| When several research teams around the world | | | | A protocol that takes advantage of the unusual |
| announced plans in the fall of 1996 for full-length | | | | nucleotide "cap" on the 5' end of mRNAs requires |
| cDNA (gene) sequencing, investigators felt that the | | | | that the first cDNA strand's extension be long |
| highly beneficial infrastructure provided since 1994 by | | | | enough to protect the cap as a contingency for final |
| the international Integrated Molecular Analysis of | | | | cDNA clone production. Soares reported, however, |
| Genome Expression (I.M.A.G.E.) consortium [HGN 6(6), | | | | that about one-third of cDNA transcripts begin within |
| 3] should be extended to the challenges of complete | | | | the mRNA, as contrasted with preferred starts at |
| cDNA sequencing. A subsequent workshop for | | | | the mRNA's 3' end, thus giving rise to3' truncations. |
| I.M.A.G.E. participants was held in May 1997 in | | | | This problem can be alleviated substantially by size |
| Gaithersburg, Maryland. The meeting was organized | | | | fractionating the mRNAs and later selecting out the |
| and chaired by Greg Lennon [then at Lawrence | | | | cDNA products with lengths equal to the size-sorted |
| Livermore National Laboratory (LLNL) and now at | | | | mRNA templates. Hans Lehrach (Max Planck Institut |
| Gene Logic Inc.] with Marvin Stodolsky coordinating | | | | für Molekulare Genetik, Germany) related the |
| for the meeting sponsor, the DOE Office of Biological | | | | value of massively parallel oligomer fingerprinting of |
| and Environmental Research. Scientists attended from | | | | cDNAs. This is an economical way to screen a library |
| France, Germany, Italy, Japan, Sweden, the United | | | | for novel and longer, potentially full-length cDNAs. |
| Kingdom, and the United States. | | | | Optimal candidate cDNAs chosen by the Lehrach |
| Several workshop participants are members of the | | | | team at the Resource Center of the German |
| subgroup EURO-IMAGE, whose goals include | | | | Genome Project are being sequenced in the |
| generating and sequencing a master set of unique | | | | laboratory of Annemarie Poustka (Deutsches |
| full-length cDNA clones (based on I.M.A.G.E. | | | | Krebsforschungszentrum). |
| consortium resources) representing 3000 transcripts | | | | More than one sequencing read commonly is |
| and 6 Mb of finished sequence. Other EURO-IMAGE | | | | necessary to display the complete sequence for |
| goals are to obtain high-resolution and comparative | | | | cDNAs longer than a few hundred bases. Strategies |
| functional mapping in human and model organisms of | | | | for economical full-length sequencing were discussed |
| 1000 master-set genes and to develop the I.M.A.G.E. | | | | by Lennon and Richard Gibbs (Baylor College of |
| consortium database for easy access to an | | | | Medicine). Sequence reads beyond 1000 bases now |
| integrated view of the sequence, map, and | | | | are being obtained with improvements to sequencing |
| expression data generated. | | | | systems by Wilhelm Ansorge's team at the European |
| U.S. funding agencies represented at the workshop | | | | Molecular Biology Laboratory. Ansorge suggested |
| included DOE, NIH, and the recently established | | | | that, for cDNAs shorter than 2 kb, good coverage |
| nonprofit Merck Genome Research Institute [HGN | | | | could be achieved by two overlapping reads on |
| 8(3-4), 9]. Selected highlights follow of technical | | | | complementary strands. |
| progress in complete cDNA sequencing, as reported | | | | Giuseppe Borsani (Telethon Institute of Genetics and |
| at the workshop. | | | | Medicine) reported on the benefits of the easily |
| Highlights of Technical Progress | | | | manipulated Drosophila model for studies of |
| Attendees addressed a wide range of topics, | | | | development and function to reveal roles |
| including the status of cDNA sequencing projects, | | | | represented by human cDNAs. |
| future targets, data- and clone-release policies, quality | | | | Mark Boguski (National Center for Biotechnology |
| criteria and assessment, and mouse and other model | | | | Information) discussed the status of the dbEST |
| organism cDNAs. Speakers projected that, with | | | | cDNA sequence database and made |
| adequate support from funding agencies, participating | | | | recommendations for the evolution needed to meet |
| laboratories could generate up to 15,000 full-length | | | | the impending new demands of complete DNA |
| cDNA sequences in the following year. With average | | | | sequencing. He observed that each group will have its |
| cDNA lengths of 2 kb, this represents some 30 Mb | | | | own selection criteria and sequencing priorities, such |
| of total sequence. | | | | as finding cancer genes, genes with Drosophila |
| Researchers have long recognized that expression of | | | | homologs, or genes that already have been mapped. |
| a single gene may culminate in the production of | | | | Boguski coined the expression "the slicing problem" to |
| several different messenger RNA (mRNA) transcripts, | | | | describe the difficulties in avoiding undesirable |
| depending both on the gene and the source tissue. | | | | duplication and redundancy due to overlapping choice |
| Added to this biological complexity are the technical | | | | categories. A possible solution would be to establish a |
| challenges of converting fragile mRNAs to the | | | | registration and tracking database modeled after the |
| sturdier cDNAs. Standard methods involve use of | | | | successful European Bioinformatics Institute's (EBI) |
| poly dT as a primer on the 3' poly A end of purified | | | | RHAlloc-RHdb approach used in constructing the |
| mRNAs, with reverse transcriptase enzymes of viral | | | | human transcript map. Patricia Rodriguez-Tomé |
| origin polymerizing the synthesis of a single-stranded | | | | (EBI) has accepted this responsibility. This data will |
| DNA complement of the mRNA. These initial DNA | | | | include an investigator or center name and contact |
| transcripts often fail to extend to the 5' end of | | | | information, identifiers for the physical cDNA clones |
| longer mRNAs. With the use of more routine | | | | being sequenced and associated EST accession |
| biochemistries, the single-stranded DNA is converted | | | | numbers, and sequencing status. When participants |
| into duplex DNA and combined with a DNA vector to | | | | registered a clone that they intended to sequence, |
| support its propagation and maintenance as a DNA | | | | the database would detect and report overlaps with |
| clone. The double-stranded DNAs produced are much | | | | clones selected by other groups. |
| more stable and less susceptible to degradative | | | | Attendees agreed that the I.M.A.G.E. consortium |
| processes than their single-stranded mRNA | | | | should convene every 6 months to maintain |
| predecessors. However, because the initial reverse | | | | necessary coordination and efficiency. A subsequent |
| transcription is often shortened, cDNA libraries with | | | | meeting, organized by Quackenbush, was held in |
| abundant truncated products are the common result, | | | | September 1997 in conjunction with the Ninth |
| particularly for the longer source mRNAs. Strategies | | | | International Genome Sequencing and Analysis |
| devised for alleviating this truncation problem were | | | | Conference in Hilton Head, South Carolina. Washington |
| described by Takao Isogai (Helix Research Institute, | | | | University scientists will organize the next meeting, |
| Japan), Nobuo Nomura (Kazusa DNA Research | | | | tentatively planned to concur with the May 1998 |
| Institute, Japan), John Quackenbush [The Institute | | | | Human Genome Workshop at Cold Spring Harbor |
| for Genomic Research (TIGR)], and M. Bento Soares | | | | Laboratory. |
| (University of Iowa). | | | | |