The NIH Director
PRIORITY SETTING MEETING FOR MOUSE GENOMICS AND GENETICS RESOURCES
FIRST FOLLOW-UP MEETING
OCTOBER 5, 1998
SUMMARY OF MEETING: POINTS OF CONSENSUS AND ACTION ITEMS
Revised December 14, 1998
A. STRUCTURAL GENOMICS
The major part of the discussion centered around the optimal strategy for an integrated approach to producing detailed maps and genomic sequence for the mouse. There was strong support for producing at least a "working draft" sequence as soon as possible.
The following resources are currently available or under development:
- Two BAC libraries with greater than 10X redundancy. The C57BL6/J library has 170,634 clones with average insert size of 197 kilobases. The 129/SvEvTACfBr library contains 211,953 clones with average insert size of 154 kilobases.
- A low resolution RH map under construction by an international consortium using 2,000 genetic markers and 33,000 STSs derived from ESTs.
- A low resolution physical map generated by screening a 129/SvEvTACfBR library with many of the same markers used to construct the low resolution RH map.
There are several ongoing activities that will enhance these resources: The 3’ ends of additional ESTs are being sequenced to generate additional gene-rich STSs for mapping and through the NCI’s Mouse Cancer Genome Anatomy Project , funds are being provided to generate additional ESTs.
1. The Mouse Strain to be Sequenced
At the March meeting, the 129 SvEvTac strain was recommended as the strain of choice for sequencing, primarily because it is the strain used by most individuals who were pursuing gene targeting experiments. As a result of further discussion in the mouse community, the recommendation is now to use the C57BL6/J strain for genomic sequencing. The reasons for recommending a change from the 129 strain to the C57BL6/J strain are: the C57BL6/J is the most widely studied mouse strain; the origins of the 129 SvEvTac strain are unclear; and knockouts are increasingly being made on BL/6 or BALB/c backgrounds.
2. Sequencing Strategy
The recommended strategy for sequencing the mouse is to build the physical map as the sequence is generated. A three-phase strategy was proposed:
Phase I (Random): Researchers would sequence individual BAC clones at random until 30 percent to 50 percent of the mouse genome was sequenced. The BAC clones would be selected at random or based on scientific interest. BACs would be sequenced and STSs from sequenced BACs would then be mapped onto a common high resolution RH mapping panel. All information would be entered into a common database.
Phase II (Directed): The sequencing strategy would change over to extending sequencing off the ends of BACs using information from the BAC fingerprinting or the BAC end sequencing projects to direct the extension. This phase would be in place until about 90 % of the genome had been sequenced.
Phase III (Closure): This might require special libraries (library made with different enzymes, smaller insert size libraries, etc) for gap filling.
3. Mapping Resources Needed for This Strategya. High Resolution Radiation Hybrid (RH) Mapping Panel. The background for these new cell lines should be something other than rodent. A set of 15,000 common markers should be cross-mapped on this panel as well as the low resolution panel currently being used. As BACs are sequenced, they should be placed on the high resolution panel in order to order and orient them.
b. Additional BAC Libraries. In addition to increasing the genomic equivalency of the current C57BLJ/6 library, additional libraries (i) using multiple enzymes and (ii) with smaller inserts should be generated.
c. Resources to Facilitate Contig Building and Gap Closure of Genomic Sequence. A fingerprinted BAC library with BAC ends sequenced would facilitate contig building and gap closing.
4. Capacity for Large-Scale Sequencing of the Mouse Genome
To ensure that at least a "working draft" of the mouse genomic sequence is produced in a timely manner, it will be necessary to increase the U.S.’s capacity to generate genomic sequence. Three models were proposed: expand existing centers; allow some of the existing centers to switch to more mouse genomic sequence; and start up new large-scale sequencing centers. Any new center should have a capacity to ramp up to producing at least one million high quality reads per year at a cost of $.10 to $.12 per base (for the "working draft"), have a reasonable ramping plan, and an efficient plan to convert the "working draft" to the finished product. It was estimated that at least four to six new centers should be started. This number would allow for the likelihood that some centers might not be competitive in the long-term. It was acknowledged that the learning curve for these new centers would be very steep and that the cost per base pair of sequence might not initially be competitive with the existing major large-scale genome sequencing centers.
The experienced large-scale sequencers were of the opinion that for new groups starting up, the management issues would present more of a challenge than implementing the technology. There was also a willingness on the part of experienced sequencers to share information and technology with the new groups in order to shorten the learning curve.
5. Cost of The "Working Draft"
The cost of producing the "working draft" will depend on the quality that is decided upon, which in turn will depend on the funding available. One estimate based on a coverage of approximately 10X was $350 million (1st year--$40M; 2nd year--$80M; 3rd year--$100M; and 4th year--$130M).
PROPOSED ACTION ITEMS
1. Explore with the mouse BAC screening consortium the possibility of switching the library to be screened from the 129SvEvTac strain to the C57BL6/J strain.
2. Support the development of a high resolution radiation hybrid (RH) mapping panel using a non-rodent cell line for background.
3. Enhance the C57BL6/J library by increasing the redundancy from 11.2X to 15X and making different additional libraries using different enzymes and various insert sizes.
4. Generate a fingerprinted BAC library and support BAC-end sequencing of the clones.
5. Increase the capacity for large-scale sequencing to accommodate the delivery of a "working draft" of the mouse sequence by 2003. It is essential that new centers, intramural and extramural, be established that are capable, within a very short time period, of producing large-scale, high quality genomic sequence.
6. Develop a strategy to ensure that the information and technology developed at the existing large-scale centers are rapidly deployed to the new centers.
7. Develop a central database for tracking progress in BAC mapping and sequencing.
B. FUNCTIONAL GENOMICS
1. Mutagenesis and Phenotyping Centers
One of the recommendations from the March meeting was the establishment of three mutagenesis and phenotyping centers that would serve the needs of the mouse community. In response to these recommendations, the NIH proposed a series of thematic centers, such as, behavior and the nervous system, developmental defects, embryonic development, cardiopulmonary, blood and sleep disorders, and immunologic disorders. The group offered the following suggestions:(1) the community should have flexibility to respond to these initiatives with a view toward maximizing local resources and interests. (2) pilot projects should be supported to develop simple screens that are high throughput, easy to use and transportable; this activity could go on within centers or could be funded as R01-type research; (3) many of these mutants will be pleiotropic and screening mechanisms need to be developed that would fully characterize mutants; (4) NIH awarding components should be flexible enough to co-fund parts of another ICDs’ phenotyping initiative, if the research is relevant to the co-funders mission; (5) NIH should support adequate numbers of centers so as to respond to the scientific interests of the majority of researchers and (6) NIH should make a commitment to support these centers as long as they are productive and serve a scientifically useful purpose.
2. Access to Mutants and Intellectual Property Issues
The group was unanimous in stating that: (a) the mutants must be freely available to the community and (b) the organizations generating the mutants should be discouraged from patenting them. This can be monitored by having, as part of the application process, a description of how and when the mutants will be made available to the community. In addition, once awards are made, the projects would be monitored for compliance.
Database needs were not explored in great detail, but databases were considered essential to the full utilization of mutants. A public database should be developed to capture a common set of features about each mutant with links to the generating laboratory for additional information and the ability to query the database. GENBANK was considered a model for a community database. There was some discussion about the ongoing activities of the Mouse Genome Database, but there was a sense that the database issues need further discussion and input from the community about what is needed.
PROPOSED ACTION ITEMS:
1. Refine initiatives for mutagenesis and phenotyping with special attention to allowing local scientific interest to drive the type of center.
2. Develop a uniform policy across the institutes and centers for obtaining access to mutants.
3. Convene a group to discuss what common features are necessary for the public database and how to link to primary data.
C. RESOURCES AND TRAINING
1. Training of pathobiologists.
There is an urgent need to increase the number of trained mouse pathobiologists, however, the size of the pipeline needs to be determined. The mechanisms proposed by NIH for training pathobiologists as clinicians and as researchers were considered appropriate.
Cryopreservation is an important technology because maintaining colonies of mice is very expensive. Additional research is needed to improve this technology and to explore efficient methods to eliminate pathogens from animals. The dissemination of this technology to other laboratories should also be considered by sponsoring courses.
3. Mutant Mouse Regional Resource Centers
The NIH proposal to establish additional resource centers that would be complementary to The Jackson Laboratory’s activity was endorsed. Any new centers would be expected to partner/collaborate with The Jackson Laboratory in order to avoid excessive duplication.
PROPOSED ACTION ITEMS
1. Refine the initiatives for (a) training and career development programs and (b) mutant mouse regional resource centers.
2. Support the development of courses in cryopreservation.
As with human genome sequencing, the mouse sequencing initiative is viewed as an international effort. Representatives of the Department of Energy and the United Kingdom's Medical Research Council were present at the meeting and described their plans with respect to sequencing the mouse genome. Both expressed a strong interest in collaborating with the NIH in developing a coordinated strategy and sharing expertise. The NIH is committed to collaborating with other national and international groups and programs.