Full Text HG-95-005


NIH GUIDE, Volume 24, Number 9, March 10, 1995

RFA:  HG-95-005

P.T. 34

  Human Genome 
  Nucleic Acid Sequencing 

National Center for Human Genome Research

Letter of Intent Receipt Date:  June 1, 1995
Application Receipt Date:  August 4, 1995


The National Center for Human Genome Research (NCHGR) invites
applications to develop and implement pilot projects to test
strategies that have the potential to lead to full-scale production
sequencing of mammalian DNA, resulting in achieving the goal of the
complete, accurate, finished sequence of human DNA by the year 2005.


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 Request
for Applications (RFA), Pilot Projects for Sequencing of Human DNA,
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
Summary Report:  Stock No. 017-001-00473-1) through the
Superintendent of Documents, Government Printing Office, Washington,
DC 20402-9325 (telephone 202-783-3238).


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.  Applications from
social/ethnic minority individuals, women, and persons with
disabilities are encouraged.  Applications from foreign institutions
will not be accepted.   However, subcontracts to foreign institutions
are allowable, with sufficient justification.

Collaborations and consortia are encouraged.  In such collaborations,
the respective contributions should be well-integrated into the
design of the application to encourage cross-fertilization of ideas
and rapid application of the research to practical purposes.


This RFA will use the National Institutes of Health (NIH) individual
research project grant (R01), pilot project/feasibility study (R21),
research program project (P01), exploratory grant (P20), and center
grant (P50) mechanisms.  The R21 mechanism is used to support highly
creative approaches for which substantial preliminary data are not
yet available.  Each R21 award will be limited to $100,000 direct
costs per year for a maximum of two years.  The P20 mechanism is used
to support groups of outstanding investigators who wish to develop
interdisciplinary research programs.  P20 awards will be limited to
$750,000 direct costs per year for a maximum of three years.  R21 and
P20 grants are not renewable, but future project continuation is
possible through other grant mechanisms such as R01 or P01.
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.  The total project period for
an application submitted in response to this RFA may not exceed three
years (with the exception of the R21 noted above).

Future unsolicited competing continuation applications will compete
with all investigator-initiated applications and be reviewed
according to the customary peer review procedures. The anticipated
award date is April 1, 1996.


Given the importance of both further technology improvement for DNA
sequencing and the need to gain more experience in large-scale
sequencing of human DNA, the NCHGR is implementing a bipartite plan
to advance the capability of large-scale sequencing of human DNA and
to strike a balance between these two program areas.  This RFA
represents one part of this plan and solicits applications to
initiate pilot projects for large-scale sequencing that will increase
the experience in production issues such as improved strategies,
substrate preparation, data analysis, and project management and
organization.  The complementary part of the plan is announced in RFA
HG-95-004, "Improved Electrophoretic DNA Sequencing Technology."  The
scientific goals of these two RFAs are very different but awards
applications submitted for either RFA will be funded out of a single
set-aside of $20 million.  Applicants may respond to both RFA, if
desired, but should not address the two RFAs in a single application.
The anticipated outcome of the plan is to support a set of
applications that will provide the balance needed in the NCHGR
program to enhance progress toward attainment of the sequence of the
human genome.

NCHGR anticipates that projects of widely varying size may be
responsive to this RFA and therefore expects to consider for funding
projects ranging in cost from $100,000 to several million dollars per
year.  Only applications found to be of high scientific merit will be
considered for funding and all of the funds will not be spent if
there are not enough highly meritorious applications.  Funding in
future years is subject to the availability of funds.



The NCHGR sponsors basic and applied research concerned with 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)
in which the goals for DNA sequencing were two-fold:  (1) development
of new technologies to facilitate DNA sequencing and (2) the
production of DNA sequence, primarily that of model organisms.

In the past year, there has been significant progress in developing
the capability for large-scale DNA sequencing.  Several laboratories
have generated at least one, and as many as ten, megabases of DNA
sequence each.  Through this experience, strategic and organizational
lessons, such as how to manage large laboratory efforts devoted to
rapid data production, have been learned.  The capacity of automated
sequencing instruments has increased, and newer, higher throughput
sequencing instruments are close to a stage at which they could be
introduced into a large-scale sequencing environment.  However,
sequencing technology and sequencing capability must still be
improved considerably to achieve the DNA sequencing capability
necessary to determine the sequence of the three billion base pairs
of human DNA within the time and cost goals for the HGP.  The
technological progress and project management experience that have
already been achieved warrant the initiation of human DNA sequencing
at increased scale.  Obtaining more experience in larger scale
sequencing of human DNA is also necessitated by the recognition that
important issues will be encountered in large-scale sequencing of
human DNA that will not be addressed in current large-scale projects
directed to sequencing non-human DNA.  One such issue is the
large-scale generation of sequencing substrates, which in most cases
will be based on high resolution maps.  The current human physical
mapping goal calls for maps with STSs every 100 kb, on average.
Current DNA sequencing methods require maps of considerably higher
resolution but, except for relatively short intervals, such high
resolution maps are not available for human DNA.  The quantity and
type of repetitive sequences present in human DNA, and the sheer size
of the genome, will present challenges to map construction.
Therefore, large-scale human DNA sequencing requires that strategies
to convert low resolution maps to sequencing substrates be developed
and tested.  However, preparation of high resolution physical maps is
not in and of itself, a goal of the NCHGR; for reasons of efficiency
and quality control, it is noted in the extended five-year plan, that
"preparation of sequence-ready sets of clones should be closely
associated with an imminent intent to sequence".

A second issue requiring study is the effect of highly conserved,
highly repetitive sequences on the assembly of DNA sequence.  Large
data sets of human DNA sequence will be useful in studying this
question.  Such data sets will also be useful in developing improved
base-calling algorithms.

In recognition of the importance of both further technology
improvement and the need to gain more experience in large-scale
sequencing of human DNA, the NCHGR is implementing a bipartite plan
to advance the capability of large-scale sequencing of human DNA and
to strike a balance between these two program areas.  This RFA
represents one part of this plan and solicits applications to
initiate pilot projects for large-scale sequencing that will increase
the experience in production issues such as improved strategies,
substrate preparation, data analysis, project management and
organization.  The other, complementary part of the plan is announced
in RFA HG-95-004, "Improved Electrophoretic DNA Sequencing
Technology".  Its purpose is to invite 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 methods that utilize Sanger
sequencing reactions coupled with electrophoretic separation of

Objectives and Scope

The purpose of this RFA is to solicit applications for pilot projects
to increase the capacity of the NCHGR program for the sequencing of
human DNA, i.e., to develop realistic paths that have the potential
to lead to full-scale production sequencing of human DNA, resulting
in achieving the goal of the complete, accurate, finished sequence of
human DNA by the year 2005.  Applications are sought that will
propose an integrated strategy that will span the entire large-scale
sequencing problem, from the up-front preparation of sequencing
substrates through the assembly and analysis of the data.  The
applicant must propose to sequence at least 1 Mb of contiguous DNA
over the three year pilot project period.

If the models tested in these pilot projects are to truly contribute
to the ultimate goal of the HGP, they will have to be capable of
scaling up effectively.  Therefore, applicants should clearly address
the question of how the proposed approach will scale for efficient,
rapid large-scale human DNA sequencing.  Importantly, the issues of
organization and managing large-scale sequencing should be addressed
in so far as is possible, i.e., the pilot project and a tentative
plan for a scaled-up project should be presented.

The primary focus of this RFA is to gain experience in sequencing
human DNA.  However, the inclusion of parallel sequencing of mouse
DNA is acceptable, with appropriate justification.

Applicants should address the following issues:

o  How the pilot-scale project will scale up and lead to the
efficient, cost-effective sequencing of the human genome and
attainment of the year 2005 goal;

o  The "up-front" aspects of high throughput sequencing,
specifically, the preparation of sequencing substrate.  At present,
the density of the markers (STSs) on the human physical mapping
varies widely on different chromosomes.  The HGP goal for the
physical map, to be completed by 1998, is to have one STS marker
every 100 kb, on average.  While such a map is extremely valuable for
many uses, additional effort will be needed to either produce a much
higher resolution map in regions to be sequenced or to develop a
strategy that can use the low resolution map directly in order to
generate the DNA substrates for sequencing.  Thus it is critical for
the applicant to address the transition from the relatively low
resolution map information that is available now to a map of the
resolution required to support sequencing, or to propose an
alternative means of substrate (template) generation.  The proposed
strategy for doing so should be tested on a region of not less than 5
Mb of DNA.  This requirement is intended to ensure that the problem
of converting 100 kb maps to sequencing substrates will be done on a
region large enough to provide information about the problems that
will be encountered in dealing with large-scale substrate
preparation.  If, on the other hand, the project proposes to sequence
regions where only minimal substrate preparation is needed, because
the high resolution map has already been constructed, applicants must
address how substrate preparation for sequencing the entire human
genome will be accomplished.

o  New Technological Developments.  Successful applications will need
a clear-cut scientific and budgetary strategy for maintaining
state-of-the-art sequencing capability.  Applicants must address the
ways in which the proposed strategy will allow incorporation of the
new technological developments in DNA sequencing that will surely
arise during the term of the grant.  The exportability of any
technology or strategies to be developed in the pilot project must
also be addressed.

o  Sequence quality.  While the ultimate goal of the HGP is to
produce DNA sequence of high quality, some applicants may wish to
propose, as a viable path toward attaining the final high quality
sequence, scenarios that produce sequence that is of lower quality
than that expected for the year 2005 product, such as sequence that
has gaps and/or a relatively high error rate.  In such cases, the
applicants must indicate how the pilot project will contribute to
attaining the ultimate goal of accurate and complete sequence.
Applicants must also estimate the value of any such "lower accuracy"
product to the community.  Thus, an applicant's overall vision and
strategy for how the HGP will accomplish its goal of producing an
accurate and complete sequence, is of central importance.
Additionally, the applicant must discuss the cost-effectiveness of a
strategy that involves an investment in lower accuracy sequencing.

Another aspect of sequence quality that applicants must address is
the description of sequence accuracy for the sequence produced in the
pilot project.

o  Cost factors.  It is recognized that the cost of sequencing in
different pilot projects will vary, depending upon several factors
including the laboratory's strategy, state of the starting map, and
current level of experience in sequencing.  However, as the
long-range vision of successful pilot projects must be toward
sequencing the entire human genome, applicants must address both
their costs through the life of the proposed (3 year) project and
projected long-term costs, as part of their overall vision for
efficiently accomplishing the goal of a complete and accurate human

o  Selection of the genomic region(s) to serve as substrate for the
pilot project.  Because of the small number of projects that will be
funded, it will be important that the region(s) chosen for sequencing
accurately represent the breadth of "problems" likely to be
encountered in the sequence of the entire genome.

The U.S. HGP has adopted a policy of encouraging rapid release of
mapping and sequencing data into public databases.  Guidelines
developed by NCHGR and DOE advisors recommend that data be made
publicly available within six months of the time they are verified.
Applicants should fully describe their plans for data release.

Post-Award Management

During the course of the grant period, mapping and cloning
technologies will improve, genomic technologies will evolve, and the
rate of progress and focus of work supported by the grant(s) may
change.  It is expected that the Principal Investigator will make any
necessary adjustment in scientific direction to accommodate the
changing environment.  In order to ensure that the project(s) remains
focused on appropriate goals, incorporates new technological advances
and makes sufficient progress, scientific and programmatic visits to
the grantee will be conducted at a frequency to be negotiated with
the awardee.


Prospective applicants are asked to submit, by June 1, 1995, 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.  Any applicant planning to
submit an application for more than $500,000 direct cost per year
must have contacted staff, listed under INQUIRIES, before submitting
the application or it will be returned to the applicant.

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 Dr. Jane L. Peterson or Dr.
Jeffery A. Schloss at the address listed under INQUIRIES.


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 and from the Office of
Grants Information, Division of Research Grants, National Institutes
of Health, 5333 Westbard Avenue, Room 449, Bethesda, MD 20892,
telephone 301/710-0267.

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
6701 Rockledge Drive, Room 1040 - MSC 7710
Bethesda, MD  20892-7710
Bethesda, MD  20817 (for express mail)

At the time of submission, two additional copies of the application
must also be sent to:

Ms. Linda Engel
Office of Scientific Review
National Center for Human Genome Research
Building 38A, Room 604
38 Library Drive MSC 6050
Bethesda, MD  20892-6050

Applications must be received by August 4, 1995.  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.


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.
Site visits will not be performed as part of the initial review and
applicant interviews will only be conducted in exceptional cases.
Therefore, applicants must present a complete and well-justified
written application. As part of the initial merit review, all
applications will receive a written critique and may undergo a
process in which only those applications deemed to have the highest
scientific merit will be discussed and assigned a priority score.
All applications will receive a second level of review by the
National Advisory Council for Human Genome Research.

Review criteria are the following:

o  scientific and technical merit of the proposed research to meet
the objectives of this RFA, including:
1.  novelty of the strategies and methods proposed;
2.  exportability of the strategies and technologies under
investigation, or quality of the plan describing how the genome will
be sequenced if the strategies and technologies are not exportable;
3.  plan for incorporating new technologies (e.g., a new base-calling
instrument) at reasonable cost;

o  significance and originality of the research and methodological

o  feasibility of the research and adequacy of the experimental

o  likelihood that the pilot project will be able to scale to a
successful system for sequencing the entire human genome;

o  qualifications and research experience of the Principal
Investigator and staff, particularly, but not exclusively, in the
area of the proposed research;

o  quality of integration of the proposed solutions to critical
large-scale sequencing issues (upstream to base- calling to

o  quality of the management plan;

o  availability of the facilities, resources 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.

Additional factors to be considered in the evaluation of competing
renewal applications will be:

o  prior demonstrated success in increasing throughput, decreasing
cost, and maintaining accuracy in DNA sequencing;

o  timeliness with which data have been placed in the public domain;


The earliest anticipated date of award is April 1, 1996.
Applications submitted in response to this RFA and RFA HG-95-004 will
compete for the same pool of funds.  Factors that will be used to
make award decisions are as follows:

o  Quality of the proposed project as determined by peer review;

o  Promise of the proposed program to contribute to the goals of RFAs
HG-95-004 and HG-95-005, and to the balance between further
technology development and pilot sequence production in the NCHGR
sequencing program;

o  Overall contribution of the pilot project to knowledge and
experience required to sequence the human genome in its entirety;

o  Selection of region(s) for study that will present the array of
problems likely to be encountered in sequencing the entire human

o  Nature and extent of the plans for placing DNA sequence data in
the public domain and disseminating information on technological
developments in a timely manner;

o  Availability of funds.


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:

Jane L. Peterson, Ph.D. or Jeffery A. Schloss, Ph.D.
Mammalian Genomics 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
Email:  jane_peterson@nih.gov
Email:  jeff_schloss@nih.gov

Direct inquiries regarding fiscal matters to:

Ms. Jean Cahill
Grants Management Officer
National Center for Human Genome Research
Building 38A, Room 613
38 Library Drive MSC 6050
Bethesda, MD  20892-6050
Telephone:  (301) 402-0733
Email:  jean_cahill@nih.gov


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 Part 74.  This program is not subject to the intergovernmental
review requirements of Executive Order 12372 or Health Systems Agency

The PHS 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 routing education,
library, day care, health care or early childhood development
services are provided to children.  This is consistent with the phs
mission to protect and advance the physical and mental health of the
american people.


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