NIH has recently engaged in community discussion of the idea of sequencing the totality of all microbes in or on the human body (the "human microbiome";). With the cost of sequencing declining and the impact of genomic approaches to biomedical research growing, the time is right to consider the opportunities that a Human Microbiome Project will offer in promoting better approaches to diagnosing disease, developing new therapeutics and therapeutic strategies, and in maintaining human health. To further this discussion, NIH organized an informal meeting of NIH staff from several Institutes and Centers with a group of scientists interested in exploration of the human microbiome to discuss the current state of knowledge, of the plans being developed internationally and of the challenges facing the organization of a Human Microbiome Project.
Why would we want to sequence all the microbes in the human body? (Jeff Gordon)
What we know
Dr. Jeff Gordon described what we currently know about the human microbiome. He also set the context for a potential project that would attempt to define, more fully, the human microbiome. A summary of his comments:
Context for an HMP
- The microbiome is an integral part of the human genetic landscape and human evolution. Sequencing the human microbiome would give us an "extended view of ourselves";. Members of the phyla Bacteroidetes and Firmicutes dominate the human bacterial "flora,"; but there is considerable variation in the composition of indigenous bacterial communities among individual humans.
- Host selection plays a very significant role in the composition of the microbiota, so that microbiomes differ greatly from organism to organism. Generally mammals with similar diets have more closely related microbial communities. The microbial communities in omnivores, carnivores and herbivores cluster differently from each other. (From Ruth Ley's study). When the gut flora from zebra fish is transferred to a germ free mouse, within 10 days the flora becomes "mouseified";, i.e., it looks more like the expected normal mouse flora than that of zebrafish.
- Experiments with germ-free and colonized animals show that microbiota direct myriad biotransformations (production of essential vitamins, xenobiotic metabolism) and affect energy balances (microbes can manipulate host genes resulting in alterations in the deposition of energy into different sites), postnatal development, gut epithelial renewal rates, cardiac size/output and blood pressure.
- The composition of the gut microbiome is affected by or affects obesity. As individuals become leaner through diet, the fraction of Firmicutes in the gut population decreases, while the fraction of Bacteroidetes increases.
- Role of archaea in human health - up to 10% of the gut microbiome is comprised of Methanobrevibacter smithii. Other experiments with germ-free mice have shown that the presence of this organism can increase the efficiency of the fermentation of the gut contents and energy storage in the host.
- Xenologs abound in the genomes of organisms in the Bacteroidetes class, indicating there is tremendous exchange of genes within and across species.
Summary of the November 2005 meeting in Paris, France entitled The Human Metagenome Project
- Interest is increasing in the scientific community in identifying the gene content and functional role of variation of microbial communities.
- There is a paradigm shift going on in the field of microbiology from the study of individual organisms to the study of microbial communities. Understanding the operation of communities and the dynamics in the composition of the communities with respect to the environmental changes is now a topic of interest.
- Microbiota could potentially form the basis of a 21st century pharmacopeia. Microbes have the capacity to synthesize many novel chemical entities that sustain mutually beneficial relationships with us. Through the identification of natural products, exploration of the effects on host signaling, examination of metabolic pathways and gene manipulation, we have the opportunity to identify and develop many possible new biologically active compounds.
George Weinstock presented a summary of this international meeting:
What organisms should be included in a HMP? What body sites?
- The goal of the meeting was to bring together scientists and funders to forge an international alliance to initiate a human metagenome project, with its first effort being focused on the intestinal microbiome. It was attended by scientists and funders from Europe, Asia and the U.S.
The attendees recommended that a metagenome project should be undertaken. This metagenome project should be organized in two components:
- Determination of a set of reference or scaffold sequences from a core set of species.
- Sample (metagenomic) sequencing utilizing high throughout technologies to characterize the microbial flora from different body sites.
- There was a lot of enthusiasm at the meeting. Some institutions (NHGRI, The Sanger Institute, The Joint Genome Institute (DOE) and possibly Genoscope (France)) indicated that they would be willing to commit or are already committed to sequencing a number of reference genomes. Representatives from China and Japan indicted that they need a concrete description of the project to take to their funding agencies to seek support. The EU would be interested in supporting efforts in this field in its next call for proposals. The NSF already funds significant microbial sequencing, but not necessarily from the human. One of the next steps is to publish the meeting report/recommendations so that partners can use it to encourage their funding agencies to participate.
- The meeting did not address the many challenges and the several fundamental issues that need to be addressed before initiating the project. There was not a good sense among the attendees of what coordinating a large international project would entail.
- There was agreement that an international consortium and its attendant attention to coordination could bring the benefits of a division of labor, economies of scale, standardization of procedures, quality control, rapid data release and reduction of redundancy to the project.
What new technology is needed?
- Ideally, the genomic sequences of all microorganisms (including bacteria, archaea, fungi, viruses, and phage) found on and in the human body should be determined. Efforts should be made to include rare organisms.
- All relevant body sites, including skin, the oral cavity, the intestinal tract, the female urogenital tract, the ear, and the upper respiratory tract, should be characterized. The effects of locale within each of the body sites should be addressed.
- Sampling should be done to address a number of variables:
- A very important question is whether there is a "normal"; "core microbiome."; The idea that there is a "normal"; flora may be a fallacy. Instead, there may be a spectrum of normalcy and several models of health.
Implementation Topics - Relative roles and timing of the sequencing of the genomes of cultivable organisms and sample sequencing
- Culture technology. Most bacteria are not culturable at present, and research is needed to develop new approaches to grow them.
- Single-cell analysis. Devices are needed that can sort single cells from microbial communities for individual examination. The ability to do so could advance the understanding of rare individual organisms that cannot be studied today.
- Data analysis - New analytical tools are needed to enable searching of the large datasets that will be produced.
- Databases - Databases are needed to link clinical annotation and other meta-data to the sequences for analysis.
- The role of metabolomics in the project was discussed. It would be ideal to do metagenomic projects in parallel with metabolomic projects. Metagenomic projects can be undertaken now because the technology is available, but metabolomics technology needs to be developed.
Data management - Do we need a central data coordinating center?
- A dataset of sequences of the genomes of a collection of reference organisms is needed for each body site
- Technology development is needed to enable efficient metagenomic sampling
- Metagenomic sampling is needed to define the cohort of organisms present within each of the chosen sites - what's there at any one time, how it changes over time, and how it is affected by the anatomical locale within the site (e.g., different parts of the mouth).
- Standardizing of sample annotation and processing will be needed. Different types of sampling protocols will need to be examined to measure their effects on the data collected.
Rough cost estimates for a possible pilot project:
- See above in technology development.
- There will need to be tools and ways to make data accessible to researchers who are not used to working with large data sets.
- A definition of standards and level of consistency will need to be defined.
The following cost estimate for a potential pilot project was developed before the meeting. It gives a rough estimate of scientific and funding needs to launch a pilot. Estimates in red italic are those for which funding is likely already in place at a number of different sequencing centers as noted:
- Sequencing 1000 reference genomes across 5 body sites:
- $18.5M with Sanger sequencing (ABI technology) or
- $12.5 M with 454 Technology.
- The NHGRI, Sanger and JGI have previously indicated that funding may already be available to cover these costs.
- Microbial Diversity: 16S rRNA analyses-- $2/clone or $200k/100,000 clones
- Technology Development ~$5M/year for 3 years:
- Sequencing technology development. NHGRI is already funding the $1000 and $100,000 genome programs. The HMP projects will drive this new technology. Further investment not currently needed.
- Sample sequencingâa pilot as described below may be possible within existing funding.
- Sample preparation.
- Culturing currently unculturable organisms.
- BAC libraries as a sampling technique.
- Normalization of the community DNA.
- Whole genome amplification (wga)âthe sequencing centers are already working on wga as part of the NHGRI medical sequencing program. Further investment may not be needed.
- Single cell analysisâengage the microfabrication, microfluidics community.
- Bioinformaticsâdata bases and data analysis tools.
- Sample (metagenomic) sequencing pilot: 300,000 reads per body site in 100 individuals (5 body sites) ~$2M/year over 3 years
- Sequencing centers are likely to contribute existing funding to this effort if it is technically feasible.
- Overall, new funding is needed primarily for technology development to enable to full metagenomic exploration once the reference genomes are finished. Larger scale metagenomic projects which examine microbial communities under different variables may be appropriately done as individual research projects if sequencing costs are sufficiently reduced in the near future.
- NIH staff will take steps to form a trans-NIH working group to work on developing a plan that will be of interest across the NIH and for laying the groundwork for the NIH's participation in an international project.
- Part of the working group's effort will be to identify components that can already be tapped into - sequencing is potentially already funded so new funding is likely not needed for that part of this project.
- NIH staff will explore the role of OPASI in promoting this project.
- A "next step"; meeting may be needed with the international research community interested in the HMP. This will help coalesce an international project.
- Finally, it will be important to have an effective plan to communicate with the general population about the importance of this project. The outcome, as it relates to health, must be well articulated. There was general agreement that the project can be tied to the ideology of disease and stress; that it is looking at the "normal"; or healthy state. The argument can be made that not until we understand the nature of health can we more thoughtfully define the target for treating someone who is sick. Right now we can only remove the signs of illness instead of actually restoring people to health. Many illnesses are ecological diseases rather than the result of exposure to a rare pathogen.
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