RFA-HG-01-003: COMPARATIVE GENOMIC SEQUENCING OF DROSOPHILA DNA

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COMPARATIVE GENOMIC SEQUENCING OF DROSOPHILA DNA Release Date: June 11, 2001 RFA: RFA-HG-01-003 National Human Genome Research Institute (http://www.nhgri.nih.gov/) Letter of Intent Receipt Date: July 20, 2001 Application Receipt Date: August 21, 2001 PURPOSE The National Institute of Human Genome Research (NHGRI) announces its interest in receiving proposals to obtain, within one year, sequence coverage and an initial assembly of the genome of Drosophila pseudoobscura. The sequence information is intended primarily to be used in conjunction with the nearly complete sequence of D. melanogaster to identify conserved regions that might be of biological significance, such as genes, intron/exon boundaries, and cis-regulatory regions, among others. HEALTHY PEOPLE 2010 The Public Health Service (PHS) is committed to achieving the health promotion and disease prevention objectives of "Healthy People 2010," a PHS-led national activity for setting priority areas. This Request for Applications (RFA), "COMPARATIVE GENOMIC SEQUENCING OF DROSOPHILA", is related to one or more of the priority areas. Potential applicants may obtain a copy of "Healthy People 2010" at http://www.health.gov/healthypeople/. ELIGIBILITY REQUIREMENTS Applications may be submitted by domestic non-profit and for-profit organizations, private and public, such as universities, colleges, private companies, hospitals, laboratories, units of state or local governments, and eligible agencies of the Federal government. Applications from foreign institutions will not be accepted, however subcontracts to foreign institutions will be considered. Racial/ethnic minority individuals, women, and persons with disabilities are encouraged to apply as Principal Investigators. The goals of this RFA can be met in the expected time frame only by groups that have already established a capability for high-throughput DNA sequencing. Therefore, applications will be accepted only from organizations that have demonstrated the ability to produce 2 million high quality sequencing reads within 12 months. MECHANISM OF SUPPORT This RFA will use the National Institutes of Health (NIH) Cooperative Agreement (U01), an "assistance" mechanism, which is distinguished from a regular research grant in that substantial scientific and/or programmatic involvement by NHGRI staff with the awardee is anticipated. The cooperative agreement is used when participation by NIH staff is warranted to support and/or stimulate the recipient"s activity by involvement in and otherwise working jointly with the award recipient in a partner role, NIH staff will not assume direction, prime responsibility, or a dominant role in the activity. Details of the responsibilities, relationships, and governance of the studies funded under cooperative agreement(s) are discussed later in this document under the section "Terms and Conditions of Award". This initiative will be managed by the staff of NHGRI. The length of the project period will be one year. FUNDS AVAILABLE Up to $5 million (total costs) will be available to support a single, one year award under the terms of this RFA. The actual level of support will depend on the receipt of an application of high scientific merit and the availability of funds. RESEARCH OBJECTIVES Many of the objectives that were initially defined for the Human Genome Project (HGP) have been obtained, and essentially all of the rest will be met within the next two to three years. In particular, the DNA sequences of Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster, have been determined, and significant progress has been made towards the sequencing of the human genome (an initial "working draft" version was published in February, 2001). Sequencing of the genomes of several other important laboratory organisms, including the mouse, rat, and zebrafish have also begun. The finished human sequence is expected by 2003, a low-coverage version of the mouse sequence has been generated, a more detailed version will be available within the next twenty-four months, and a finished sequence no later than 2005, similarly, a low-coverage version of the rat sequence will be generated by the end of 2003. The sequences of these genomes, and of the many other small genomes that have also been generated in the past few years, are important data sets that will be used, in combination with additional information about these organisms, to increase our understanding of basic biology and disease. One important use of genomic sequence data is to aid in the identification of genes and other functional DNA sequence elements, and to help predict gene function. However, even the best current methods for predicting gene structure and function directly from the sequence of a single organism are no more than moderately successful. Methods that compare sequence from two or more sources, on the other hand, are considerably more effective in finding genes and other functional elements in DNA sequence data, and can also provide some insight into some important facets of gene function. Depending on the evolutionary distance between the sequences being compared, such analyses can reveal either similarities, as conserved features (e.g., coding regions, intron/exon boundaries, functional non-coding regions), or differences (e.g. Snaps, insertions, deletions), both of which can be important for addressing specific biological questions. A short list of insights that can be gained from appropriate comparative sequence studies includes: o confirmation of gene identification, o definition of accurate gene structure, o identification of small genes and genes containing single exons that are missed by other methods, o identification of non-protein coding genes, o identification of conserved protein domains, o identification of alternative splice products, o identification of orthologs by synteny, o information about RNA secondary structure, o identification of cis-acting control regions, o information on transcriptional networks, o information about evolutionary relationships. Over 100 years of research has established the fruit fly as one of the most important and best understood experimental systems for studying many biological problems, including the molecular and genetic basis of development, and population genetics. Experimental studies of Drosophila melanogaster benefit from some of the most sophisticated and powerful genetic techniques and resources available, including chemical and insertional mutagenesis, highly detailed physical and meiotic map resources, and the ability to readily transform an embryo, among many others. Furthermore, it is now well established that conclusions based on the substantial molecular understanding of D. melanogaster development can often be informative about vertebrate development and disease. With the genomic sequence of D. melanogaster nearly completed, the Drosophila system is poised to become a prime testing ground for comparative sequence analysis at the genomic level. Thus, the availability of the sequence from a second Drosophila species would significantly increase the value of the initial D. melanogaster sequence, by enabling the comparative approach to the identification and study of conserved regions for gene function, regulation, and chromosome organization and mechanics. Further, hypotheses generated from results based on computational comparisons in Drosophila can rapidly be confirmed experimentally. The selection of a second Drosophila species to sequence involves many factors. Perhaps the most important is the evolutionary distance between the second species and D. melanogaster as this, to a large extent, will determine the kinds of insights that can be gained from comparative analyses. Comparison with the sequence of a relatively close species might be expected to tend to provide more insight into mechanisms of speciation, whereas comparisons that identified sequences that are similar in two more diverged genomes might be expected to generate important information about functional components of the genome. In addition to evolutionary distance, other factors that will influence the choice of the second Drosophila genome to sequence include genome size (which affects the total cost of the project), availability, and prevalence and ease of use in the laboratory. The primary intent of this RFA is to obtain sequence information that will be useful for comparative studies to identify regions of the Drosophila genome that have been conserved for important functional reasons. The choice of a target for comparative studies should be at an appropriate evolutionary distance from D. melanogaster---50 to 60 million years---for this purpose. This evolutionary distance is thought to be sufficient for sequences that are not functionally constrained to have been substituted, but not so distant that functionally constrained regions have been substituted to the extent that they are undetectable. A number of Drosophila species that satisfy this condition are routinely maintained in the laboratory. These include D. virilis, D. funebris, D. willistoni, and D. pseudoobscura. Of these, D. pseudoobscura has the smallest genome size (120-150 Mb of euchromatic DNA, approximately the same as that of D. melanogaster). For these reasons, the Revised Drosophila White Paper 2001 (http://www.nih.gov/science/models/fly/whitepaper2001.html)---an assessment by the Drosophila community of future research resource needs---identifies D. pseudoobscura as a strong candidate for the second Drosophila species to be sequenced. There are also several resources useful for a genomic sequencing project already available for D. pseudoobscura, including a 15X deep BAC library made from sheared D. pseudoobscura embryo DNA, and a fosmid library. Thus, this solicitation targets the genomic sequence of D. pseudoobscura. RESEARCH SCOPE The goal of this RFA is to generate an assembled genomic sequence of D. pseudoobscura of sufficiently high quality to form a basis for sequence comparison with the sequenced genome of D. melanogaster, and eventually that of yet other species. The quality of the assembled D. pseudoobscura sequence should be sufficient to facilitate insights such as those provided in the short list above, but especially those relating to gene finding and confirmation of gene structure, and identification of conserved non-coding regions. In practical terms, this means an assembly with few enough gaps so that the average contig size is greater than the size of the average gene. There are alternate strategies to generating genome coverage and assembly of the quality sought by this RFA. As one example, it has been reported that a genome of the size and complexity of D. melanogaster can be assembled to a high degree, even using as low as 6.5X sequence coverage, by a whole genome shotgun strategy (see M.D. Adams, et al., Science 287, p. 2185-2195, 2000, and E.W. Myers et al., Science 287, p. 2196-2203, 2000). Similarly, clone-based strategies have also been successful in assembling complete sequences of genomes of this size (see The C. elegans Sequencing Consortium Science 282, p. 2012-2018, 1998, The Arabidopsis Initiative Nature 408, p. 796 815, 2000). This RFA does not specify the strategy to be used for sequencing the genome of D. pseudoobscura, leaving up to the applicant to propose the strategy that s/he prefers. It should be understood that the assembly sought by this RFA is not expected to represent a finished sequence, but rather a version of the sequence that is of maximal utility and optimal quality within the constraints of the funds available. The applicant should justify the appropriateness of the sequencing strategy proposed in terms of the quality of the assembly and the utility of that assembly for comparative sequence studies. Because of the small size of the D. pseudoobscura genome relative to contemporary sequencing capacity, only one award will be made. Applicants must address the items listed in Application Guidance for Production Sequencing under Application Procedures. NHGRI POLICIES CONCERNING DNA SEQUENCING Over the past several years, NHGRI has established a number of policies related to large-scale sequencing as described below. These policies will apply to the award made as a result of this RFA. Thus, where applicable, applicants must present plans to adhere to the policies: Intellectual Property. In NHGRI"s opinion, in the absence of specific biological information, genomic DNA sequence information should not be patented but released into the public domain where it will be freely available for use by the entire research community (see web site: http://www.nhgri.nih.gov/Grant_info/Funding/Statements/RFA/intellectual_property.html). Applicants are reminded that the grantee institution is required to disclose each subject invention to the Federal Agency providing research funds within two months after the inventor discloses it in writing to grantee institution personnel responsible for patent matters (see http://www.iedison.gov/). NHGRI will monitor adherence to this policy through the use of the appropriate existing databases, to learn whether or not attempts are being made to patent large blocks of D. pseudoobscura DNA sequence. Data Release. NHGRI policy calls for grantees to release DNA sequence assemblies of 2,000 base pair units or larger within 24 hours of assembly (see http://www.nhgri.nih.gov/Grant_info/Funding/Statements/). NHGRI has developed an extension of that policy to accommodate public release of data from other sequencing approaches on a similarly rapid basis. Current policy (contact NHGRI Program Staff listed below for details) is that read and trace data, such as those from whole genome shotgun efforts, are to be deposited weekly in the NCBI trace repository (http://www.ncbi.nlm.nih.gov/Traces/). Sequence Quality. An important component of the HGPs sequencing program has been the establishment of quality standards for both finished and working draft sequence. Just as important has been the on-going assessment of the quality of the sequence data produced by the HGP participants, and NHGRI regularly assesses the quality of sequence data produced under its large scale-sequencing program. The standards and the results of previous quality assessment exercises are described at http://www.nhgri.nih.gov:80/Grant_info/Funding/Statements/RFA/quality_standard.html. As the strategy that will be used for sequencing the D. pseudoobscura genome is not specified in this RFA, it is not possible to specify quality standards. Rather, the applicant should propose a set of quality standards appropriate to the strategy and product that will be generated. If necessary, the standards will be negotiated prior to making the award, and will then be included in the Terms and Conditions. The quality standards should be stated in terms of factors such as read quality, data tracking, and assembly quality (see "Application Guidance for Production Sequencing" under "Application Procedures"). The results of any previous quality assessment activities should also be described. The applicant should discuss the results of recent NHGRI-conducted quality assessment of his/her group"s sequence data, if such an assessment has been done. If an applicant has not participated in an NHGRI-sponsored quality assessment exercise, s/he should be prepared to submit data for independent analysis during the review of the application, if the reviewers request such information. SPECIAL REQUIREMENTS FOR COOPERATIVE AGREEMENTS 1. Definitions ARBITRATION PANEL: A panel that is formed to review scientific or programmatic disagreement (within the scope of the award) that may arise between the award recipient and NHGRI. It will be composed of three members: (a) a designee of the awardee, (b) one NHGRI designee, and (c) a third designee with relevant expertise who is chosen by the other two. The Arbitration Panel will help resolve any scientific or programmatic issues that may develop during the course of work and that restrict progress. AWARDEE: The institution to which the cooperative agreement is awarded. COOPERATIVE AGREEMENT: An assistance mechanism in which there is anticipated substantial involvement by NIH program staff with the recipient organization during the performance of the planned activity. NHGRI PROGRAM DIRECTOR: Scientist of the NHGRI extramural staff who provides normal stewardship for the award and who, in addition, has substantial scientific/programming involvement during conduct of this activity, as defined in the terms and conditions of award. The nature of this involvement is described below. PRINCIPAL INVESTIGATOR (P.I.): The person who assembles the project, is responsible for submitting the application in response to this RFA, and is responsible for the performance of the project. The Principal Investigator will coordinate project activities scientifically and administratively. SCIENTIFIC ADVISORY PANEL (SAP): A panel that evaluates the progress of the Genome Sequencing Network and provides recommendations to the Director, NHGRI, about continued support of the components of the NHGRI large-scale sequencing program. The SAP is composed of four to six senior scientists with relevant expertise who are not P.I.s of a cooperative agreement involved in the NHGRI large-scale sequencing program. If it is deemed necessary, a subcommittee to the SAP may be appointed to deal with any specific issues that might arise with the D. pseudoobscura genome sequencing project. 2. Terms and Conditions of Award The following terms and conditions will be incorporated into the award statement and will be provided to the Principal Investigator, as well as the appropriate institutional official, at the time of award. The following special terms of award are in addition to, and not in lieu of, otherwise applicable OMB administrative guidelines, HHS grant administration regulations at 45 CFR Parts 74 and 92 [Part 92 is applicable when State and local Governments are eligible to apply], and other HHS, NIH, and NIH grant administration policies: A. The administrative and funding instrument used for this program will be the Cooperative Agreement (U01, U54). The cooperative agreement is an "assistance" mechanism (rather than an "acquisition" mechanism), in which substantial NIH scientific and/or programmatic involvement with the awardee is anticipated during the performance of the activity. Under the Cooperative Agreement, the NIH purpose is to support and/or stimulate the recipient"s activity by involvement in and otherwise working jointly with the award recipient in a partner role, but it is not to assume direction, prime responsibility, or a dominant role in the activity. Consistent with this concept, the dominant role and prime responsibility for the activity resides with the awardee for the project as a whole, although specific tasks and activities in carrying out the study will be shared among the awardee and the NHGRI Program Director. B. P.I. Rights and Responsibilities: The P.I. will have the primary responsibility for defining the details for the project within the guidelines of the RFA and for performing the scientific activity. The P.I. will agree to accept close coordination, cooperation, and participation of NHGRI staff in those aspects of scientific and technical management of the project as described under "NHGRI Program Staff Responsibilities." The P.I. of a sequence production center will: o Determine experimental approaches, design protocols, set project milestones and conduct experiments, o Ensure that the genomic sequence produced meets the quality standards and cost agreed upon at the time of award, o Ensure that data resources developed as part of this project are released according to NHGRI policies and that results are published and submitted to GenBank, o Adhere to the NHGRI policies regarding intellectual property, data release and other policies that might be established during the course of this activity, o Submit data for quality assessment in any manner specified by the reviewers, the NHGRI Program Staff, or the subcommittee of the Scientific Advisory Panel identified in the definition above, o Submit quarterly progress reports in a standard format, as agreed upon at the time of the award, o Accept and implement the guidelines and procedures common to all NHGRI large-scale sequencing program awardees. C. NHGRI Program Staff Responsibilities: The NHGRI Program Director will have substantial scientific/programmatic involvement during the conduct of this activity through technical assistance, advice and coordination. However, the role of the NHGRI Program Director will be to facilitate and not to direct the activities. The Program Director will: o Participate in the process of setting research priorities, deciding optimal research approaches and protocol designs, and contributing to the adjustment of research protocols or approaches as warranted. The NHGRI Program Director will assist and facilitate the process and not direct it, o Serve as liaison, helping to coordinate activities between the awardee, the NHGRI and other Institutes and Centers of the NIH, and if necessary, the subcommittee of the SAP. Serve as an information resource about extramural genome research activities. The Program Director will also assist in any necessary coordination of the efforts of the awardee with other U.S. large-scale sequencing efforts and with the international sequencing community. o Assist in developing operating guidelines, quality control procedures, and consistent policies for dealing with recurrent situations that require coordinated action. The Program Director will report progress to the NHGRI Director, and other interested NIH Institutes and Centers periodically. o Assist in promoting the availability of the D. pseudoobscura genome sequence and related resources developed in the course of this project to the scientific community at large. o Retain the option to recommend the withholding or reduction of support from the awardee if it substantially fails to achieve its sequencing goals at the quality and cost agreed to at the time of award, fails to remain state of the art in its production sequencing capabilities, or fails to comply with the Terms and Conditions of the award. o Participate in data analyses, interpretations, and where warranted, co-authorship of the publication of results. D. Scientific Advisory Panel The Scientific Advisory Panel will be responsible, through a subcommittee it appoints as described in the Definitions Section above, for reviewing and evaluating the progress of the awardee toward producing an assembled sequence of the D. pseudoobscura genome. The subcommittee of the Scientific Advisory Panel may meet with the awardee (via conference call or in person) after the second quarterly report and make recommendations regarding progress, and present advice about changes, if any, which may be necessary in the D. pseudoobscura program to the Director, NHGRI. E. Arbitration Process Any disagreement that may arise on scientific/programmatic matters (within the scope of the award), between award recipients and the NHGRI may be brought to arbitration. An Arbitration Panel, whose composition is described in the Definitions (above) will be convened. This special arbitration procedure in no way affects the awardee"s right to appeal an adverse action that is otherwise appealable in accordance with NIH regulations 42 CFR Part 50, Subpart D and HHS regulation at 45 CFR Part 16. F. Quarterly Milestones At the time of award, the awardee will define quarterly milestones for the D. pseudoobscura sequencing project and will update these milestones as appropriate during the course of the project. These milestones will be made a condition of the award. Quarterly reports will address the attainment of these milestones. URLS IN NIH GRANT APPLICATIONS OR APPENDICES All applications and proposals for NIH funding must be self-contained within specified page limitations. Unless otherwise specified in an NIH solicitation, internet addresses (URLs) should not be used to provide information necessary to the review because reviewers are under no obligation to view the Internet sites. Reviewers are cautioned that their anonymity may be compromised when they directly access an Internet site. LETTER OF INTENT Prospective applicants are asked to submit 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 a subsequent application, the information that it contains allows IC staff to estimate the potential review workload and plan the review. The letter of intent should be sent by July 20, 2001 to: Dr. Adam L. Felsenfeld Program Director, Large Scale Sequencing National Human Genome Research Institute National Institutes of Health Building 31, Room B2B07, MSC 2033 Bethesda, MD 20892-2033 APPLICATION PROCEDURES The research grant application form NIH 398 (rev. 4/98) is to be used in applying for these grants. These forms are available at most institutional offices of sponsored research and may be obtained from the Division of Extramural Outreach and Information Resources, National Institutes of Health, 6701 Rockledge Drive, MSC 7910, Bethesda, MD 20892-7910, telephone 301/710-0267, E-mail: GrantsInfo@nih.gov. The RFA label available in the NIH 398 (Rev 4/98) application form must be affixed to the bottom of the face page of the application. Type the RFA number on the label. 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 2 of the face page of the application form and the YES box must be marked. The sample RFA label available at http://grants.nih.gov/grants/funding/phs398/label-bk.pdf has been modified to allow for this change. Please note this is in pdf format. Submit a signed, typewritten original of the application, including the Checklist, and three signed photocopies, in one package to: CENTER FOR SCIENTIFIC REVIEW NATIONAL INSTITUTES OF HEALTH 6701 ROCKLEDGE DRIVE, SUITE 1040 - MSC 7710 BETHESDA, MD 20892-7710 or BETHESDA, MD 20817 (for express/courier service) At the time of submission, two additional copies of the application must also be sent to: Dr. Ken Nakamura Scientific Review Administrator Office of Scientific Review Building 31, Room B2B37, MSC 2032 National Human Genome Research Institute National Institutes of Health Bethesda, MD 20982-2032 Telephone: (301) 402-0838 Applications must be received by August 21, 2001. If an application is received after that date, it will be returned to the applicant without review. The Center for Scientific Review (CSR) will not accept any application that is essentially the same as one currently pending initial review, unless the applicant withdraws the pending application. The CSR will not accept any application that is essentially the same as one already reviewed. This does not preclude the submission of a substantial revision of an application already reviewed, but such an application must follow the guidance in the PHS 398 applications instructions for the preparation of revised applications, including an introduction addressing the previous critique. SPECIAL APPLICATION GUIDANCE FOR PRODUCTION SEQUENCING Applicants must consider and address the following in preparing applications for this RFA: 1. Progress Report The NHGRI has conducted several competitions for large-scale sequencing projects in the past few years, and has learned that there are specific information items that are central to the review of large-scale sequencing proposals. It has been our experience that the applications that have been most highly rated have provided such information clearly and succinctly. Section A. Text. The total length for this section should not exceed 10-15 pages (5000-7500 words). Brief, concise answers are encouraged. Please focus these answers on your past accomplishments, rather than future plans, unless specifically asked for. A. Sequence production. How does your group"s past performance support its ability to successfully accommodate the D. pseudoobscura sequencing project? Discussion should include, but is not limited to: 1) Prior experience in sequence production. What type of genomic sequence (e.g., BAC-based, whole genome shotgun, draft, finished, etc.) has your group produced in the past year? How much of each type was produced? How much of each type, if any, was deposited in GenBank? 2) Based on an average of the last three months of sequencing, what is the total current capacity of your group? Please include the number of attempted lanes per month, the number of successful lanes per month, and the number of base pairs per lane at of least phred 20--or equivalent--quality. Please include the capacity used for sequencing genomic DNA of all organisms and supported by funding from all sources. 3) If you have produced paired end reads, describe your record in tracking the paired ends as well as your success in retrieving the data. 4) Describe your experience in assembly of genomes of (at least) the size and complexity of D. pseudoobscura. What assembly software and hardware resources do you have available? 5) Prior experience in attaining milestones. What example(s) can you provide that you have proposed milestones for a sequencing project and then met them on schedule? What internal metrics have you used to evaluate progress in the past and what internal metrics (for example, reads/month, failed lanes, base pairs per lane in GenBank, etc.) are the most useful to you in managing your project"s sequencing performance? 6) Cost analysis. Cost analyses should be expressed in terms of total costs, which include all equipment and indirect costs. What is your current cost per lane for shotgun sequencing? Do you anticipate being able to reduce that further in the next year? In the next two years? If so, please explain how. How do you monitor costs internally? 7) Integration. Do you anticipate that there will be any issues associated with expanding your group"s program to include sequencing the genome of a new organism? If not, please explain why. If so, please discuss how those integration problems will be addressed. B. Discuss how your center checks the quality of the sequence it produces. C. How do you expect your management plan to accommodate the needs of this project? D. State your data release policy. Section B. Graphical and Tabular Material Please provide the following material. A. A graph indicating, for the past year, the number of lanes attempted per week, the number of successful lanes per week, and the weekly success rate. B. Please provide a graph showing shotgun sequence output per month for at least the last 12 months. (This should be a non-cumulative monthly total). 2. Research Proposal Sequence Production Plan. The applicant must present a plan to implement and evaluate a strategy for sequencing and assembling the D. pseudoobscura genome. The plan must include milestones for achieving the proposed sequence production. The plan must thoroughly discuss and justify the applicant"s specific choices pertinent to all phases of the sequence pipeline, appropriate to the strategy proposed, starting with obtaining D. pseudoobscura DNA or a large-insert clone library through the release of the sequence data to the trace repository and the assembly to GenBank. The applicant should also discuss whether the data to be generated could be used to produce the finished D. pseudoobscura sequence if such an outcome were to be called for in the future and, if so, how. It will be important to discuss potential bottlenecks or other problems that may be anticipated and how they will be addressed. A plan for assembly (including a description of computational issues) of the genomic sequence must be provided, and the expected characteristics of the proposed assembly should be discussed including, for example, expected contig size and distribution, expected gap size and distribution, amount of genome covered in contigs of various size. Finally, the applicant should describe the anticipated utility of the expected assembly for comparative sequence studies (as outlined in the Research Scope above). This should be supported by reference to the literature or by preliminary results. Sequence Cost. The cost of shotgun sequencing must be thoroughly addressed. Both past sequencing costs (monthly costs for the six complete months prior to the application submission date) and projected costs for D. pseudoobscura sequence production must be discussed. It is not necessary to address finishing costs, the discussion of costs should reflect the production of only the sequence assembly proposed in the application. The calculated costs of sequencing (both prior and projected sequencing costs) must take into account all of the expenses associated with sequence production, beginning with the DNA obtained to start the project through the assembly of the sequence to the depth proposed, as well as the cost of data submission. The total cost of sequencing must also include any production-related technology development (see below) that has been or will be supported by the project. However, the applicant must also provide a breakdown of costs so that the reviewers can evaluate the contribution of different cost elements, such as production-related technology development, to the reported total cost. Sequence Quality: The applicant should describe existing quality assessment protocols and an analysis of the quality of sequence produced in the period in the six full months prior to the application submission date. Internal quality control programs should be described, including quality assessment criteria, which may include (for example) read length at Phred-20 (or comparable) quality, paired- end rate, mis-pair rate, data tracking, insert size distribution, etc. Evidence of the usefulness of such programs should be included, as well as any changes being proposed for the D. pseudoobscura sequencing project. The applicant must also be prepared to submit sequence data produced in the last six months, including sequence traces, success rates, and information about data tracking, prior to review if NHGRI and the reviewers decide that data quality needs to be assessed in more detail. This decision will be made after the application has been seen by the reviewers. If an applicant"s data have been evaluated by an NHGRI quality assessment exercise in the past six months, this may be sufficient to fulfill this requirement. Management Plan. The management of a sequencing center requires a significant commitment by the P.I. of the project. A P.I. for a large- scale project is expected to devote at least 30% effort to the project. If a P.I. is already devoting more than 30% effort to his or her existing center, an additional 10% effort should be committed to the D. pseudoobscura sequencing project. A description of how the sequencing project will be integrated into the other sequencing activities of the center should be provided. REVIEW CONSIDERATIONS General Considerations Upon receipt, applications will be reviewed for completeness by CSR and for responsiveness by the NHGRI. Incomplete applications will be returned to the applicant without further consideration. If NHGRI staff find that the application is not responsive to the RFA, it will be returned without further consideration. Applications that are complete and responsive to the RFA will be evaluated for scientific and technical merit by an appropriate peer review group convened by the NHGRI in accordance with the review criteria stated below. As part of the initial merit review, a process will be used by the initial review group in which applications receive a written critique and undergo a process in which only those applications deemed to have the highest scientific merit, generally the top half of the applications under review, will be discussed, assigned a priority score, and receive a second level review by the National Advisory Council for Human Genome Research. All applications will be judged on the basis of the scientific and technical merit of the proposed projects and the documented ability of the investigators to meet the RESEARCH OBJECTIVES of the RFA. Review Criteria The application must be directed toward attaining the programmatic goals as stated under RESEARCH OBJECTIVES AND SCOPE. The following criteria will be used by peer review groups to evaluate these applications: o Likelihood that the project will produce an assembled sequence of D. pseudoobscura, of high quality and utility for comparative sequence studies, within the constraints of the funds provided in the RFA. o Prior experience and quality of the proposed plans for: a) producing high quality sequence data, b) producing a high-quality genome assembly, relative to the need for comparative sequence studies. o Quality of the proposed plan for sequence production and identifying and solving critical integration problems, including adequacy of the informatics activities. o Sequence quality: a) merit of sequence quality assessment plans, including monitoring and minimizing sequencing errors, and other QA/QC plans, b) results from NHGRI sequence quality assessment exercises o Track Record of the P.I. and other key personnel o Quality of the center"s existing management, including workflow, division of labor/responsibility among components, coordination between components, appropriate staffing, training, etc. o Plans for release of data and resources developed through this project. o Plans to coordinate efforts with other U.S. and international groups with a critical role in Drosophila genomics, e.g., FlyBase, etc. o Availability of the facilities, resources, expertise and technology necessary to perform the research, and the level of institutional commitment. o Appropriateness of the proposed budget and time-line in relation to the proposed research. AWARD CRITERIA Awards will be made on the basis of scientific and technical merit as determined by peer review, program needs and balance, plans for data release and intellectual property, and the availability of funds. INQUIRIES Written and telephone inquiries concerning this RFA are encouraged. The opportunity to clarify issues or questions about the RFA from potential applicants is welcome. Direct inquiries regarding programmatic issues to: Dr. Adam L. Felsenfeld Division of Extramural Research National Human Genome Research Institute National Institutes of Health Building 31, Room B2B07, MSC 2033 Bethesda, MD 20892-2033 Telephone: (301) 496-7531 FAX: (301) 480-2770 E-mail: Adam_Felsenfeld@nih.gov For review issues: Dr. Ken Nakamura Scientific Review Administrator Office of Scientific Review National Human Genome Research Institute National Institutes of Health Building 31, Room B2B37, MSC 2032 Bethesda, MD 20982-2032 Telephone: (301) 402-0838 FAX: (301) 435-1580 E-mail: Ken_Nakamura@nih.gov Direct inquiries regarding fiscal matters to: Ms. Jean Cahill Grants Administration Branch National Human Genome Research Institute Building 31, Room B2B34, MSC 2031 Bethesda, MD 20892-2031 Telephone: (301) 402-0733 FAX: (301) 402-1951 E-mail:Jean_Cahill@nih.gov Schedule Letter of Intent Receipt Date: July 20, 2001 Application Receipt Date: August 21, 2001 Scientific Review Date: November 5, 2001 Advisory Council Date: February, 2002 Anticipated Award Date: February, 2002 AUTHORITY AND REGULATIONS This program is described in the catalog of Federal Domestic Assistance No. 93.172. Awards are made under the authority 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 NIH grants policies and Federal Regulations 42 CFR Part 52 and 45 CFR Parts 74 and 92. This program is not subject to the intergovernmental review requirements of Executive Order 122372 or Health Systems Agency review. The NIH strongly encourages all grant and contract recipients to provide a smoke-free workplace and promote the non-use of all tobacco products. In addition, Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking in certain facilities (or in some cases, any portion of a facility) in which regular or routine education, library, day care, health care or early childhood development services are provided to children. This is consistent with the NIH mission to protect and advance the physical and mental health of the American people.

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