RELEASE DATE:  February 12, 2004
RFA Number:  RFA-HG-04-002 (This RFA has been reissued, see RFA-HG-05-003)

EXPIRATION DATE:  October 15, 2004

Department of Health and Human Services (DHHS)
National Institutes of Health (NIH)

National Human Genome Research Institute (NHGRI)


LETTER OF INTENT RECEIPT DATE:  March 15, 2004, September 14, 2004
APPLICATION RECEIPT DATE:  April 15, 2004, October 14, 2004

o Purpose of this RFA
o Research Objectives
o Mechanism(s) of Support 
o Funds Available
o Eligible Institutions
o Individuals Eligible to Become Principal Investigators
o Special Requirements
o Where to Send Inquiries
o Letter of Intent
o Submitting an Application
o Supplementary Instructions
o Peer Review Process
o Review Criteria
o Receipt and Review Schedule
o Award Criteria
o Required Federal Citations


The purpose of this Request for Applications (RFA) is to solicit grant 
applications to develop novel technologies that will substantially 
reduce the cost of genomic DNA sequencing.  Current technologies are 
able to produce the sequence of a mammalian-sized genome of the desired 
data quality for $10 to $50 million; the goal of this initiative is to 
reduce costs by at least two orders of magnitude.  It is anticipated 
that emerging technologies are sufficiently advanced that, with 
additional investment, it may be possible to achieve proof of principle 
or even early stage commercialization for genome-scale sequencing 
within five years.  A parallel RFA HG-04-003  
solicits grant applications to develop technologies to meet the longer-
term goal of achieving four-orders of magnitude cost reduction in about 
ten years.



The ability to sequence complete genomes and the free dissemination of 
the sequence data have dramatically changed the nature of biological 
and biomedical research.  Sequence and other genomic data have the 
potential to lead to remarkable improvement in many facets of human 
life and society, including the understanding, diagnosis, treatment and 
prevention of disease; advances in agriculture, environmental science 
and remediation; and the understanding of evolution and ecological 

The ability to sequence many genomes completely has been made possible 
by the enormous reduction of the cost of sequencing in the past two 
decades, from tens of dollars per base in the 1980s to a few cents per 
base today.  However, even at current prices, the cost of sequencing a 
mammalian-sized genome is tens of millions of dollars and, accordingly, 
we must still be very selective when choosing new genomes to sequence.  
In particular, we remain very far away from being able to afford to use 
comprehensive genomic sequence information in individual health care.  
For this, and many other reasons, the rationale for achieving the 
ability to sequence entire genomes very inexpensively is very strong.

There are many areas of high priority research to which genomic 
sequencing at dramatically reduced cost would make vital contributions.

o Expanded comparative genomic analysis across species, which will 
yield great insights into the structure and function of the human 
genome and, consequently, the genetics of human health and disease.  
Studies to date that have been able to compare small regions of several 
genomes, and “draft” versions of full genomes, have clearly 
demonstrated the need for much more complete data sets.  While some of 
the needed data will be obtained over the next two or three years using 
existing DNA sequencing technology, and while costs will continue their 
gradual decline, the cost of current approaches to sequence acquisition 
will continue to limit the amount of useful data that can be produced. 

o Studies of human genetic variation and the application of such 
information to individual health care, which will also require much 
cheaper sequencing technology.  Today, genetic variation must be 
assessed by genotyping the relatively few known differences at a 
relatively small number of loci within the human population.  A richer 
and better characterized catalog of such variable sites is being 
generated to support more detailed and powerful analyses.  While these 
methods are, and will become even more, powerful and likely to provide 
a significant amount of important new information, they are 
nevertheless only a surrogate for determining the full, contiguous 
sequence of individual human genomes, and are not as informative as 
sequencing would be.  For example, current genotyping methods are 
likely to miss rare differences between people at any particular 
location in the genome and have limited ability to determine long-range 
information (e.g., genomic rearrangements).  Therefore, new methods 
based on complete genomic sequencing will be needed to use genomic 
information for individual health care in the most effective manner 

o While the genomes of a few agriculturally important animals and 
plants have been sequenced, the most informative studies will require 
comparisons between different individuals, different domesticated 
breeds and several wild variants of each species.

o Sequence analysis of microbial communities, many members of which 
cannot be cultured, would provide a rich source of medically and 
environmentally useful information.  And accurate, rapid sequencing may 
also be the best approach to microbial monitoring of food and the 
environment, including rapid detection and mitigation of bioterrorism 

Given the broad utility and high importance of dramatically reducing 
DNA sequencing costs, NHGRI is launching two parallel technology 
development programs.  The first, described in this RFA, has the 
objective of reducing the cost of producing a high quality sequence of 
a mammalian-sized genome by two orders of magnitude.  The goal of the 
second program (see accompanying RFA HG-04-003) is the development of 
technology to sequence a genome for a cost that is reduced by four 
orders of magnitude.  For both programs, the cost targets are defined 
in terms of a mammalian-sized genome, about 3 gigabases (Gb), with a 
target sequence quality equivalent to, or better than, that of the 
mouse assembly published in December 2002 (Nature 420:520, 2002).  

The ultimate goal of this program is to obtain technologies that can 
produce assembled sequence (i.e., de novo sequencing).  However, an 
accompanying shorter-term goal is to obtain highly accurate sequence 
data at the single base level, i.e., without assembly information, that 
can be overlaid onto a reference sequence for the same organism (i.e., 
re-sequencing).  This could be achieved, for example, with short reads 
that have no substantial information linking them to other reads.  
While the sequence product of this kind of technology would lack some 
important information, such as information about genomic 
rearrangements, it would nevertheless potentially be available more 
rapidly and produce data of great value for certain uses in studying 
disease etiology and pharmacogenomics, and for comparative genomics 
between closely-related organisms.  Therefore, both programs’ 
objectives include a balanced portfolio of projects developing both de 
novo and re-sequencing technologies.

Sequencing strategy and quality 

State-of-the-art technology (i.e., fluorescence detection of 
dideoxynucleotide-terminated DNA extension reactions resolved by 
capillary array electrophoresis [CAE]) allows the determination of 
sequence “read” segments approximately 1000 nucleotides long.  If all 
of the DNA in a 2-3 Gb genome were unique, it would be possible to 
determine the sequence of the entire genome by generating a sufficient 
number (millions) of randomly-overlapping thousand-base reads and align 
them by overlaps.  However, the human and the majority of other 
interesting genomes contain a substantial amount of repetitive DNA 
(short [tens to thousands of nucleotides], nearly or completely 
identical sequences present in multiple [tens to thousands of] copies).  
To cope with the complexities of repetitive DNA elements and to 
assemble the thousand-base reads in the correct long-range order across 
the genome, current genomic sequencing methods involve a variety of 
additional strategies, such as the sequencing of both ends of cloned 
DNA fragments, use of libraries of cloned fragments of different 
lengths, incorporation of map information, achievement of substantial 
redundancy (multiple reads of each nucleotide from overlapping 
fragments) and application of sophisticated assembly algorithms to 
align and filter the read information.

The “gold standard” for genomic sequencing is 99.99% accuracy (not more 
than one error per 10,000 nucleotides) with essentially no gaps 
(  At present, the final steps in 
achieving that very high sequence quality cannot be automated and 
require substantial hand-crafting.  However, recent experience suggests 
that the majority of comparative sequence information can be obtained 
from automatically generated sequence assemblies that have been 
variously identified as “high-quality draft” or “comparative grade.” 
Therefore, while the ultimate goal is sequencing technology that 
produces perfect accuracy, the goal of the current program is to 
develop technology for producing automatically generated sequence of at 
least the quality of the mouse draft genome sequence that was published 
in December 2002 (Nature 420:520, 2002).

Emerging technologies, collectively characterized as sequencing-by-
synthesis or sequencing-by-extension, may be able to achieve large 
numbers of sequence reads by extending very large numbers of different 
DNA templates simultaneously, but generally only for a few tens of 
bases as currently practiced.  Even if it is possible to extend these 
reads to several hundred bases, it will still be necessary to link 
those reads to achieve long-range sequence contiguity.  For some 
purposes, long-range sequence contiguity may not be required.  For 
example, the re-sequencing of genomes (determination of the DNA 
sequence for many individuals of a species after a reference sequence 
for that species has been determined), such as might be used for 
medical diagnostic purposes, could be achieved by aligning individual 
reads on the reference sequence.  However, short reads, particularly 
ones with lower per-base quality, can be very difficult to align given 
the nature of repetitive DNA and of closely-related gene families in 
complex genomes.  Also, chromosomal rearrangements may be difficult to 
detect without high quality sequence information bridging the 
breakpoints with enough sequence to know in which repeat the breakpoint 
lies.  The determination of single nucleotide polymorphisms (SNPs) and 
their phase (for haplotypes) also requires contiguity of varying 
length.  The ultimate goal and a high priority for the NHGRI’s 
sequencing technology development efforts, as exemplified in these two 
RFAs, continues to be de novo, assembled sequence.  However, because of 
the value of re-sequencing for many future purposes, these RFAs also 
solicit the development of very inexpensive technology for very high 
quality re-sequencing (without assembly).

Technology path

Most investigators interested in reducing DNA sequencing costs 
anticipate that a few additional two-fold decreases in cost can yet be 
achieved with the current CAE-based technology, with a realistic lower 
limit of perhaps $5 million per mammalian-sized genome.  However, it is 
likely that this efficiency will only be achieved in a few very large, 
well-capitalized, experienced, automated laboratories.  To achieve the 
broadest benefit from DNA sequencing technology for biology and 
medicine, systems that are not only substantially more efficient but 
also more usable by the average research laboratory are needed.

One set of current technology development efforts is aimed at 
increasing parallel sample processing while integrating the sample 
preparation and analysis steps on a single platform.  Thus, in one 
approach, lithography is used to create a large number of microchannels 
on a single device and to integrate an efficient sample injector with 
each separation channel.  Chambers for on-chip DNA amplification, cycle 
sequencing reactions and sample clean-up have been also developed, and 
experiments to integrate these steps, an approach that effectively 
places much of the actual process and process control onto the device, 
are being conducted in several laboratories.  Attendant improvements in 
separation polymers and in fluorescent dyes will facilitate these 
developments.  As these approaches are based largely on the experience 
of currently successful high-throughput CAE-based methods, they have 
potential to produce cost savings in the range of several factors of 
two beyond the CAE-based system itself.  They also have the potential 
to widen the user base for the technology, as the infrastructure and 
knowledge needed to conduct relatively high-throughput sequencing, or 
clinical diagnostic sequencing, would be substantially reduced and 

Other approaches to improving sequencing technology involve methods 
that are independent of the Sanger dideoxynucleotide chain termination 
reaction or of electrophoretic separation of the termination products.  
Two methods that were proposed in the early days of the HGP involve the 
use of mass spectrometry and sequencing by hybridization.  Both methods 
have been pursued, with some limited success for sequencing, but 
substantial success for other types of DNA analysis.  Both continue to 
hold additional potential utility for sequencing, although certain 
inherent limitations will need to be overcome.

More recently, additional methodologies have been investigated. These 
may be classified into two approaches. One is sequencing-by-extension, 
in which template DNA is elongated stepwise and each extension product 
is detected.  Extension is generally achieved by the action of a 
polymerase that adds a deoxynucleotide, followed by detection of a 
fluorescent or chemiluminescent signal; the cycle is then repeated.  
Modifications of this approach rely on other types of enzymes and 
detection of hybridization of labeled oligonucleotides.  To obtain 
sufficient throughput, the method is implemented at a high level of 
multiplexing, e.g., by arraying large numbers of sequencing extension 
reactions on a surface.  A key factor in this general approach is the 
manner in which the fluorescent signal is generated and the system 
requirements thus imposed.  Depending on the specific approach, 
challenges of template extension methods include the synthesis of 
labeled nucleotide analogues; identification of processive polymerases 
that can incorporate nucleotide analogs with high fidelity; 
discrimination of fluorescent nucleotides that have been incorporated 
into the growing chain from those present in the reaction mix 
(background); distinction of subsequent nucleotide additions from 
previous ones; accurate enumeration of homopolymer runs (multiple 
sequential occurrence of the same nucleotide); maintenance of synchrony 
among the multiple copies of DNA being extended to generate a 
detectable signal, or achievement of sensitivity that detects extension 
of individual DNA molecules; and development of fluidics, surface 
chemistry, and automation to build and run the system.  Current efforts 
to develop such methods have produced, at best, short sequence reads 
(less than or equal to 100 bases), so a continuing challenge is to 
extend read length and develop sequence assembly strategies.  NHGRI 
anticipates that the state of the art for this approach is sufficiently 
advanced that, with additional investment, it may be possible to 
achieve proof of principle or even early commercialization for genome-
scale sequencing within five years.  It is anticipated that the cost of 
genome sequencing with this technology could be reduced by two orders 
of magnitude from today’s costs.  It is important to note that 
sequencing by extension is one prototype for achieving these time and 
cost goals, but other technological approaches may also be viable.  
Developing technology with which to reduce the cost of genome 
sequencing by 100-fold is the subject of this RFA.

A second alternative to CAE sequencing seeks to read out the linear 
sequence of nucleotides without copying the DNA and without 
incorporating labels, relying instead on extraction of signal from the 
native DNA nucleotides themselves.  The most familiar model for this 
approach, but almost certainly not the only way to achieve 10,000-fold 
reduction in sequencing costs, is nanopore sequencing, first introduced 
in the mid-1990s.  Generally, this approach requires a sensor, perhaps 
comparable in size to the DNA molecule itself, that interacts 
sequentially with individual nucleotides in a DNA chain and 
distinguishes between them on the basis of chemical, physical or 
electrical properties.  Optimal implementation of such a method would 
analyze intact, native genomic DNA molecules isolated from biological, 
medical or environmental samples without amplification or modification, 
and would provide very long sequence reads (tens of thousands to 
millions of bases) rapidly and at sufficiently high redundancy to 
produce assembled sequence of high quality.  NHGRI anticipates that the 
science and technology needed to reduce sequencing costs by four orders 
of magnitude, whether by the nanopore or some other approach, will 
require substantial basic research and development, and may take as 
long as ten years to achieve.  Reaching this goal is the subject of a 
parallel RFA, HG-04-003


The goal of research supported under this RFA is to develop or improve 
technology to enable rapid, efficient genomic DNA sequencing.  The 
specific goal is to reduce sequencing costs by at least two orders of 
magnitude -- $100,000 serves as a useful target cost for a mammalian-
sized genome because the availability of complete genomic sequences at 
that cost would revolutionize biological research and medicine.  While 
not in a cost range that would enable the use of sequencing in 
individualized medicine, such technology would permit the sequencing of 
many genomes for a small fraction of current costs.  A 100-fold cost 
reduction would make possible extensive studies of human variation for 
disease gene studies, substantially expanded comparative genomics to 
understand the human genome, and many other studies relevant to NIH, 
other federal agencies and the private sector. Entirely new lines of 
investigation would be enabled by making “large-scale sequencing” 
accessible to the diverse interests of many research laboratories and 

Many projects aimed at next-generation DNA sequencing technologies 
require substantial advances in a combination of fields such as signal 
detection, enzymology, chemistry, engineering, bioinformatics, etc.  It 
is therefore anticipated that research programs responding to this RFA 
will involve multidisciplinary teams of investigators.  The guidance 
for budget requests accommodates the formation of groups having 
investigators at several institutions, in cases where that is needed to 
assemble a team of the appropriate balance, breadth and experience.

The scientific and technical challenges inherent in achieving a 100-
fold reduction in sequencing costs are considerable.  Achieving this 
goal may require research projects that entail substantial risk.  That 
risk should be balanced by an outstanding scientific and management 
plan designed to achieve the very high payoff goals of this 

Although the ultimate goal of this RFA is to develop full-scale 
sequencing systems, independent research on essential components will 
also be considered to be responsive.  However, it will be important for 
applicants proposing research on system components or concepts to 
describe how the knowledge gained as a result of their project would be 
incorporated into a full system that they might subsequently propose to 
develop, or that is being developed by other groups.  Such independent 
proposals are an important path for pursuing novel, high risk/high pay-
off ideas.

Research conducted under this RFA may include development of the 
computational tools associated with the technology, e.g., to extract 
sequence information, including signal processing, and to evaluate 
sequence quality and assign confidence scores.  It may also address 
strategies to assemble the sequence from the information being obtained 
from the technology or by merging the sequence data with information 
from parallel technology.  However, this RFA will not support 
development of sequence assembly software independent of technology 
development to obtain the sequence.

The quality of sequence to be generated by the technology is of 
paramount importance for this solicitation.  Two major factors 
contributing to genomic sequence quality are per-base accuracy and 
contiguity of the assembly.  Much of the utility of comparative 
sequence information will derive from characterization of sequence 
variation between species, and between individuals of a species.  
Therefore, per-base accuracy must be high enough to distinguish 
polymorphism at the single-nucleotide level (substitutions, insertions, 
deletions).  Experience and resulting policy have established a target 
accuracy of not more than one error per 10,000 bases.  All applications 
in response to this RFA, whether to develop re-sequencing or de novo 
sequencing technologies, must propose achieving per-base quality at 
least to this standard.  

Assembly information is needed for determining sequence of new genomes, 
and ultimately also for genomes for which a reference sequence exists, 
to detect rearrangements, insertions and deletions.  Rearrangements are 
known to cause diseases; knowledge of rearrangement can reveal new 
biological mechanisms.  The phase of single nucleotide polymorphisms to 
define haplotypes is important in understanding and diagnosing disease.  
Achieving a high level of sequence contiguity will be essential to 
achieve the full benefit from the use of sequencing for individualized 
medicine, e.g., to evaluate genomic contributions to risk for specific 
diseases and syndromes, and drug responsiveness.  Nevertheless, it is 
recognized that perfect sequence assembly from end to end of each 
chromosome is unlikely to be achievable with most technologies in a 
fully automated fashion and without adding considerable cost.  
Therefore, for the purpose of this solicitation, grant applications 
proposing technology development for de novo sequencing shall describe 
how they will achieve, for about $1000, a draft-quality assembly that 
is at least comparable to that represented by the mouse draft sequence 
produced by December 2002:  7.7-fold coverage, 6.5-fold coverage in Q20 
bases, assembled into 225,000 sequence contigs connected by at least 
two read-pair links into supercontigs [total of 7,418 supercontigs at 
least 2 kb long], with N50 length for contigs equal to 24.8 kb and for 
supercontigs equal to 16.9 Mb (Nature 420:520, 2002).

The grant applications will be evaluated, and funding decisions made, 
in such a way as to develop a balanced portfolio that has strong 
potential to develop both robust re-sequencing and de novo sequencing 
technologies.  If the estimate that achieving the goal of 100-fold 
reduction in cost for genome sequencing incorporating substantial 
assembly information will require about 5 years to achieve is correct, 
then re-sequencing technologies might be expected to be demonstrated in 
a shorter time.  Grant applications that present a plan to achieve high 
quality re-sequencing while on the path to high quality de novo 
sequencing will receive high priority.   

The major focus of this RFA is on the development of new technologies 
for detection of nucleotide sequence.  However, any new technology will 
eventually have to be effectively incorporated into the entire 
sequencing workflow, starting with a biological sample and ending with 
sequence data of the desired quality, and this issue should be 
addressed.  Given that sample preparation requirements are a function 
of the detection method and the sample detection method affects the way 
in which output data are handled, these aspects of the problem are 
clearly relevant and should be addressed in an appropriate timeframe.  
However, NHGRI is interested in seeing that the most critical and 
highest-risk aspects of the project, on which the rest of the project 
is dependent, are addressed and proven as early as possible.

NHGRI anticipates that successful projects funded through this RFA may 
be sufficiently advanced as to be approaching early stages of 
commercialization within about five years.  Therefore, practical 
implementation issues related to workflow and process control for 
efficient, high quality, high-throughput DNA sequencing should be 
considered early.  Some technology development groups lack practical 
experience in high throughput sequencing, and in testing of methods and 
instruments for robust, routine operation.  Applicants may therefore 
wish to include such expertise as they develop their suite of 
collaborations and capabilities.

The goal of this research is to develop technology to produce sequence 
from entire genomes.  It is conceivable that sequence from selected 
important regions (e.g., all of the gene regions) could be determined 
in the near future, using more conventional technologies, at very low 
cost.  However, that is not the purpose of this initiative, and grant 
applications that propose to meet the cost targets by sequencing only 
selected regions of a genome will be considered unresponsive.

This RFA will use NIH R21, R21/R33, R01 and P01 award mechanism(s).  As 
an applicant you will be solely responsible for planning, directing, 
and executing the proposed project.

Applicants may request an R01 or P01 (depending on the organization of 
the proposed project) if sufficient preliminary data are available to 
support such an application.  A fully integrated management and 
research plan should use the R01 mechanism.  The P01 mechanism should 
be used if multiple projects under different leadership must proceed in 
parallel; however, the issue of synergy in a multi-focal effort is of 
great importance and must be addressed in the application.  

Applicants requiring support to demonstrate feasibility may apply for 
either an R21 pilot/exploratory project or an R21/R33 award, which 
offers single submission and evaluation of both a feasibility/pilot 
phase (R21) and an expanded development phase (R33) in one application.  
The R21/R33 should be used when both quantitative milestones for the 
feasibility demonstration, and a research plan for the follow-on 
research, can be presented. The transition from the R21 award to the 
R33 award will be expedited by administrative review.  The R21 alone is 
appropriate when the possible outcomes of the proposed feasibility 
study are unclear and it is not possible to propose sufficiently clear-
cut and quantitative milestones for administrative evaluation, nor 
would it be possible to describe the R33 phase of the research in 
sufficient detail to allow adequate initial review. 

This RFA uses just-in-time concepts.  It also uses the modular 
budgeting as well as the non-modular budgeting formats (see  
Specifically, if you are submitting an application with direct costs in 
each year of $250,000 or less, use the modular budget format.  
Otherwise follow the instructions for non-modular budget research grant 
applications.  This program does not require cost sharing as defined in 
the current NIH Grants Policy Statement at  
However, cost-sharing is permitted as a component of institutional 


The NHGRI intends to commit approximately $8 million in FY 2004 to fund 
4 to 10 new and/or competitive continuation grants in response to this 
RFA, and an additional $5 million in FY 2005.  For the R21 mechanism, 
an applicant may request a project period of up to 2 years and a budget 
of up to $200,000 direct costs per year.  For R21/R33 applications, the 
total project period may not exceed 3 years, distributed as required 
for the project; the R21 phase may request a budget up to $200,000 
direct cost and the R33 phase up to $2 million direct cost per year.  
For R01 and P01 mechanisms, an applicant may request a project period 
of up to 3 years and a budget of up to $2 million direct cost per year.  
Budgets may exceed this guidance only to accommodate indirect costs to 
subcontracts.  Applicants should be aware that NHGRI intends to fund as 
many promising projects, of varying scope, as possible in order to 
pursue multiple approaches to solving these difficult problems and 
mitigate risk.  Therefore, awards may not be made at the maximum budget 
level.  Instead, the nature and scope of the proposed research will 
vary, and it is anticipated that the size and duration of awards will 
also vary.  Although the financial plans of NHGRI provide support for 
this program, awards pursuant to this RFA are contingent upon the 
availability of funds and the receipt of a sufficient number of 
meritorious applications.  
You may submit (an) application(s) if your institution has any of the 
following characteristics:
o For-profit or non-profit organizations 
o Public or private institutions, such as universities, colleges,             
hospitals, and laboratories 
o Units of State and local governments
o Eligible agencies of the Federal government  
o Domestic or foreign institutions/organizations

Any individual with the skills, knowledge, and resources necessary to 
carry out the proposed research is invited to work with his/her 
institution to develop an application for support.  Individuals from 
underrepresented racial and ethnic groups, as well as individuals with 
disabilities are always encouraged to apply for NIH programs.   

NHGRI anticipates that the development of technologies needed to meet 
these goals will require approximately five years.  Therefore, a 
timeline for a 5 year project should be presented, culminating in the 
demonstration of sequencing a substantial amount of DNA (e.g., at least 
0.5 – 1 gigabase) at the target cost and quality; other measures may be 
proposed by the applicant.  The applicant must also present a much more 
detailed timeline for the initial grant period, which may vary from 1 
year to 3 years.  This detailed timeline should be accompanied by 
quantitative milestones (see below) that address the key scientific and 
technical challenges central to the approach under investigation.  The 
timeline and milestones will be essential for use by both the grantee 
and NHGRI for planning the research projects and assessment of progress 
toward goals, and by the reviewers for evaluating the proposal.   

Timelines and quantitative milestones are essential for development of 
a realistic research plan; they provide a basis for project leaders to 
make decisions, assess their own progress, set priorities, and 
redistribute resources when needed.  It will be particularly important 
to establish quantitative milestones in cases where subsequent steps in 
technology development depend upon threshold performance 
characteristics of earlier developments.  Elaboration of timelines and 
milestones is primarily the responsibility of applicant, and the 
quality and utility of the proposed timelines and milestones will be a 
review criterion, because they reflect the insights and judgment of the 
applicant concerning key challenges and how best to conduct the 
research.  NHGRI appreciates that these projects will require research, 
not just engineering; progress toward milestones will be evaluated 
accordingly.  However, if the proposed timeline and milestones are not 
adequate in the case of an otherwise meritorious proposal, reviewers of 
the application may make recommendations to NHGRI regarding improved 
timelines and milestones.  

In all cases, prior to funding an application, NHGRI will negotiate the 
milestones with the applicant, incorporating recommendations from the 
review panel and the National Advisory Council for Human Genome 
Research.  The negotiated milestones will become a condition of the 
award, including appropriate language to recognize that the project 
includes research whose outcomes are unpredictable.  In the case of 
research programs projected to require longer than the initial grant 
period, the decision to fund beyond the initial period, for a total of 
up to five years, will be based on a competitive renewal process that 
will take into account overall progress in the field as well as 
progress on the individual research effort, as compared to the 
negotiated milestones.  

For R21/R33 awards, the transition from the R21 to the R33 is dependent 
upon completion of negotiated milestones.  Once these milestones have 
been achieved, the investigator will submit a progress report to NHGRI.  
Receipt of this report will trigger a review to determine if the R33 
should be awarded.  The release of R33 funds will be based on 
successful completion of negotiated milestones, negotiation of revised 
milestones for the R33 phase, program priorities, and availability of 

Applicants must plan to submit progress reports twice per year – once 
at the time of the non-competing continuation and once at a time to be 
determined by NHGRI staff.  The latter may coincide with grantee 
meetings or meetings of advisors to NHGRI.  NHGRI will use information 
from reports, site visits, etc. to evaluate each grantee’s progress and 
the success of the overall program; this information will be used to 
determine if funding levels should be increased or decreased for future 
years, for each grant and for the program.

It is anticipated that five years will be required to achieve the goals 
of this program.  Progress during the first two years will be assessed 
by NHGRI and its advisors, and P.I.s and their team leaders will 
receive advice about the NHGRI’s interest in accepting a competing 
application to extend the initial award.

To accelerate progress in the field of advanced DNA sequencing 
technology development, grantees will be expected to participate 
actively and openly in at least one grantee meeting per year.  
Substantial information sharing will be required and is a condition of 
the award; failure to openly share information will be grounds for 
discontinuation of funding.  It is understood that some information 
developed under the grants will be proprietary and cannot be shared 
immediately without damaging the commercialization potential of the 
technology.  Applicants should describe their plans for participating 
in the grantee meetings and for managing the intellectual property 
concerns in the context of those meetings and other opportunities for 
information sharing.  Other investigators in the field (i.e., not 
supported under this program) may be invited to participate in these 
workshops; their agreement to share information substantially will be a 
prerequisite to their participation.  Applicants should request travel 
funds in their budgets for the principal investigator and two 
additional lead investigators to attend the annual meeting.

Grantees will be asked to host the annual grantee meetings on a 
rotating basis.  At the time of award, NHGRI will negotiate a schedule 
for the grantee meetings and will adjust budgets to accommodate these 
meetings.  Holding these meetings at grantee sites will facilitate 
information sharing and participation of a larger portion of the 
research staff than would otherwise occur.

Applicants may include funds for an internally appointed advisory 
board.  However, they should not contact potential advisors, nor should 
potential advisors be named in the grant application, because of the 
conflicts of interest this may produce in the review process.

All applicants must describe their plan for providing access to the 
technology developed under this grant support.  For example, the 
technology might be made available as a fee-for-service, through sale 
of instruments and/or reagents, through collaboration, through 
publication and posting of results, plans and methods, or by other 

Applicants should describe any institutional commitment being offered 
to the support of the project.  Institutional commitment may take many 
forms, including space, equipment and other resources devoted to and 
improved for the project, time and effort of investigators, etc.  This 
information should be incorporated into the management plan (see 


We encourage inquiries concerning this RFA and welcome the opportunity 
to answer questions from potential applicants.  Inquiries may fall into 
three areas:  scientific/research, peer review, and financial or grants 
management issues:

o Direct your questions about scientific/research issues to:

Jeffery A. Schloss, Ph.D.
Division of Extramural Research
National Human Genome Research Institute
Building 31, Room B2B07
Bethesda, MD  20892-2033
Telephone:  (301) 496-7531
FAX:  (301) 480-2770

o Direct your questions about peer review issues to:

Ken Nakamura, Ph.D.
Scientific Review Branch
National Human Genome Research Institute
Building 31, Room B2B37
31 Center Drive, MSC 2032
Bethesda, MD  20892-2032
Telephone:  (301) 402-0838
FAX:  (301) 435-1580

o Direct your questions about financial or grants management matters 

Jean Cahill
Grants Administration Branch
National Human Genome Research Institute
Building 31, Room B2B34
31 Center Drive
Bethesda, MD  20892-2031
Telephone:  (301) 402-0733
FAX: (301) 402-1951

Prospective applicants are asked to submit a letter of intent that 
includes the following information:

o Descriptive title of the proposed research
o Name, address, and telephone number of the Principal Investigator
o Names of other key personnel 
o Participating institutions
o Number and title of this RFA

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 is to be sent by the date listed at the beginning 
of this document.  The letter of intent should be sent to:

Jeffery A. Schloss, Ph.D.
Division of Extramural Research
National Human Genome Research Institute
Building 31, Room B2B07
31 Center Drive
Bethesda, MD  20892-2033
Telephone:  (301) 496-7531
FAX:  (301) 480-2770


Applications must be prepared using the PHS 398 research grant 
application instructions and forms (rev. 5/2001). Applications must 
have a DUN and Bradstreet (D&B) Data Universal Numbering System (DUNS) 
number as the Universal Identifier when applying for Federal grants or 
cooperative agreements. The DUNS number can be obtained by calling 
(866) 705-5711 or through the web site at The DUNS number should be entered on 
line 11 of the face page of the PHS 398 form. The PHS 398 document is 
available at in 
an interactive format.  For further assistance contact GrantsInfo, 
Telephone (301) 710-0267, Email:

SUPPLEMENTARY INSTRUCTIONS:  Follow page limitations described in the 
PHS 398 form 
_instructions.htm#Page_Limitations)with the following modifications:

All mechanisms:  Research to meet the goals of this program is 
anticipated to require about five years.  All applications that propose 
long-term projects should present an outline plan to achieve the 
overall goal in that time.  The research plan must be well developed 
for all years for which a budget is requested and should include a well 
laid out research plan, a description of the level of risk of key 
technical challenges, alternative approaches, go/no-go decision points, 
etc.  Using this approach, it should be possible to prepare well-
justified plans in succinct text that complies with the following page 

R21:  Items a - d of the Research Plan (Specific Aims, Background and 
Significance, Preliminary Studies, and Research Design and Methods) may 
not exceed a total of 15 pages.  No preliminary data are required but 
they may be included if available. 

R21/R33:  The R21/R33 Phased Innovation Award application must be 
submitted as a single application with one face page.  Although it is 
submitted as a single application, it should be clearly organized into 
two phases.  To achieve a clear distinction between the two phases, 
applicants should submit Sections a, b, c and d for the R21 phase, then 
the R21 milestones, and then sections a and d (but not b and c) for the 
R33 phase (including R33 milestones).  A discussion of the milestones 
relative to the progress of the R21 phase, as well as the implications 
of successful completion of the milestones for the R33 phase, should be 
included.  The PHS 398 form Table of Contents should be modified to 
show the sections for each phase as well as the milestones.  There is a 
page limit of 35 pages for the composite research plan.  Section a-d of 
the R21 research plan must not be longer than 15 pages, with the 
remainder available for the milestones and the a and d sections for the 
R33 phase.

R01:  Sections a-d are not to exceed 35 pages.  Up to three additional 
pages explicitly labeled “Management Plan” and inserted between 
sections d and e should describe all aspects of project management, 
incorporating, e.g., how multiple investigators/disciplinary approaches 
will be coordinated, coordination between distant sites, decision-
making processes, use of progress toward milestones as inputs to 
decision-making, etc.

P01:  The combined sections a-d for all of the projects and cores are 
not to exceed 35 pages so that P01, R01 and R21/R33 applicants will be 
on an even playing field for describing their research progress and 
plans.  P01 applicants may refer to background and significance 
sections of one subproject in the text for other projects, to save 
space.  Up to three additional pages explicitly labeled “Management 
Plan” and inserted at the beginning of the Experimental Design section 
should describe all aspects of project management, incorporating, e.g., 
how multiple investigators/disciplinary approaches will be coordinated, 
coordination between distant sites, decision-making processes, use of 
progress toward milestones as inputs to decision-making, etc.

For P01 applications, separate budgets should be provided for each 
project, preceded by a composite budget for the entire grant.
USING THE RFA LABEL: The RFA label available in the PHS 398 (rev. 
5/2001) 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 RFA 
label is also available at:
SENDING AN APPLICATION TO THE NIH: 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, Room 1040, MSC 7710
Bethesda, MD  20892-7710
Bethesda, MD  20817 (for express/courier service)
At the time of submission, two additional copies of the application and 
all copies of the appendix material must be sent to:

Ken Nakamura, Ph.D.
Scientific Review Branch
National Human Genome Research Institute
Building 31, Room B2B37
31 Center Drive, MSC 2032
Bethesda, MD  20892-2032
Telephone:  (301) 402-0838

APPLICATION PROCESSING: Applications must be received on or before the 
application receipt date listed in the heading of this RFA.  If an 
application is received after that date, it will be returned to the 
applicant without review. 

Although there is no immediate acknowledgement of the receipt of an 
application, applicants are generally notified of the review and 
funding assignment within 8 weeks.
The Center for Scientific Review (CSR) 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.  However, when a previously unfunded application, 
originally submitted as an investigator-initiated application, is to be 
submitted in response to an RFA, it is to be prepared as a NEW 
application.  That is, the application for the RFA must not include an 
Introduction describing the changes and improvements made, and the text 
must not be marked to indicate the changes from the previous unfunded 
version of the application.  

Upon receipt, applications will be reviewed for completeness by the CSR 
and responsiveness by the NHGRI. Incomplete applications will not be 

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, all 
applications will:

o 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 and assigned a priority score
o Receive a written critique
o Receive a second level review by the National Advisory Council for 
Human Genome Research.

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 evaluate the 
application in order to judge the likelihood that the proposed research 
will have a substantial impact on the pursuit of these goals. The 
scientific review group will address and consider each of the following 
criteria in assigning the application’s overall score, weighting them 
as appropriate for each application. 

o Significance 
o Approach 
o Innovation
o Investigator
o Environment

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.

SIGNIFICANCE: Does this study address the problem outlined in the RFA, 
the development of technology that has the potential to reduce the cost 
of sequencing a complete genome by two orders of magnitude? 

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?  Are key scientific and technological 
issues on which the rest of the approach depends, identified and 
addressed early in the project?  Does the proposed technology 
incorporate assessment of sequence quality and address the sequencing 
of entire genomes?  Does the application clearly state whether the goal 
is to develop re-sequencing, or de novo sequencing technology, and if 
the latter, is there an adequate plan for evaluating the achieved long-
range contiguity?  Is the analysis of sequencing costs well developed 
and accurate?
INNOVATION: Does the project employ novel concepts, approaches or 
methods?  Are the aims original and innovative? Does the project 
challenge existing paradigms or develop new methodologies or 

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)?  Do the P.I. and other lead investigators have sufficient 
experience to manage a project of the proposed complexity?  Are plans 
to integrate activities and set priorities across the multiple 
disciplines and investigators (and institutions, if appropriate), 
adequately developed and explained?

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?  

ADDITIONAL REVIEW CRITERIA: In addition to the above criteria, the 
following items will be considered in the determination of scientific 
merit and the priority score:

o  Are the plans sufficiently bold to constitute a substantial advance, 
if they can be achieved, toward the demanding goals of the RFA?  Are 
the bold plans counterbalanced to manage the inherent risk, for example 
by firm theoretical basis, reasonable preliminary data, the track 
record of the lead investigators, and an outstanding scientific and 
management plan?

o  Are the timeline and milestones logical and realistic?  Do they 
identify key technology barriers and dependencies?  Are they adequately 
developed and quantitative, to serve as effective guidance for 
assessment of progress by the investigators and the NHGRI?  

o  Are plans to participate actively and openly in grantee meetings 
sufficiently clear and forthcoming so as to contribute substantially to 
advancement of the field?

o  Is the plan for technology dissemination appropriate and adequate?

human subjects and protections from research risk relating to their 
participation in the proposed research will be assessed. (See criteria 
included in the section on Federal Citations, below).
of plans to include subjects from both genders, all racial and ethnic 
groups (and subgroups), and children as appropriate for the scientific 
goals of the research.  Plans for the recruitment and retention of 
subjects will also be evaluated. (See Inclusion Criteria in the 
sections on Federal Citations, below).

are to be used in the project, the five items described under Section f 
of the PHS 398 research grant application instructions (rev. 5/2001) 
will be assessed.


BUDGET:  The reasonableness of the proposed budget and the requested 
period of support in relation to the proposed research.


Letter of Intent Receipt Date:  March 15, 2004, September 14, 2004
Application Receipt Date:  April 15, 2004, October 14, 2004
Peer Review Date:  June-July, 2004, Feb-March, 2005
Council Review:  September 2004, May 2005
Earliest Anticipated Start Date:  September 30, 2004, June 1, 2005


Award criteria that will be used to make award decisions include:

o Scientific merit (as determined by peer review)
o Availability of funds
o Programmatic priorities, including maximizing the possibility for 
successfully achieving the goal of substantial reduction in the cost of 
genomic DNA sequencing.

HUMAN SUBJECTS PROTECTION:  Federal regulations (45CFR46) require that 
applications and proposals involving human subjects must be evaluated 
with reference to the risks to the subjects, the adequacy of protection 
against these risks, the potential benefits of the research to the 
subjects and others, and the importance of the knowledge gained or to 
be gained. 

SHARING RESEARCH DATA:  Starting with the October 1, 2003 receipt date, 
investigators submitting an NIH application seeking $500,000 or more in 
direct costs in any single year are expected to include a plan for data 
sharing or state why this is not possible.  Investigators should 
seek guidance from their institutions, on issues related to 
institutional policies, local IRB rules, as well as local, state and 
Federal laws and regulations, including the Privacy Rule. Reviewers 
will consider the data sharing plan but will not factor the plan into 
the determination of the scientific merit or the priority score.

policy of the NIH that women and members of minority groups and their 
sub-populations must be included in all NIH-supported clinical research 
projects unless a clear and compelling justification is provided 
indicating 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 

All investigators proposing clinical research should read the "NIH 
Guidelines for Inclusion of Women and Minorities as Subjects in 
Clinical Research - Amended, October, 2001," published in the NIH Guide 
for Grants and Contracts on October 9, 2001 
a complete copy of the updated Guidelines are available at
The amended policy incorporates: the use of an NIH definition 
of clinical research; updated racial and ethnic categories in 
compliance with the new OMB standards; clarification of language 
governing NIH-defined Phase III clinical trials consistent with the new 
PHS Form 398; and updated roles and responsibilities of NIH staff and 
the extramural community.  The policy continues to require for all NIH-
defined Phase III clinical trials that: a) all applications or 
proposals and/or protocols must provide a description of plans to 
conduct analyses, as appropriate, to address differences by sex/gender 
and/or racial/ethnic groups, including subgroups if applicable; and b) 
investigators must report annual accrual and progress in conducting 
analyses, as appropriate, by sex/gender and/or racial/ethnic group 

SUBJECTS:  The NIH maintains a policy 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, 

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 is available at

NIH policy requires education on the protection of human subject 
participants for all investigators submitting NIH proposals for 
research involving human subjects.  You will find this policy 
announcement in the NIH Guide for Grants and Contracts Announcement, 
dated June 5, 2000, at

HUMAN EMBRYONIC STEM CELLS (hESC):  Criteria for federal funding of 
research on hESCs can be found at 
and at
Only research using hESC lines that are registered in the 
NIH Human Embryonic Stem Cell Registry will be eligible for Federal 
funding (see   It is the responsibility of the 
applicant to provide, in the project description and elsewhere in the 
application as appropriate, the official NIH identifier(s) for the hESC 
line(s)to be used in the proposed research.  Applications that do not 
provide this information will be returned without review. 

The Office of Management and Budget (OMB) Circular A-110 has been 
revised to provide public access to research data through the Freedom 
of Information Act (FOIA) under some circumstances.  Data that are (1) 
first produced in a project that is supported in whole or in part with 
Federal funds and (2) cited publicly and officially by a Federal agency 
in support of an action that has the force and effect of law (i.e., a 
regulation) may be accessed through FOIA.  It is important for 
applicants to understand the basic scope of this amendment.  NIH has 
provided guidance at

Applicants may wish to place data collected under this PA in a public 
archive, which can provide protections for the data and manage the 
distribution for an indefinite period of time.  If so, the application 
should include a description of the archiving plan in the study design 
and include information about this in the budget justification section 
of the application. In addition, applicants should think about how to 
structure informed consent statements and other human subjects 
procedures given the potential for wider use of data collected under 
this award.

The Department of Health and Human Services (DHHS) issued final 
modification to the “Standards for Privacy of Individually Identifiable 
Health Information”, the “Privacy Rule,” on August 14, 2002.  The 
Privacy Rule is a federal regulation under the Health Insurance 
Portability and Accountability Act (HIPAA) of 1996 that governs the 
protection of individually identifiable health information, and is 
administered and enforced by the DHHS Office for Civil Rights (OCR). 
Those who must comply with the Privacy Rule (classified under the Rule 
as “covered entities”) must do so by April 14, 2003 (with the exception 
of small health plans which have an extra year to comply).  

Decisions about applicability and implementation of the Privacy Rule 
reside with the researcher and his/her institution. The OCR website 
( provides information on the Privacy Rule, 
including a complete Regulation Text and a set of decision tools on “Am 
I a covered entity?”  Information on the impact of the HIPAA Privacy 
Rule on NIH processes involving the review, funding, and progress 
monitoring of grants, cooperative agreements, and research contracts 
can be found at

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.   Furthermore, we caution reviewers that their 
anonymity may be compromised when they directly access an Internet 

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 RFA is related to one or more of the priority areas. 
Potential applicants may obtain a copy of "Healthy People 2010" at

AUTHORITY AND REGULATIONS: This program is described in the Catalog of 
Federal Domestic Assistance at (No. 93.172) and is 
not subject to the intergovernmental review requirements of Executive 
Order 12372 or Health Systems Agency review.  Awards are made under the 
authorization of Sections 301 and 405 of the Public Health Service Act 
as amended (42 USC 241 and 284)  and under Federal Regulations 42 CFR 
52 and 45 CFR Parts 74 and 92. All awards are subject to the terms and 
conditions, cost principles, and other considerations described in the 
NIH Grants Policy Statement.  The NIH Grants Policy Statement can be 
found at

The PHS strongly encourages all grant recipients to provide a smoke-
free workplace and discourage the 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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