| veral research teams around the world announced | | | | Genomic Research (TIGR)], and M. Bento Soares |
| plans in the fall of 1996 for full-length cDNA (gene) | | | | (University of Iowa). |
| sequencing, investigators felt that the highly | | | | A protocol that takes advantage of the unusual |
| beneficial infrastructure provided since 1994 by | | | | nucleotide "cap" on the 5' end of mRNAs requires |
| the international Integrated Molecular Analysis of | | | | that the first cDNA strand’s extension be |
| Genome Expression (I.M.A.G.E.) consortium [HGN | | | | long enough to protect the cap as a contingency |
| 6(6), 3] should be extended to the challenges of | | | | for final cDNA clone production. Soares reported, |
| complete cDNA sequencing. A subsequent | | | | however, that about one-third of cDNA |
| workshop for I.M.A.G.E. participants was held in | | | | transcripts begin within the mRNA, as contrasted |
| May 1997 in Gaithersburg, Maryland. The meeting | | | | with preferred starts at the mRNA's 3' end, thus |
| was organized and chaired by Greg Lennon [then | | | | giving rise to3' truncations. This problem can be |
| at Lawrence Livermore National Laboratory | | | | alleviated substantially by size fractionating the |
| (LLNL) and now at Gene Logic Inc.] with Marvin | | | | mRNAs and later selecting out the cDNA products |
| Stodolsky coordinating for the meeting sponsor, | | | | with lengths equal to the size-sorted mRNA |
| the DOE Office of Biological and Environmental | | | | templates. Hans Lehrach (Max Planck Institut |
| Research. Scientists attended from France, | | | | für Molekulare Genetik, Germany) related the |
| Germany, Italy, Japan, Sweden, the United | | | | value of massively parallel oligomer fingerprinting |
| Kingdom, and the United States. | | | | of cDNAs. This is an economical way to screen a |
| Several workshop participants are members of | | | | library for novel and longer, potentially full-length |
| the subgroup EURO-IMAGE, whose goals include | | | | cDNAs. Optimal candidate cDNAs chosen by the |
| generating and sequencing a master set of unique | | | | Lehrach team at the Resource Center of the |
| full-length cDNA clones (based on I.M.A.G.E. | | | | German Genome Project are being sequenced in |
| consortium resources) representing 3000 | | | | the laboratory of Annemarie Poustka (Deutsches |
| transcripts and 6 Mb of finished sequence. Other | | | | Krebsforschungszentrum). |
| EURO-IMAGE goals are to obtain high-resolution | | | | More than one sequencing read commonly is |
| and comparative functional mapping in human and | | | | necessary to display the complete sequence for |
| model organisms of 1000 master-set genes and | | | | cDNAs longer than a few hundred bases. |
| to develop the I.M.A.G.E. consortium database for | | | | Strategies for economical full-length sequencing |
| easy access to an integrated view of the | | | | were discussed by Lennon and Richard Gibbs |
| sequence, map, and expression data generated. | | | | (Baylor College of Medicine). Sequence reads |
| U.S. funding agencies represented at the | | | | beyond 1000 bases now are being obtained with |
| workshop included DOE, NIH, and the recently | | | | improvements to sequencing systems by Wilhelm |
| established nonprofit Merck Genome Research | | | | Ansorge’s team at the European Molecular |
| Institute [HGN 8(3-4), 9]. Selected highlights follow | | | | Biology Laboratory. Ansorge suggested that, for |
| of technical progress in complete cDNA | | | | cDNAs shorter than 2 kb, good coverage could |
| sequencing, as reported at the workshop. | | | | be achieved by two overlapping reads on |
| Highlights of Technical Progress | | | | complementary strands. |
| Attendees addressed a wide range of topics, | | | | Giuseppe Borsani (Telethon Institute of Genetics |
| including the status of cDNA sequencing projects, | | | | and Medicine) reported on the benefits of the |
| future targets, data- and clone-release policies, | | | | easily manipulated Drosophila model for studies of |
| quality criteria and assessment, and mouse and | | | | development and function to reveal roles |
| other model organism cDNAs. Speakers projected | | | | represented by human cDNAs. |
| that, with adequate support from funding | | | | Mark Boguski (National Center for Biotechnology |
| agencies, participating laboratories could generate | | | | Information) discussed the status of the dbEST |
| up to 15,000 full-length cDNA sequences in the | | | | cDNA sequence database and made |
| following year. With average cDNA lengths of 2 | | | | recommendations for the evolution needed to |
| kb, this represents some 30 Mb of total | | | | meet the impending new demands of complete |
| sequence. | | | | DNA sequencing. He observed that each group will |
| Researchers have long recognized that expression | | | | have its own selection criteria and sequencing |
| of a single gene may culminate in the production | | | | priorities, such as finding cancer genes, genes with |
| of several different messenger RNA (mRNA) | | | | Drosophila homologs, or genes that already have |
| transcripts, depending both on the gene and the | | | | been mapped. |
| source tissue. Added to this biological complexity | | | | Boguski coined the expression "the slicing problem" |
| are the technical challenges of converting fragile | | | | to describe the difficulties in avoiding undesirable |
| mRNAs to the sturdier cDNAs. Standard methods | | | | duplication and redundancy due to overlapping |
| involve use of poly dT as a primer on the 3' poly | | | | choice categories. A possible solution would be to |
| A end of purified mRNAs, with reverse | | | | establish a registration and tracking database |
| transcriptase enzymes of viral origin polymerizing | | | | modeled after the successful European |
| the synthesis of a single-stranded DNA | | | | Bioinformatics Institute's (EBI) RHAlloc-RHdb |
| complement of the mRNA. These initial DNA | | | | approach used in constructing the human |
| transcripts often fail to extend to the 5' end of | | | | transcript map. Patricia Rodriguez-Tomé (EBI) |
| longer mRNAs. With the use of more routine | | | | has accepted this responsibility. This data will |
| biochemistries, the single-stranded DNA is | | | | include an investigator or center name and |
| converted into duplex DNA and combined with a | | | | contact information, identifiers for the physical |
| DNA vector to support its propagation and | | | | cDNA clones being sequenced and associated EST |
| maintenance as a DNA clone. The double-stranded | | | | accession numbers, and sequencing status. When |
| DNAs produced are much more stable and less | | | | participants registered a clone that they intended |
| susceptible to degradative processes than their | | | | to sequence, the database would detect and |
| single-stranded mRNA predecessors. However, | | | | report overlaps with clones selected by other |
| because the initial reverse transcription is often | | | | groups. |
| shortened, cDNA libraries with abundant truncated | | | | Attendees agreed that the I.M.A.G.E. consortium |
| products are the common result, particularly for | | | | should convene every 6 months to maintain |
| the longer source mRNAs. Strategies devised for | | | | necessary coordination and efficiency. A |
| alleviating this truncation problem were described | | | | subsequent meeting, organized by Quackenbush, |
| by Takao Isogai (Helix Research Institute, Japan), | | | | was held in September 1997 in conjunction with |
| Nobuo Nomura (Kazusa DNA Research Institute, | | | | the Ninth International Genome Sequencing and |
| Japan), John Quackenbush [The Institute for | | | | Analysis Conference in Hilton Head, South Carolina. |