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 301/435-0714, 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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	Office of Extramural Research (OER)			National Institutes of Health (NIH) 9000 Rockville Pike Bethesda, Maryland 20892			Department of Health and Human Services (HHS)
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