This Program Announcement expires three years from the Release Date shown 
directly below.

TECHNOLOGIES FOR CLOSING DNA SEQUENCE GAPS AND IMPROVING METHODS FOR 
OBTAINING THE SEQUENCE OF DIFFICULT-TO-SEQUENCE REGIONS

Release Date:  June 27, 2000

PA NUMBER:  PAS-00-112

National Human Genome Research Institute

THIS PA USES THE "MODULAR GRANT" AND "JUST-IN-TIME" CONCEPTS.  IT INCLUDES 
DETAILED MODIFICATIONS TO STANDARD APPLICATION INSTRUCTIONS THAT MUST BE USED 
WHEN PREPARING APPLICATIONS IN RESPONSE TO THIS PA.

PURPOSE

The National Human Genome Research Institute (NHGRI) invites applications to 
develop strategies and technologies for obtaining DNA sequence in the gaps 
that, due to limitations in available cloning and sequencing technology, will 
remain in essentially finished genomic sequence.  Such gaps may arise from an 
inability to clone a region in any available vector system or to an inability 
to obtain sequence from all or part of an available clone. Such gaps that 
remain have been encountered in every large genome sequencing effort to date.  
NHGRI is encouraging development of novel approaches that will allow 
completion of the DNA sequence within the gaps that are left by current 
sequencing methods and that will improve the efficiency of sequencing in 
genomic regions that have proved difficult to sequence.  

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 Program Announcement 
(PA), Technology for Closing DNA Sequence Gaps, 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 and foreign 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.  Racial/ethnic minority individuals, 
women, and persons with disabilities are encouraged to apply as principal 
investigators. 

MECHANISM OF SUPPORT

This PA will use the National Institutes of Health (NIH) regular research 
grant (R01) and exploratory/developmental grant (R21) mechanisms.  
Responsibility for the planning, direction, and execution of the proposed 
project will be solely that of the applicant.  The total project period for 
an application submitted in response to this PA may not exceed three years.  

Specific application instructions have been modified to reflect "MODULAR 
GRANT" and "JUST-IN-TIME" streamlining efforts.  Complete and detailed 
instructions and information on Modular Grant applications can be found at 
http://grants.nih.gov/grants/funding/modular/modular.htm.

FUNDS AVAILABLE

Approximately $2 million is available for funding grants directed at this 
problem and five to ten awards may be made during the first year of the 
program, contingent upon the availability of funds and receipt of a 
sufficient number of high quality applications. The anticipated award dates 
are December 1, April 1 and July 1.  This program announcement will be in 
effect for three years, additional announcements to continue this program may 
be issued in the future.

RESEARCH SCOPE AND OBJECTIVES

BACKGROUND

The NHGRI is currently engaged, along with several other federal, private, 
and international organizations, in a multi-year research program called the 
Human Genome Project (HGP).  Many of the initial goals of the HGP, including 
genetic and physical maps of the mouse and human, and the DNA sequences of E. 
coli, S. cerevisiae, C. elegans and D. melanogaster, have been realized. A 
working draft version of at least 90% of the euchromatic part of the human 
genome will be completed in 2000, and the complete high quality human 
sequence will follow within the next couple of years.  By the end of May 
2000, more than 20% of the human sequence had been finished, including all of 
chromosomes 21 and 22, and approximately 90% of the genome was in "working 
draft" form.  Mouse genome sequencing has also begun, an intermediate version 
will be generated within the next couple of years and the complete sequence 
will follow by 2005 or sooner.

In the course of trying to complete the sequence of some of the larger 
genomes such as that of C. elegans and human chromosomes 21 and 22, 
investigators have encountered a number of regions that they have not been 
able to finish with their current capabilities, resulting in gaps in the 
otherwise finished, contiguous high-quality sequence.  There are a number of 
problems that lead to gaps. Certain genomic regions apparently cannot be 
cloned in any of the large-insert vectors routinely used in high-throughput 
sequencing (BACs, PACs, or cosmids) or the other large-insert vectors 
sometimes used to complement these systems (e.g., YACs or fosmids).  Gaps may 
also originate from the inability of shorter regions to be cloned into the 
smaller sequencing vectors (plasmids, M13) routinely used for subcloning or 
in whole-genome shotgun approaches. Finally, small gaps may arise when 
currently available sequencing chemistries are unable to completely read 
through the sequence of an available clone.  The reasons for these small gaps 
may include the presence of repetitive elements, high GC content, high AT 
content, or other sequence features. Thus, despite the best efforts of genome 
sequencing centers to use all available methods, some gaps have remained 
refractory to closure, apparently due to the limits imposed by the current 
technology.  In recognition that such problematic regions exist, the standard 
that has been adopted by the international sequencing effort for declaring 
that the sequence of a genome is "essentially complete" is that the sequence 
of all regions that can be cloned in standard vectors has been determined, 
and that all remaining gaps have been mapped, sized, and annotated.  Thus, 
for example, the published chromosome 22 sequence (Nature 402:6761 (1999)) 
actually contains eleven gaps for which sequence could not be obtained, 
because clones covering the regions could not be found despite considerable 
effort to find them in several clone libraries. These gaps were sized and 
shown to be small.  The published chromosome 21 sequence included three clone 
gaps and seven sequencing gaps (Nature 405: 311 - 319 (2000)).  

In addition, genomic sequencing projects have also encountered numerous 
regions that, although they can be closed, require significant and prolonged 
efforts to do so.  The genomic sequencers have developed numerous 
techniques/strategies that can be applied to these regions.  These include 
directed sequencing using oligonucleotide primers, the construction and 
sequencing of very short insert shotgun libraries ("shatter" libraries), 
transposon-based approaches, PCR sequencing across gaps, and the use of 
alternative sequencing chemistries.  Similarly, a certain amount of 
experience in sequencing highly repeated regions has been obtained.  This has 
allowed the sequencing of the large tandem repeats in the C. elegans genome 
that are similar to centromeres in other organisms, although even in these 
cases it has not been possible to determine if the repeats have been entirely 
covered.  Other focused efforts to sequence centromeres in Arabidopsis and 
Drosophila have found that some sequencing of centromeric DNA has been 
possible and have revealed very interesting results. The existence of genes 
in such regions has been documented, and data that have contributed to the 
understanding of centromere function have been obtained.  

SCOPE

Overall, there are at present no methods that can be successfully used to 
resolve all difficult-to-sequence regions, and there are still regions that 
cannot be sequenced by any available method in almost every large genome 
sequenced to date.  The purpose of this solicitation is to stimulate the 
development of novel, innovative approaches to the sequencing of regions that 
are currently refractory, or very difficult, to sequence by existing methods.  
In order to provide access to clones containing regions that contain gaps or 
DNA that was difficult to sequence, the large-scale sequencing centers have 
provided lists of clones containing such regions that are posted at 
http://www.nhgri.nih.gov/About_NHGRI/Der/gapPA.html.  Investigators applying 
for this PA may use this information to find clones on which they may develop 
and demonstrate their strategy/technology.

This PA is limited to proposals to develop and obtain proof of principle for 
new technologies, rather than to large-scale application of any method.  
Support for production-scale application of methods developed through this PA 
should be sought through a subsequent, separate program.  

INCLUSION OF WOMEN AND MINORITIES IN RESEARCH INVOLVING HUMAN SUBJECTS

It is the policy of the NIH that women and members of minority groups and 
their subpopulations must be included in all NIH supported biomedical and 
behavioral research projects involving human subjects, unless a clear and 
compelling rationale and justification is provided that inclusion is 
inappropriate with respect to the health of the subjects or the purpose of 
the research.  This policy results from the NIH Revitalization Act of 1993 
(Section 492B of Public Law 103-43).

All investigators proposing research involving human subjects should read the 
"NIH Guidelines For Inclusion of Women and Minorities as Subjects in Clinical 
Research," which have been published in the Federal Register of March 28, 
1994 (FR 59 14508-14513) and in the NIH Guide for Grants and Contracts, Vol. 
23, No. 11, March 18, 1994, available on the web at the following URL 
address:  http://grants.nih.gov/grants/guide/notice-files/not94-100.html

INCLUSION OF CHILDREN AS PARTICIPANTS IN RESEARCH INVOLVING HUMAN SUBJECTS

It is the policy of NIH that children (i.e., individuals under the age of 21) 
must be included in all human subjects research, conducted or supported by 
the NIH, unless there are scientific and ethical reasons not to include them.  
This policy applies to all initial (Type 1) applications submitted for 
receipt dates after October 1, 1998.

All investigators proposing research involving human subjects should read the 
"NIH Policy and Guidelines on the Inclusion of Children as Participants in 
Research Involving Human Subjects" that was published in the NIH Guide for 
Grants and Contracts, March 6, 1998, and is available at
http://grants.nih.gov/grants/guide/notice-files/not98-024.html.

Investigators also may obtain copies of these policies from the program staff 
listed under INQUIRIES.  Program staff may also provide additional relevant 
information concerning the policy.

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.

APPLICATION PROCEDURES

Applications are to be submitted on the grant application form PHS 398 (rev. 
4/98) is to be used in applying for these grants, with the modifications 
noted below.  Applications will be accepted at the standard application 
deadlines as indicated in the application kit.  Application kits 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, email: [email protected].

Applicants planning to submit an investigator-initiated new (type 1), 
competing continuation (type 2), competing supplement, or any amended/revised 
version of the preceding grant application types requesting $500,000 or more 
in direct costs for any year are advised that he or she must contact the 
Institute program staff before submitting the application, i.e., as plans for 
the study are being developed.  Furthermore, the application must obtain 
agreement from the Institute staff that the Institute will accept the 
application for consideration for award.  Finally, the applicant must 
identify, in a cover letter sent with the application, the staff member and 
Institute who agreed to accept assignment of the application.

This policy requires an applicant to obtain agreement for acceptance of both 
any such application and any such subsequent amendment.  Refer to the NIH 
Guide for Grants and Contracts, March 20, 1998, at 
http://grants.nih.gov/grants/guide/notice-files/not98-030.html.

The modular grant concept establishes specific modules in which direct costs 
may be requested as well as a maximum level for requested budgets.  Only 
limited budgetary information is required under this approach.  The just-in-
time concept allows applicants to submit certain information only when there 
is a possibility for an award.  It is anticipated that these changes will 
reduce the administrative burden for the applicants, reviewers and Institute 
staff.  The research grant application form PHS 398 (rev 4/98) is to be used 
in applying for these grants, with modifications noted below.

SPECIFIC INSTRUCTIONS FOR MODULAR GRANT APPLICATIONS

BUDGET INSTRUCTIONS

Modular Grant applications will request Direct Costs in $25,000 modules, up 
to a total direct cost request of $250,000 per year.  (Applications that 
request more than $250,000 direct costs in any year must follow the 
traditional PHS 398 application instructions).  The total direct costs must 
be requested in accordance with the program guidelines and the modifications 
made to the standard PHS 398 application instructions described below:

PHS 398

o FACE PAGE- Items 7a and 7b should be completed, indicating Direct Costs (in 
$25,000 increments up to a maximum of $250,000) and Total Costs [Modular 
Total Direct plus Facilities and Administrative (F&A) Costs] for the initial 
budget period.  Items 8a and 8b should be completed indicating the Direct and 
Total Costs for the entire proposed period of support.

o DETAILED BUDGET FOR THE INITIAL BUDGET PERIOD - Do not complete Form Page 4 
of the PHS 398.  It is not required and will not be accepted with the 
application.

o BUDGET FOR THE ENTIRE PROPOSED PERIOD OF SUPPORT - Do not complete the 
categorical budget table on Form Page 5 of the PHS 398.  It is not required 
and will not be accepted with the application.

o NARRATIVE BUDGET JUSTIFICATION - Prepare a Modular Grant Budget Narrative 
Page. (See http://grants.nih.gov/grants/funding/modular/modular.htm for 
sample pages.)  At the top of the page, enter the Total Direct Costs 
requested for each year.  This is not a Form Page.

o PERSONNEL - List key project personnel, including their names, percent of 
effort, and roles on the project.  No individual salary information should be 
provided.  However, the applicant should use the NIH appropriation language 
salary cap and the NIH policy for graduate student compensation in developing 
the budget request.

o CONSORTIUM/CONTRACTUAL COSTS - Provide an estimate of  total costs (direct 
plus F&A costs) for each year, each rounded to the nearest $1,000.  List the 
individuals/organizations with whom consortium or contractual arrangements 
have been made, the percent effort of key personnel, and their role on the 
project.  Indicate whether the collaborating institution is foreign or 
domestic.  The total cost for a consortium/contractual arrangement is 
included in the overall requested modular direct cost amount.  Include the 
Letter of Intent to establish a consortium.

o Provide an additional narrative budget justification for any variation in 
the number of modules requested.

o BIOGRAPHICAL SKETCH - The Biographical Sketch provides information used by  
reviewers in the assessment of each individual"s qualifications for a 
specific role in the proposed project, as well as to evaluate the overall 
qualifications of the research team.  A biographical sketch is required for 
all key personnel, following the instructions below.  No more than three 
pages may be used for each person.  A sample biographical sketch may be 
viewed at:  http://grants.nih.gov/grants/funding/modular/modular.htm 

o Complete the educational block at the top of the form page,
o List position(s) and any honors,
o Provide information, including overall goals and responsibilities, on 
research projects ongoing or completed during the last three years,
o List selected peer-reviewed publications, with full citations.

o CHECKLIST - This page should be completed and submitted with the 
application.  If the F&A rate agreement has been established, indicate the 
type of agreement and the date.  All appropriate exclusions must be applied 
in the calculation of the F&A Costs for the initial budget period and all 
future budget years.

o The applicant should provide the name and phone number of the individual to 
contact concerning fiscal and administrative issues if additional information 
is necessary following the initial review. 

The title and number of this program announcement must be typed on line 2 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 five signed photocopies in one package to:

CENTER FOR SCIENTIFIC REVIEW
NATIONAL INSTITUTES OF HEALTH
6701 ROCKLEDGE DRIVE, ROOM 1040, MSC 7710
BETHESDA, MD  20892-7710
BETHESDA, MD  20817 (for express/courier service)

REVIEW CONSIDERATIONS

Applications will be assigned on the basis of established PHS referral 
guidelines. Applications will be evaluated for scientific and technical merit 
by an appropriate scientific review group convened in accordance with the 
standard NIH peer review procedures. As part of the initial merit review, all 
applications will 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 applications under review, will be discussed, 
assigned a priority score, and receive a second level review by the 
appropriate national advisory council or board.

Review Criteria

The goals of NIH-supported research are to advance our understanding of 
biological systems, improve the control of disease, and enhance health. In 
the written comments reviewers will be asked to discuss the following aspects 
of the application in order to judge the likelihood that the proposed 
research will have a substantial impact on the pursuit of these goals. Each 
of these criteria will be addressed and considered in assigning the overall 
score, weighting them as appropriate for each application. Note that the 
application does not need to be strong in all categories to be judged likely 
to have major scientific impact and thus deserve a high priority score. For 
example, an investigator may propose to carry out important work that by its 
nature is not innovative but is essential to move a field forward.

(1) Significance: Does this study address an important problem? If the aims 
of the application are achieved, how will scientific knowledge be advanced? 
What will be the effect of these studies on the concepts or methods that 
drive this field?

(2) Approach: Are the conceptual framework, design, methods, and analyses 
adequately developed, well integrated, and appropriate to the aims of the 
project? Does the applicant acknowledge potential problem areas and consider 
alternative tactics?

(3) Innovation: Does the project employ novel concepts, approaches or method? 
Are the aims original and innovative? Does the project challenge existing 
paradigms or develop new methodologies or technologies?

(4) Investigator: Is the investigator appropriately trained and well suited 
to carry out this work? Is the work proposed appropriate to the experience 
level of the principal investigator and other researchers (if any)?

(5) Environment: Does the scientific environment in which the work will be 
done contribute to the probability of success? Do the proposed experiments 
take advantage of unique features of the scientific environment or employ 
useful collaborative arrangements? Is there evidence of institutional 
support?

In addition to the above criteria, in accordance with NIH policy, all 
applications will also be reviewed with respect to the following:

o The adequacy of plans to include both genders, minorities and their 
subgroups, and children as appropriate for the scientific goals of the 
research. Plans for the recruitment and retention of subjects will also be 
evaluated.

o The reasonableness of the proposed budget and duration in relation to the 
proposed research

o The adequacy of the proposed protection for humans, animals or the 
environment, to the extent they may be adversely affected by the project 
proposed in the application.

o For R21 applications, preliminary data are not required. However, the 
applicant does have the responsibility for developing a sound research plan 
and for presenting any other information that can be considered as evidence 
of feasibility. 

Award Criteria

Applications will compete for available funds with all other recommended 
applications.  The following will be considered in making funding decisions:  
quality of the proposed project as determined by peer review, availability of 
funds, and program priority. 

INQUIRIES

Inquiries 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. 
Division of Extramural Research 
National Human Genome Research Institute 
31 Center Drive, Room B2B07
Building 31, MSC 2033
Bethesda, MD 20892-2033
Telephone: (301) 496-7531 
FAX: (301) 480-2770 
Email:  [email protected]

Direct inquiries regarding fiscal matters to: 

Ms. Jean Cahill 
Grants Management Office 
National Human Genome Research Institute 
31 Center Drive, Room B2B34
Building 31, MSC 2031
Bethesda, MD 20892-2031
Telephone: (301) 402-0733 
FAX: (301) 402-1951 
E-mail: [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 Part 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 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 PHS mission to protect and advance the physical and mental health of 
the American people.

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