Daily Archives: July 9, 2015

Biology for the post-genomic era

Fully Open Access. Genome Biology covers all areas of biology and biomedicine studied from a genomic and post-genomic perspective. Content includes research, new methods and software tools, and reviews, opinions and commentaries. Areas covered include, but are not limited to: sequence analysis; bioinformatics; insights into molecular, cellular and organismal biology; functional genomics; epigenomics; population genomics; proteomics; comparative biology and evolution; systems and network biology; genomics of disease; and clinical genomics. All content is open access immediately on publication.

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Enter exitrons

Dorothee Staiger and Gordon Simpson discuss the discovery of exon-like introns and their contributions to proteome complexity and phenotypic diversity

Focus on splicing

Chris Burge and Daniel Dominguez discuss how splicing-regulatory proteins modulate assembly of the spliceosome to activate and repress splicing

Diagnosing Mendelian diseases

Advantages over clinical exome sequencing can be achieved by using an NGS-based multiplexing assay involving comprehensive gene panels.

Read length cost-benefit

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Genome Biology

HGP at the start

The HGP began officially in October 1990, but its origins go back earlier. In the mid-1980s, three scientists independently came up with the idea of sequencing the entire human genome: Robert Sinsheimer, then chancellor of University of California at Santa Cruz, as a way to spend $30 million donated to his institution to build a telescope when that project fell through; Salk Institute researcher Rene Dulbecco as a way to understand the genetic origins of cancer and other diseases; and the Department of Energy's (DOE's) Charles DeLisi as a way to detect radiation-induced mutations, an interest of that agency since the atomic bombings of Hiroshima and Nagasaki. Such a project had become technically feasible due to advances made during the previous decade or two: in the early 1970s, recombinant DNA technologies (use of restriction enzymes to splice DNA, reverse transcriptase to make DNA from RNA, viral vectors to carry bits of DNA into cells, bacterial cloning to multiply quantities of DNA); in the late 1970s, DNA sequencing and use of RFLP (restriction fragment length polymorphism) markers for gene mapping; and in the early to mid-1980s, DNA synthesis, pulsed-field gel electrophoresis, polymerase chain reaction (PCR), and automated DNA sequencing.

Sinsheimer's, Dulbecco's, and DeLisi's idea found supporters among a number of prominent molecular biologists and human geneticistsfor example, Walter Bodmer, Walter Gilbert, Leroy Hood, Victor McKusick, and James D. Watson. However, many molecular biologists expressed misgivings. Especially through 1986 and 1987, there were concerns about the routine nature of sequencing and the amount of junk DNA that would be sequenced, that the expense and big science approach would drain resources from smaller and more worthy projects, and that knowledge of gene sequence was inadequate to yield knowledge of gene function.[1] In September 1986, committees were established to study the feasibility of a publicly-funded project to sequence the human genome: one by the National Research Council (NRC) on scientific merit, and one by the Office for Technology Assessment (OTA) as a matter of public policy. Both committees released reports in 1988. The OTA report, Mapping Our Genes: Genome Projects: How Big, How Fast? downplayed the concerns of scientist critics by emphasizing that there was not one but many genome projects, that these were not on the scale of the Manhattan or Apollo projects, that no agency was committed to massive sequencing, and that the study of other organisms was needed to understand human genes. The NRC report, Mapping and Sequencing the Human Genome, sought to accommodate the scientists concerns by formulating recommendations that genetic and physical mapping and the development of cheaper, more efficient sequencing technologies precede large-scale sequencing, and that funding be provided for the mapping and sequencing of nonhuman (model) organisms as well.

It was the DOE that made the first push toward a Big Science genome project: DeLisi advanced a five-year plan in 1986, $4.5 million was allocated from the 1987 budget, and recognizing the boost the endeavor would provide to national weapons laboratories, Senator Pete Domenici from New Mexico introduced a bill in Congress. The DOE undertaking produced consternation among biomedical researchers who were traditionally supported by the NIH's intramural and extramural programsfor example, Caltech's David Botstein referred to the initiative as DOE's program for unemployed bomb-makers (in Cook-Deegan 1994, p. 98). James Wyngaarden, head of the NIH, was persuaded to lend his agency's support to the project in 1987. Funding was in place in time for fiscal year (FY) 1988 with Congress awarding the DOE $10.7 million and the NIH $17.2 million.[2] The DOE and NIH coordinated their efforts with a Memorandum of Understanding in 1988 that agreed on an official launch of the HGP on October 1, 1990 and an expected date of completion of 2005. Total cost estimated by the NRC report was $3 billion.

The project's specific goals at the outset were: (i) to identify all genes of the human genome (initially estimated to be 100,000); (ii) to sequence the approximately 3 billion nucleotides of the human genome; (iii) to develop databases to store this information; (iv) to develop tools for data analysis; (v) to address ethical, legal, and social issues; and (vi) to sequence a number of model organisms, including the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae, the roundworm Caenorhabditis elegans, the fruitfly Drosophila melanogaster, and the mouse Mus musculans. The DOE established three genome centers in 198889 at Lawrence Berkeley, Lawrence Livermore, and Los Alamos National Laboratories; as Associate Director of the DOE Office of Health and Environmental Research (OHER), David Galas oversaw the DOE's genome project from April 1990 until he left for the private sector in 1993. The NIH instituted a university grant-based program for human genome research and placed Watson, co-discoverer of the structure of DNA and director of Cold Spring Harbor Laboratory, in charge in 1988. In October 1989, the Department of Health and Human Services established the National Center for Human Genome Research (NCHGR) at the NIH with Watson at the helm. During 1990 and 1991, Watson expanded the grants-based program to fund seven genome centers for five-year periods to work on large-scale mapping projects: Washington University, St. Louis; University of California, San Francisco; Massachusetts Institute of Technology; University of Michigan; University of Utah; Baylor College of Medicine; and Children's Hospital of Philadelphia.

As the HGP got underway, a number of philosophers weighed in on its scientific meritin terms of cost, potential impact on other areas of research, ability to lead to medical cures, and the usefulness of sequence data (Kitcher 1995; Rosenberg 1995; Tauber and Sarkar 1992; Vicedo 1992). However, of particular interest to philosophers is goal (v) concerning ethical, legal, and social issues. At an October 1988 news conference called to announce his appointment, Watson, in an apparently off-the-cuff response to a reporter who asked about the social implications of the project, promised that a portion of the funding would be set aside to study such issues (Marshall 1996c). The result was the NIH/DOE Joint Working Group on Ethical, Legal, and Social Implications (ELSI) of Human Genome Research, chaired by Nancy Wexler, which began to meet in September 1989.[3] The Joint Working Group identified four areas of high priority: quality and access in the use of genetic tests; fair use of genetic information by employers and insurers; privacy and confidentiality of genetic information; and public and professional education (Wexler in Cooper 1994, p. 321). The NIH and DOE each established ELSI programs: philosopher Eric T. Juengst served as the first director of the NIH-NCHGR ELSI program from 1990 to 1994. ELSI was funded initially to the tune of three percent of the HGP budget for both agencies; this was increased to four and later five percent at the NIH.

Map first, sequence later

As the NRC report had recommended, priority at the outset of the project was given to mapping rather than sequencing the human genome. HGP scientists sought to construct two kinds of maps. Genetic maps order polymorphic markers linearly on chromosomes; the aim is to have these markers densely enough situated that linkage relations can be used to locate chromosomal regions containing genes of interest to researchers. Physical maps order collections (or libraries) of cloned DNA fragments that cover an organism's genome; these fragments can then be replicated in quantity for sequencing. The joint NIH-DOE five-year plan released in 1990 set specific benchmarks: a resolution of 2 to 5 centimorgans (cM) for genetic linkage maps and physical maps with sequence-tagged site (STS) markers (unique DNA sequences 100200 base pairs long) spaced approximately 100 kilobases (kb) apart and 2-megabase (Mb) contiguous overlapping clones (contigs) assembled for large sections of the genome. Sequencing needed to be made more efficient and less costly: aims were to reduce sequencing costs to $.50 per base and to complete 10 million bases of contiguous DNA (0.3 percent of the human genome) but otherwise to focus efforts on the smaller genomes of less complex model organisms (Watson 1990). HGP goals were facilitated by a number of technological developments during this initial period. For physical mapping, yeast artificial chromosomes (YACs) introduced in 1987 (Burke et al. 1987) permitted much larger segments of DNA to be ordered and stored for sequencing than was possible with plasmid or cosmid libraries. A new class of genetic markers, microsatellite repeats, was identified in 1989 (Litt and Luty 1989; Tautz 1989; Weber and May 1989); because these sets of tandem repeats of short (either dinucleotide, trinucleotide, or tetranucleotide) DNA sequences are more highly polymorphic and detectable by PCR, microsatellites quickly replaced RFLPs as markers of choice for genetic linkage mapping and furnished the STS markers which facilitated the integration of genetic and physical maps. Another technological achievementthe combined use of reverse transcription, PCR, and automated sequencing to map expressed genesled to administrative changes at the NIH when, in April 1992, Watson resigned from his position as director of the NCHGR following a conflict with NIH director Bernadine Healy over gene patenting. In 1991, while working at the NIH, J. Craig Venter sequenced small portions of cDNAs from existing libraries to provide identifying expressed sequence tags (ESTs) of 200300 bases which he then compared to already identified genes from various species found in existing databases (Adams et al. 1991).[4] Watson disagreed with Healy's decision to approve patent applications for the ESTs despite lack of knowledge of their function.[5] Soon after Watson's departure, Venter left NIH for the private sector.[6]

Francis Collins, an MD-PhD whose lab at University of Michigan co-discovered genes associated with cystic fibrosis and neurofibromatosis and contributed to efforts to isolate the gene for Huntington's disease, was appointed by Healy as Watson's replacement, and he began at the NCHGR in April 1993. Collins established an intramural research program at the NCHGR to complement the extramural program of grants for university-based research which already existed; ELSI remained a grant-funded program. The original NIH-DOE five-year plan was updated in 1993. The new five-year plan, in effect through 1998, accommodated progress that had been made in mapping, sequencing, and technological development (Collins and Galas 1993). The goal of a 25 cM genetic map was expected to be met by the 1995 target date. The deadline for a physical map with STS markers at intervals of 100 kb was extended to 1998; a map with intervals averaging 300 kb was expected by 1995 or 1996. Although the goal of $.50 per base cost of sequencing was projected to be met by 1996, it was recognized that this would be insufficient to meet the 2005 target date. The updated goal was to build up to a collective sequencing capacity of 50 Mb per year and to have 80 Mb of DNA (from both human and model organism genomes) sequenced by the end of 1998. This would be achieved by increasing the number of groups working on large-scale sequencing and heightening efforts to develop new sequencing technologies. Accordingly, in November 1995, the U.K.'s Wellcome Trust launched a $75 million, seven-year concentrated sequencing effort at the Sanger Centre in Cambridge, and in April 1996, the NCHGR awarded grants totaling $20 million per year for six centers (Houston's Baylor College of Medicine, Stanford University, The Institute for Genomic Research [TIGR], University of Washington-Seattle, Washington University School of Medicine in St. Louis, and Whitehead Institute for Biomedical ResearchMIT Genome Center) to pilot high-volume sequencing approaches (Marshall 1996a).

Although the HGP's inceptions were in the U.S., it had not taken long for mapping and sequencing the human genome to become an international venture (see Cook-Deegan 1994). France began to fund genome research in 1988 and had developed a more centralized, although not very well-funded, program by 1990. More significant were the contributions of Centre dEtudes du Polymorphisme Humain (CEPH) and Gnthon. CEPH, founded in 1983 by Jean Dausset, maintained a collection of DNA donated by intergenerational families to help in the study of hereditary disease; Jean Weissenbach led an international effort to construct a complete genetic map of the human genome using the CEPH collection; later, with funding from the French muscular dystrophy association (AFM), director Daniel Cohen set out to construct a YAC clone library for physical mapping and oversaw the launching of Gnthon in 1991 as an industrial-sized mapping and sequencing operation funded by the AFM. The U.K.'s genome project received its official start in 1989 although Sydney Brenner had commenced genome research at the Medical Research Council (MRC) laboratory several years before this. MRC funding was supplemented with private monies from the Imperial Cancer Research Fund, and later, the Wellcome Trust. The Sanger Centre, led by John Sulston and funded by Wellcome and the MRC, opened in October 1993. A combined four-year, 15-million-euro genome program by the European Community (E.C.) commenced in 1990. Germany, its citizens all too aware of abuses in the name of genetics, lagged behind other European countries: although individual researchers received government funds for genome research in the late-1980s and participated in the E.C. initiative, no actual national genome project was undertaken until 1995 (Kahn 1996). Japan, ahead of the U.S. in having funded the development of automated sequencing technologies since the early 1980s, was the major genome player outside the U.S. and Europe with several government agencies beginning small-scale genome projects in the late-1980s and early- 1990s, but a frequent target of U.S. criticism for the size of its investment relative to GNP.[7] China was the latecomer on the international scene: with 250 million yuan ($30 million) over three years from government and industry, the Chinese National Human Genome Center with branches in Beijing and Shanghai opened in July 1998, and was followed in 1999 by the Beijing Genomics Institute.[8]

More:
The Human Genome Project (Stanford Encyclopedia of Philosophy)