R01), pilot project/feasibility study (R21), research program project (P01), and exploratory grant (P20) mechanisms. The R21 mechanism is used to support highly creative approaches for which substantial preliminary data is not yet available. R21 awards will be limited to $100,000 direct costs per year and to a maximum of three years of support, although the funding limit may be waived under exceptional circumstances. The P20 mechanism is used to support groups of outstanding investigators who wish to develop interdisciplinary research programs. P20 awards will be limited to three years at $750,000 direct costs per year. R21 and P20 grants are not renewable, but future project continuation is possible through other grant mechanisms such as R01 or P01. The levels and time frames for support of R21 and P20 awards indicated here are specific to this solicitation. Responsibility for the planning, direction, and execution of the proposed project will be solely that of the applicant. Awards will be administered under PHS grants policy as stated in the Public Health Service Grants Policy Statement. This RFA is a one-time solicitation. Future unsolicited competing continuation applications will compete with all investigator- initiated applications and be reviewed according to the customary peer review procedures. The total project period for applications submitted in response to the present RFA may not exceed three years. The anticipated award date is September 30, 1995. FUNDS AVAILABLE It is anticipated that $3,000,000 (direct and indirect costs) per year for up to three years will be available beginning in fiscal year 1995. It is anticipated that between five and ten awards will be made, representing a mix of research topics and mechanisms. Because the nature and scope of the research proposed in response to this RFA may vary, it is anticipated that the size of awards will also vary. The number of awards may be increased if a large number of highly meritorious applications are received and if funds are available. The number of awards made will be contingent upon the quality of applications received and the availability of funds. RESEARCH OBJECTIVES Background The NCHGR sponsors basic and applied research concerned with the development and application of new technologies for the characterization and analysis of the human genome and the genomes of selected model organisms. The activities encompassed by the NCHGR program include genetic and physical mapping, DNA sequencing, informatics related to mapping and sequencing, gene identification, and technology development that will facilitate all of these efforts. The NCHGR, in conjunction with the Department of Energy, recently formulated a new five-year plan (Science Vol. 262, pp. 43-46, 1993) that states that significant technological advances will need to be made in the area of DNA sequencing if the demanding goals of the Human Genome Project are to be met. A major long-term goal of the Human Genome Program is to identify all the genes encoded in the three billion base pairs of human DNA. The first step in this process is to assemble detailed genetic and physical maps of the human genome. Subsequently, the complete sequence of human DNA must be determined. In order to attain this second objective, DNA sequencing technology must be further developed so that the cost will be significantly decreased and the rate significantly increased. In 1994, the cost of sequencing performed by highly skilled researchers in laboratories devoted to genomic DNA sequencing was estimated to be about $1 per finished base. The state-of-the-art approach for large-scale sequencing includes isolation of sample DNA from a biological source followed by amplification of the DNA and the synthesis of fluorescently tagged replicate ladders of the template DNA in a Sanger sequencing reaction. Biochemistry for DNA sequencing is currently performed in volumes that range from multiple microliters to hundreds of microliters, and only modest parallelization and automation has been achieved. The DNA ladders are resolved on a separation medium through slab gel electrophoresis and ladder composition is evaluated by laser-based detection technology. Software tools are then utilized for identification of the four bases, assembly of contiguous sequences from individual reads, and sequence finishing, including resolution of ambiguities. Current sequence throughput from automated electrophoresis instruments is approximately 7,000,000 bases (raw) per instrument year. Application of these instruments in sequencing approaches based on a combination of shotgun and directed strategies results in a throughput of about 700,000 finished bases per year. Even at this relatively modest throughput, sequence assembly and finishing limitations result in a systems bottleneck. Current efforts to optimize genomic sequencing technology focus on the development of a fully automated, integrated, modular system, that accounts for sample flow in a way that eliminates bottlenecks and maximally utilizes each step of the system from the front end of sample isolation through to data collection and analysis. Recent advances suggest that dramatically improved DNA sequencing technology can be developed through the application of existing miniaturization and automation technologies to state-of-the-art genomic sequencing. A continuum of approaches exists that creates a path to a substantially reduced scale for sequencing devices with associated throughput increase and cost decrease. Further development of these approaches requires several technological advances that are expected to be addressable based on existing scientific principles. Capillary Electrophoresis and Ultrathin Gels Recent work in ultrathin (capillary or slab) gel electrophoresis has demonstrated the potential for greatly increased separation speed due to improved heat transfer. These approaches are also expected to yield cost savings by reduction in sample size if small volume robotic reaction systems are developed concurrently. Although preliminary data suggest that these formats will be successful in improving sequence throughput, technological challenges remain to the development of dependable, exportable systems. Remaining challenges include the need to identify new separation matrices that will allow for increased reproducibility of separation and easy manipulation in these formats, small volume loading technology, reduced volume sample preparation chemistry, improved detection technology, and systems automation to attain maximal sustainable throughput. Microfabricated Devices and MEMS It is anticipated that further reductions in scale and increases in throughput could be achieved through the application of existing microfabrication and MEMS to DNA sequencing. It is anticipated that successful application of this technology could increase the speed of sequencing by two to three orders of magnitude. Technological challenges that are likely to be encountered are related to the nature of the sample and include reduced volume sample preparation chemistries, improved sample loading technologies, optimizing surface chemistries to minimize sample and reagent loss, and microscale fluidics and detection issues. Systems Integration, Data Acquisition and Handling A key component of high-throughput automated systems will be improved software tools for base calling, sequence assembly, and sequence finishing, as well as for overall systems integration. Even in current approaches, sequence finishing is a bottleneck and requires manual intervention to achieve sequence closure as well as resolution of ambiguities. Therefore, if order of magnitude improvements in finished sequence throughput are to be achieved, this component will require substantial attention and creative approaches for full automation. Objectives The purpose of this RFA is to encourage applications from individuals and groups interested in developing novel automated sequencing technology suitable for large-scale electrophoresis-based genomic sequencing through the reduction in scale of existing approaches utilizing Sanger sequencing reactions coupled with electrophoretic separation of fragments. Applications are encouraged that will provide technology that can ultimately achieve the sequencing goals of the Human Genome Project. Therefore applications should address the development of sequencing technology that when fully developed will allow: o the large-scale sequencing of DNA at a cost significantly below 50 cents per base pair, and o increased large-scale genomic DNA sequence throughput, by at least 10-fold over current methods. Novel sequencing technologies based on reductions in scale that will be considered include, but are not limited to, capillary and ultrathin electrophoresis separation systems, and microfabricated and MEMS systems. Applications to develop such systems should consider front-end sample preparation through separation and detection, aiming for a fully automated, integrated, modular system. Support for individual components of an integrated system will be considered, given indications of a vision towards a fully integrated system or appropriate collaborations. It is important for applicants to demonstrate an appreciation of bottlenecks that arise in the application of existing technology to large-scale genomic DNA sequencing, and to indicate how these bottlenecks will be overcome in the proposed system. In this context, collaboration with large-scale genomic sequencing efforts is encouraged. It is also important for applicants to discuss plans for exportation and support of the technology developed. As the development of a fully functional integrated system is largely dependent on successful data handling, it is anticipated that proposals will deal with this issue in detail. Applications for the development of improved tools for data acquisition, data handling, sequence assembly, and finishing are encouraged, as these steps represent the greatest bottlenecks in current systems and additional improvements in these areas are required to unleash the full power of systems with a capacity for higher throughput. It is crucial that the development of new data acquisition and handling tools be closely linked to large-scale sequencing efforts using current technology and existing data sets, and should also be well connected to ongoing efforts toward reductions in scale to ensure adaptability of ensuing products to higher throughput systems under development. Applications to pursue DNA sequencing projects using state-of-the-art techniques, or improved non-electrophoretic-based technology such as hybridization-based approaches or scanning probe microscopy will not be considered responsive to this request for applications. However, the NCHGR welcomes applications in those areas, as well as other new approaches to DNA sequencing, through program announcements PA-92-50 and PA-90-21. Applications that are solely directed toward the development of databases to support large-scale sequencing projects, or development of algorithms and analytical tools for the annotation of genomic information, should be submitted under program announcement PA-92-50. LETTER OF INTENT Because of the specialized interest of this NCHGR program, it is strongly recommended that potential applicants contact NCHGR staff to discuss research objectives and appropriate mechanisms. Prospective applicants are asked to submit, by February 1, 1994, a letter of intent that includes a descriptive title of the proposed research, the name, address, and telephone number of the Principal Investigator, the identities of other key personnel and participating institutions, and the number and title of the RFA in response to which the application may be submitted. Although a letter of intent is not required, is not binding, and does not enter into the review of subsequent applications, the information that it contains allows NCHGR staff to estimate the potential review workload and to avoid conflict of interest in the review. The letter of intent is to be sent to: Sample Preparation and/or Separation Devices Carol A. Dahl, Ph.D. Sequencing Technology Branch National Center for Human Genome Research Building 38A, Room 610 38 Library Drive MSC 6050 Bethesda, MD 20892-6050 Telephone: (301) 496-7531 FAX: (301) 480-2770 E-mail: xd2@cu.nih.gov Systems Integration, Data Acquisition and Handling Robert L. Strausberg, Ph.D. Sequencing Technology Branch National Center for Human Genome Research Building 38A, Room 610 38 Library Drive MSC 6050 Bethesda, MD 20892-6050 Telephone: (301) 496-7531 FAX: (301) 480-2770 E-mail: cxr@cu.nih.gov APPLICATION PROCEDURES The research grant application form PHS 398 (rev. 9/91) is to be used in applying for these grants. These forms are available at most institutional offices of sponsored research; from the Office of Grants Information, Division of Research Grants, National Institutes of Health, 5333 Westbard Avenue, Room 449, Bethesda, MD 20892, telephone 301/435-0714; and from the NIH program administrator listed under INQUIRIES. The RFA label available in the PHS 398 (rev. 9/91) application form must be affixed to the bottom of the face page of the application. Failure to use this label could result in delayed processing of the application such that it may not reach the review committee in time for review. In addition, the RFA title and number must be typed on line 2a of the face page of the application form and the YES box must be marked. Submit a signed, typewritten original of the application, including the Checklist, and three signed, photocopies, in one package to: Division of Research Grants National Institutes of Health Westwood Building, Room 240 Bethesda, MD 20892** To expedite the review process, at the time of submission, send two additional copies of the application to: Office of Scientific Review National Center for Human Genome Research Building 38A, Room 609 38 Library Drive MSC 6050 Bethesda, MD 20892-6050 Applications must be received by March 17, 1994. If an application is received after that date, it will be returned to the applicant without review. The Division of Research Grants (DRG) will not accept any application in response to this RFA that is essentially the same as one currently pending initial review, unless the applicant withdraws the pending application. The DRG will not accept any application that is essentially the same as one already reviewed. This does not preclude the submission of substantial revisions of applications already reviewed, but such applications must include an introduction addressing the previous critique. REVIEW CONSIDERATIONS Upon receipt, applications will be reviewed for completeness by DRG and for responsiveness to the RFA by the NCHGR program staff. Incomplete applications will be returned to the applicant without further consideration. If the application is not responsive to the RFA, NCHGR staff will contact the applicant to determine whether to return the application to the applicant or submit it for review in competition with unsolicited applications at the next review cycle. Those applications that are complete and responsive will be evaluated in accordance with the criteria stated below for scientific/technical merit by an appropriate peer review group convened by the NCHGR. As part of the initial merit review, a process (triage) may be used by the initial review group in which applications will be determined to be competitive or non-competitive based on their scientific merit relative to other applications received in response to the RFA. Applications judged to be competitive will be discussed and be assigned a priority score. All applicants will receive a summary statement consisting of the reviewer's written comments essentially unedited. Summary statements for competitive applications will also contain a summary of the review committee's discussion. The second level of review will be provided by the National Advisory Council for Human Genome Research. Review criteria for RFAs are generally the same as those for unsolicited research grant applications, and include the following: o scientific, technical, or medical significance and originality of proposed research; o appropriateness and adequacy of the experimental approach and methodology proposed to carry out the research; o qualifications and research experience of the Principal Investigator and staff, particularly, but not exclusively, in the area of the proposed research; o availability of the resources necessary to perform the research; o appropriateness of the proposed budget and duration in relation to the proposed research; Additional scientific/technical merit criteria specific to this RFA include: o likelihood that the proposed technology will serve as a technology that can support the completion of the goals of the Human Genome Project for sequencing of genomic DNA. o degree to which the project contributes to a fully integrated, automated, modular system for the sequencing of genomic DNA; o degree to which the proposed technology considers and effectively overcomes existing bottlenecks in large-scale sequencing of genomic DNA; o degree to which the proposed system addresses data acquisition and handling issues; o degree to which the proposal considers exportation and support of the ensuing technology; AWARD CRITERIA The anticipated date of award is September 30, 1995. The following criteria will be considered in making funding decisions. o the quality of the proposed project as determined by peer review; o balance among the projects in addressing the issues related to the development of a fully integrated, automated, miniaturized, highly parallel system for sequencing of genomic DNA with associated tools for data acquisition and handling, as specified in this RFA; o availability of funds. INQUIRIES Written and telephone inquiries concerning this RFA are encouraged. The opportunity to clarify any issues or questions from potential applicants is welcome. Direct inquiries regarding programmatic issues to: Sample Preparation and/or Separation Devices Carol A. Dahl, Ph.D. Program Director, Sequencing Technology Branch National Center for Human Genome Research Building 38A, Room 610 38 Library Drive MSC 6050 Bethesda, MD 20892-6050 Telephone: (301) 496-7531 FAX: (301) 480-2770 E-mail: xd2@cu.nih.gov Systems Integration, Data Acquisition and Handling Robert L. Strausberg, Ph.D. Chief, Sequencing Technology Branch National Center for Human Genome Research Building 38A, Room 610 38 Library Drive MSC 6050 Bethesda, MD 20892-6050 Telephone: (301) 496-7531 FAX: (301) 480-2770 E-mail: cxr@cu.nih.gov Direct inquiries regarding fiscal matters to: Jean M. Cahill Grants and Contracts Management Section National Center for Human Genome Research Building 38A, Room 613 38 Library Drive MSC 6050 Bethesda, MD 20892-6050 Telephone: (301) 402-0733 FAX: (301) 402-1951 AUTHORITY AND REGULATIONS This program is described in the Catalog of Federal Domestic Assistance No. 93.172. Awards are made under authorization of the Public Health Service Act, Title IV, Part A (Public Law 78-410, as amended by Public Law 99-158, 42 USC 241 and 285) and administered under PHS grants policies and Federal Regulations 42 CFR 52 and 45 CFR 74. This program is not subject to the intergovernmental review requirements of Executive Order 12372 or Health Systems Agency review. The Public Health Service (PHS) strongly encourages all grant recipients to provide a smoke-free workplace and promote the non-use of all tobacco products. This is consistent with the PHS mission to protect and advance the physical and mental health of the American people. .


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