Full Text HG-95-004

IMPROVED ELECTROPHORETIC DNA SEQUENCING TECHNOLOGY

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

RFA:  HG-95-004

P.T. 34

Keywords: 
  Human Genome 
  Nucleic Acid Sequencing 


National Center for Human Genome Research

Letter of Intent Receipt Date:  June 15, 1995
Application Receipt Date:  August 29, 1995

PURPOSE

The National Center for Human Genome Research (NCHGR) invites
applications for research projects to develop novel automated
sequencing technology suitable for large-scale genomic sequencing
through the reduction in scale and increased parallelization of
existing approaches that utilize Sanger sequencing reactions coupled
with electrophoretic separation of fragments.  The Request For
Applications (RFA) encompasses front end sample preparation,
separation, detection, and data acquisition and handling.
Technologies solicited include a spectrum of approaches ranging from
capillary and ultrathin gel electrophoresis to microfabricated and
microelectro mechanical systems (MEMS) that could yield reductions in
scale and increased throughput.  This RFA is a reissuance of RFA
HG-95-001.

HEALTHY PEOPLE 2000

The Public Health Service (PHS) is committed to achieving the health
promotion and disease prevention objectives of "Healthy People 2000,"
a PHS-led national activity for setting priority areas.  This RFA,
Improved Electrophoretic DNA Sequencing Technology, is related to
several priority areas, including cancer, heart disease and stroke,
diabetes and chronic disability conditions, maternal and infant
health, and others.  Potential applicants may obtain a copy of
"Healthy People 2000" (Full Report:  Stock No. 017-001-00474-0 or
Summary Report:  Stock No. 017-001-00473-1) through the
Superintendent of Documents, Government Printing Office, Washington,
DC 20402-9325 (telephone 202-783-3238).

ELIGIBILITY REQUIREMENTS

Applications may be submitted by domestic for-profit and non- profit
organizations, public and private, such as universities, colleges,
hospitals, laboratories, units of State and local governments, and
eligible agencies of the Federal Government.  Collaborations between
scientists in academia and industry are encouraged, as are
applications from minority individuals and women.  Applications from
foreign institutions will not be accepted.  However, subcontracts to
foreign institutions are allowable, with sufficient justification.

Applications are particularly encouraged from scientists, such
as engineers and physicists, and institutions, such as for-profit
institutions and biotechnology companies, that have not traditionally
requested research support from the NCHGR.  Applicants whose
expertise is primarily nonbiological are especially encouraged to
interact closely with biologists during the development of the
research plan.

MECHANISM OF SUPPORT

Support for this program will be through the National Institutes of
Health (NIH) individual research project grant (R01), pilot
project/feasibility study (R21), research program project (P01), and
exploratory grant (P20) mechanisms.  The R21 mechanism is used to
support highly creative approaches for which substantial preliminary
data are not yet available.  Each R21 award will be limited to
$100,000 direct costs per year and to a maximum of three years of
support, although the funding limit may be waived under exceptional
circumstances.  The P20 mechanism is used to support groups of
outstanding investigators who wish to develop interdisciplinary
research programs.  Each P20 awards will be limited to three years at
$750,000 direct costs per year.  R21 and P20 grants are not
renewable, but future project continuation is possible through other
grant mechanisms such as R01 or P01.  The levels and time frames for
support of R21 and P20 awards indicated here are specific to this
solicitation.  Responsibility for the planning, direction, and
execution of the proposed project will be solely that of the
applicant. Awards will be administered under PHS grants policy as
stated in the Public Health Service Grants Policy Statement.

This RFA is a one-time solicitation.  Future unsolicited competing
continuation applications will compete with all
investigator-initiated applications and be reviewed according to the
customary peer review procedures.  The total project period for
applications submitted in response to the present RFA may not exceed
three years.  The anticipated award date is April 1, 1996.

FUNDS AVAILABLE

Given 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 scientific areas.  This RFA represents one part of
this plan and invites applications for research projects to develop
novel automated sequencing technology suitable for large-scale
genomic sequencing through the reduction in scale and increased
parallelization of existing approaches utilizing Sanger sequencing
reactions coupled with electrophoretic separation of fragments.  The
complementary part of the plan announced in RFA HG-05-005, "Pilot
Projects for Sequencing of the Human Genome," 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 scientific goals of these two RFAs are very
different, but the applications for both RFAs will be competing for
the same pool of available funds.  The anticipated outcome of the
plan is to support a set of applications that will provide the
balance needed in these two areas of the NCHGR program to enhance
progress toward attainment of the sequence of the human genome.

It is anticipated that $20,000,000 (direct and indirect costs) per
year for up to three years will be available for the combination of
this RFA and RFA HG-95-005, beginning in fiscal year 1996.  It is
anticipated that approximately 15 awards will be made from the pool
of applications received in response to this RFA and RFA HG-95-005,
representing a mix of research topics and mechanisms.  Because the
nature and scope of the research proposed in response to this RFA may
vary, it is anticipated that the size of awards will also vary.  The
number of awards may be increased if a large number of highly
meritorious applications are received and if funds are available.
The number of awards made will be contingent upon the quality of
applications received and the availability of funds.

RESEARCH OBJECTIVES

Background

The NCHGR sponsors basic and applied research concerned with the
development and application of new technologies for the
characterization and analysis of the human genome and the genomes of
selected model organisms.  The activities encompassed by the NCHGR
program include genetic and physical mapping, DNA sequencing,
informatics related to mapping and sequencing, gene identification,
and technology development that will facilitate all of these efforts.

The NCHGR, in conjunction with the Department of Energy, recently
formulated a new five-year plan (Science Vol. 262, pp. 43-46, 1993)
that states that significant technological advances will need to be
made in the area of DNA sequencing if the demanding goals of the
Human Genome Project are to be met.

A major long-term goal of the Human Genome Program is to identify all
the genes encoded in the three billion base pairs of human DNA.  The
first step in this process is to assemble detailed genetic and
physical maps of the human genome.  Subsequently, the complete
sequence of human DNA must be determined.  In order to attain this
second objective, DNA sequencing technology must be further developed
so that the cost will be significantly decreased and the rate
significantly increased.  In 1994, the cost of sequencing performed
by highly skilled researchers in laboratories devoted to genomic DNA
sequencing was estimated to be about $1 per finished base.

The state-of-the-art approach for large-scale sequencing includes
isolation of sample DNA from a biological source followed by
amplification of the DNA and the synthesis of fluorescently tagged
replicate ladders of the template DNA in a Sanger sequencing
reaction.  Biochemistry for DNA sequencing is currently performed in
volumes that range from multiple microliters to hundreds of
microliters, and only modest parallelization and automation has been
achieved. The DNA ladders are resolved on a separation medium through
slab gel electrophoresis and ladder composition is evaluated by
laser-based detection technology.  Software tools are then utilized
for identification of the four bases, assembly of contiguous
sequences from individual reads, and sequence finishing, including
resolution of ambiguities.

Current sequence throughput from automated electrophoresis
instruments is approximately 7,000,000 bases (raw) per instrument
year.  Use of these instruments in sequencing approaches based on a
combination of shotgun and directed strategies results in a
throughput of about 700,000 finished bases per year.  Even at this
relatively modest throughput, sequence assembly and finishing
limitations result in a systems bottleneck.  Current efforts to
optimize genomic sequencing technology focus on the development of a
fully automated, integrated, modular system, that accounts for sample
flow in a way that eliminates bottlenecks and maximally utilizes each
step of the system from the front end of sample isolation through to
data collection and analysis.

Recent advances suggest that dramatically improved DNA sequencing
technology can be developed through the application of existing
miniaturization and automation technologies to state-of-the-art
genomic sequencing.  A continuum of approaches exists that creates a
path to a substantially reduced scale for sequencing devices with
associated throughput increase and cost decrease.  Further
development of these approaches requires several technological
advances that are expected to be addressable based on existing
scientific principles.

Capillary Electrophoresis and Ultrathin Gels

Recent work in ultrathin (capillary or slab) gel electrophoresis has
demonstrated the potential for greatly increased separation speed due
to improved heat transfer.  These approaches are also expected to
yield cost savings by reduction in sample size if small volume
robotic reaction systems are developed concurrently.  Although
preliminary data suggest that these formats will be successful in
improving sequence throughput, technological challenges remain to the
development of dependable, exportable systems.  Remaining challenges
include the need to identify new separation matrices that will allow
for increased reproducibility of separation and easy manipulation in
these formats, small volume loading technology, reduced volume sample
preparation chemistry, improved detection technology, and systems
automation to attain maximal sustainable throughput.

Microfabricated Devices and MEMS

It is anticipated that further reductions in scale and increases in
throughput could be achieved through the application of existing
microfabrication and MEMS to DNA sequencing.  It is anticipated that
successful application of this technology could increase the speed of
sequencing by two to three orders of magnitude.  Technological
challenges that are likely to be encountered are related to the
nature of the sample and include reduced volume sample preparation
chemistries, improved sample loading technologies, optimizing surface
chemistries to minimize sample and reagent loss, and microscale
fluidics and detection issues.

Systems Integration, Data Acquisition and Handling

A key component of high-throughput automated systems will be improved
software tools for base calling, sequence assembly, and sequence
finishing, as well as for overall systems integration. Even in
current approaches, sequence finishing is a bottleneck and requires
manual intervention to achieve sequence closure as well as resolution
of ambiguities.  Therefore, if order of magnitude improvements in
finished sequence throughput are to be achieved, this component will
require substantial attention and creative approaches for full
automation.

Objectives

The purpose of this RFA is to encourage applications from individuals
and groups interested in developing novel automated sequencing
technology suitable for large-scale electrophoresis-based genomic
sequencing through the reduction in scale of existing approaches that
utilize Sanger sequencing reactions coupled with electrophoretic
separation of fragments.  Applications are encouraged that will
provide technology that can ultimately achieve the sequencing goals
of the Human Genome Project.  Therefore applications should address
the development of sequencing technology that when fully developed
will allow:

o  the large-scale sequencing of DNA at a cost significantly below 50
cents per base pair, and

o  increased large-scale genomic DNA sequence throughput, by at least
10-fold over current methods.

Novel sequencing technologies based on reductions in scale that will
be considered include, but are not limited to, capillary and
ultrathin electrophoresis separation systems, and microfabricated and
MEMS systems.  Proposals to develop such systems should consider
front-end sample preparation through separation and detection, aiming
for a fully automated, integrated, modular system.  Support for
individual components of an integrated system will be considered,
given indications of a vision towards a fully integrated system or
appropriate collaborations.  It is important for applicants to
demonstrate an appreciation of bottlenecks that arise in the
application of existing technology to large-scale genomic DNA
sequencing, and to indicate how these bottlenecks will be overcome in
the proposed system.  In this context, collaboration with large-scale
genomic sequencing efforts is encouraged.  It is also important for
applicants to discuss plans for export and support of the technology
developed.

As the development of a fully functional integrated system is largely
dependent on successful data handling, it is anticipated that
proposals will deal with this issue in detail.  Applications for the
development of improved tools for data acquisition, data handling,
sequence assembly, and finishing are encouraged, as these steps
represent the greatest bottlenecks in current systems and additional
improvements in these areas are required to unleash the full power of
systems with a capacity for higher throughput.  It is crucial that
the development of new data acquisition and handling tools be closely
linked to large-scale sequencing efforts using current technology and
existing data sets, and should also be well connected to ongoing
efforts toward reductions in scale to ensure adaptability of ensuing
products to higher throughput systems under development.

Applications to pursue DNA sequencing projects using state-of-the-art
techniques, or improved non-electrophoretic-based technology such as
hybridization-based approaches or scanning probe microscopy will not
be considered responsive to this request for applications.  However,
the NCHGR welcomes applications in those areas, as well as other new
approaches to DNA sequencing, through program announcements PA-92-50
and PA-90-21.  Applications that are solely directed toward the
development of databases to support large-scale sequencing projects,
or development of algorithms and analytical tools for the annotation
of genomic information, should be submitted under program
announcement PA-92-50.

LETTER OF INTENT

Because of the specialized interest of this NCHGR program it is
strongly recommended that potential applicants contact NCHGR staff to
discuss research objectives and appropriate mechanisms. Prospective
applicants are asked to submit, by June 15, 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.

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. Carol A. Dahl or Dr. Robert
L. Strausberg at the address listed under INQUIRIES.

APPLICATION PROCEDURES

The research grant application form PHS 398 (rev. 9/91) is to be used
in applying for these grants.  These forms are available at most
institutional offices of sponsored research; from the Office of
Grants Information, Division of Research Grants, National Institutes
of Health, 5333 Westbard Avenue, Room 449, Bethesda, MD 20892,
telephone 301/710-0267; and from the NIH program administrator listed
under INQUIRIES.

The RFA label available in the PHS 398 (rev. 9/91) application form
must be affixed to the bottom of the face page of the application.
Failure to use this label could result in delayed processing of the
application such that it may not reach the review committee in time
for review.  In addition, the RFA title and number must be typed on
line 2a of the face page of the application form and the YES box must
be marked.

Submit a signed, typewritten original of the application, including
the Checklist, and three signed, photocopies, in one package to:

Division of Research Grants
National Institutes of Health
6701 Rockledge Drive, Room 1040 - MSC 7710
Bethesda, MD  20892-7710
Bethesda, MD  20817 (for express mail)

To expedite the review process, at the time of submission, please
send two additional copies of the application to:

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 29, 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.

REVIEW CONSIDERATIONS

Upon receipt, applications will be reviewed for completeness by DRG
and for responsiveness to the RFA by the NCHGR program staff.
Incomplete applications will be returned to the applicant without
further consideration.  If the application is not responsive to the
RFA, NCHGR staff will contact the applicant to determine whether to
return the application to the applicant or submit it for review in
competition with unsolicited applications at the next review cycle.
Those applications that are complete and responsive will be evaluated
in accordance with the criteria stated below for scientific/technical
merit by an appropriate peer review group convened by the NCHGR.  As
part of the initial merit review, 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 for this RFA are generally the same as those for
unsolicited research grant applications, and are the following:

o  scientific, technical, or medical significance and originality of
proposed research;

o  appropriateness and adequacy of the experimental approach and
methodology proposed to carry out the research;

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

o  availability of the resources necessary to perform the research;

o  appropriateness of the proposed budget and duration in relation to
the proposed research;

Additional scientific/technical merit criteria specific to this RFA
include:

o  likelihood that the proposed technology will serve as a technology
that can support the completion of the goals of the Human Genome
Project for sequencing of genomic DNA.

o  degree to which the project contributes to a fully integrated,
automated, modular system for the sequencing of genomic DNA;

o  degree to which the proposed technology considers and effectively
overcomes existing bottlenecks in large-scale sequencing of genomic
DNA;

o  degree to which the proposed system addresses data acquisition and
handling issues;

o  degree to which the proposal considers exportation and support of
the ensuing technology;

AWARD CRITERIA

The anticipated date of award is April 1, 1996.  The following
criteria will be considered in making funding decisions.

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

o  balance among the projects in addressing the issues related to the
development of a fully integrated, automated, miniaturized, highly
parallel system for sequencing of genomic DNA with associated tools
for data acquisition and handling, as specified in this RFA;

o  promise of the proposed program to contribute to the goals of RFA
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  availability of funds.

INQUIRIES

Written and telephone inquiries concerning this RFA are encouraged.
The opportunity to clarify any issues or questions from potential
applicants is welcome.

Direct inquiries regarding programmatic issues to:

Carol A. Dahl, Ph.D., or Robert L. Strausberg, Ph.D.
Sequencing Technology Branch
National Center for Human Genome Research
Building 38A, Room 610
38 Library Drive MSC 6050
Bethesda, MD  20892-6050
Telephone:  (301) 496-7531
FAX:  (301) 480-2770
Email:  [email protected]
Email:  [email protected]

Direct inquiries regarding fiscal matters to:

Jean M. Cahill
Grants and Contracts Management Section
National Center for Human Genome Research
Building 38A, Room 613
38 Library Drive MSC 6050
Bethesda, MD  20892-6050
Telephone:  (301) 402-0733
FAX:  (301) 402-1951
Email:  [email protected]

AUTHORITY AND REGULATIONS

This program is described in the Catalog of Federal Domestic
Assistance No. 93.172.  Awards are made under authorization of the
Public Health Service Act, Title IV, Part A (Public Law 78- 410, as
amended by Public Law 99-158, 42 USC 241 and 285) and administered
under PHS grants policies and Federal Regulations 42 CFR 52 and 45
CFR 74.  This program is not subject to the intergovernmental review
requirements of Executive Order 12372 or Health Systems Agency
review.

The 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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