Minutes of the Conference call on March 27, 2001 of the Aspergillus fumigatus genome sequencing group

Participating in the call:

Background:

At the November, 2000, meeting at TIGR, it had been agreed that both the Sanger Centre and TIGR would attempt to make chromosome-specific libraries using DNA separated on electrophoretic gels.  If this approach failed, a whole genome shotgun approach would be adopted.  Prior to the conference call, Michael Quail of the Sanger Centre circulated a report on the status of the Chromosome I pilot project, detailing progress and problems that had been encountered.  Portions of this report are quoted below:

Two BAC libraries with average insert sizes of 80 kb have been constructed at the Sanger Centre by cloning Sau3AI and EcoRI partial digest fragments.  A 36 x 96 well plate set of clones from the Sau3AI library has been replicated and gridded onto filters. This set, representing 7.5 fold genome coverage, has been completely fingerprinted with PstI to give, at a high stringency, 273 contigs. It is planned to fingerprint at least the same number of clones again, from a different library (probably 18 x 96 from that constructed in Paris and the same number from the Sanger EcoRI library), in order to extend these contigs. The Sau3AI set are currently being end-sequenced and are being probed with genetic markers in order to anchor those to the fingerprint map. To date, 547 end-sequences have been obtained, though this number is expected to rise rapidly in the near future as we move actively to end-sequence.  Sequencing of a BAC clone covering niaD and surrounding genes is planned. After probing the library with a PCR product covering the whole of niaD, three positively hybridising clones were identified. Unfortunately these clones each fell into different contigs. Since it was clear that the whole gene probe was not specific enough, we are now probing with separate 5' and 3' probes as well as probes for neighbouring genes.

It was hoped that the smallest A. fumigatus chromosome (1.7Mb) could be sequenced within the pilot project funding, by shotgun sequencing of a library generated from fragmented, isolated chromosomal DNA. Though chromosomes I and II could be easily separated in high melting point agarose, the yield of DNA, after transfer to low-melting point agarose and extraction, was very low producing in the order of 50 - 100 ng of DNA.  This was not sufficient to make a high quality library for sequencing. An attempt was made, but produced a relatively low titre library with high non-recombinant background.  However, by probing with amplified inserts from this library against a chromosomal strip, it was estimated that the chromosomal DNA used was >80% pure, indicating that this approach could be viable should more material be obtained.  We plan to use these chromosome 1 specific inserts to map BAC contigs to that chromosome. Since chromosome I sequencing as a chromosome shotgun is unlikely to be possible within the pilot project, it is envisaged that we will revert to the original plan and sequence a BAC contig.

Conference call:

The purpose of the conference call was to discuss the change in sequencing strategy from a chromosome-by-chromosome approach to a whole genome shotgun approach.  Michael Quail briefly summarized the problems that had been outlined in his report.  Bill Nierman confirmed that TIGR had also had problems with the gel-purified DNA and had been able to isolate only 300 ng of DNA from both the large and medium sized chromosomal "blobs" and could not make a library using DNA from the large "blob." 

There was unanimous agreement that a whole genome shotgun approach should now be adopted.  The rest of the call addressed issues of funding; timetables; the distribution and coordination of efforts; and data release policies.

On the topic of funding, Michael Dunn was unable to make a definitive statement about the status of the Aspergillus fumigatus grant at the Wellcome Trust, except to say that the decision will be made in June or July 2001.  Assuming that the Sanger Center will be funded by the Wellcome Trust to continue work on A. fumigatus, the shotgun phase of sequencing would not begin until early 2002, with an expected completion in the late summer of that year. 

Dennis Dixon emphasized that the NIH would be flexible.  Thus, the NIH funding could be spread over more than two years to permit closure/finishing by both groups after the shotgun phase at Sanger was completed.  In addition, it would be possible to apply to the NIH for additional money.  Discussing the timetable at TIGR and assuming that good quality libraries of 2, 10 and 50 kb could be constructed, Tamara Feldblyum estimated that 5 - 6 x coverage would be generated by September or October 2001.  If no funding were to be forthcoming from the Wellcome Trust, then TIGR could go on to generate a 8 - 10 x coverage with some gap closure and automatic gene calling.

The Spanish contribution was outlined by Miguel Sánchez Pérez (Salamanca). Formal notification of funding is expected in April or May 2001, at a level of 600,000 Ecus, for 3 years (although it could be spent over a shorter time frame).  This would be sufficient to sequence 3 - 4 Mb and they would be able to start contributing to the shotgun phase of the sequencing in July or September 2001.  In Paris, the Pasteur's contribution is internally funded.  Frank Kunst and Jean-Paul Latgé stated that they did not plan to do any shotgun sequencing.  However, they would generate 6000 BAC-end sequences as end-pairs from the Paris library; 2000 of these BAC-end sequences were already completed.  They intend on placing these sequence reads on the Pasteur website and would distribute these data on CD-ROM to TIGR and Sanger when required.  Moreover, the Pasteur would be willing to be involved in the finishing/closure stages after TIGR and the Sanger Centre completed the shotgun sequencing.

Some time was spent discussing management and coordination of the project.  Regarding finishing/closure, there are two options: 1. for centres to close the gaps contained in the clones that they have already sequenced or 2. to assign specific regions to centres. Assigning specific regions to different centers would be complex and Michael Quail commented on the not insignificant logistical aspects of "shifting clones around the world".  Bill Nierman described the standard procedure at TIGR whereby clones are replicated robotically and are frozen in glycerol.  It was suggested that the shotgun libraries could be duplicated by the two main centres (TIGR and Sanger) and Tamara Feldblyum stated that it would be easier to do this using robotics when the clones were first picked for sequencing.  The Salamanca group would find it easier if they received specific clones from the Sanger Centre for closure rather than copies of the libraries.  TIGR and the Sanger Centre will investigate how to exchange sequence traces.  Neil Hall suggested depositing them at the EBI or at the NCBI. An alternative suggestion would be to use CD-ROMS.  Specific details will be worked out at a meeting between Sanger and TIGR this coming June.

TIGR will carry out regular assemblies, which are required to track progress.  These assemblies will be made available immediately on the TIGR website. All the centres  (TIGR, Sanger, Pasteur and Salamanca) agreed that they would release sequence information onto their websites as soon as these data were generated.   Requests for individual BAC clones from the Paris library would, however, require the signing of a Material Transfer Agreement (MTA).

There was a discussion about mapping.  No further effort will be expended on attempts to create an electrophoretic karyotype.  Given that Aspergillus fumigatus has no genetic map "and never will have one", it is essential to have a good tile path.  The Sanger Center group offered assurances that they would be able to generate a good BAC tiled path which would include the assignment of markers.  Moreover, the Sanger Centre will sign the MTA with the Pasteur so that they can use the Paris library in their fingerprinting.  A minimally tiled clone set could be built at the end of this project.  In addition, HAPPY mapping* could be carried out, if necessary.  As part of the continuing pilot project, the Sanger will sequence the largest BAC contig.  Currently, the largest contig is 400 kb. 

David Denning now solicited input from Jean-Paul Latgé, Geoff Turner and Joan Bennett.  All three enthusiastically supported both the concept of the whole genome shotgun approach and the mapping strategy.  Jean-Paul Latgé wondered about involving the Whitehead Institute, but it was agreed that only one US center (i.e. TIGR) should participate, especially given the realities of funding.  However, since the Whitehead has successfully initiated a large scale filamentous fungal genome project (Neurospora crassa), Joan Bennett suggested that it would be worthwhile to have discussions with them about annotation and gene finding in molds.

To wrap up the discussion, David Denning summarized the main points of the conference call, i.e. that a whole genome shotgun approach will be adopted, with TIGR making the three libraries, which they will share with the Sanger Centre.  Sanger will continue to map the three BAC libraries and generate end-sequences.  Salamanca will also contribute to the shotgun sequencing. 

The issue of publication was raised, albeit prematurely.  The Arabidopsis project was held up as a good model for doing annotation.   Finally, it was agreed that another meeting should be held, after firm decisions about funding from the Wellcome Trust and the Spanish government were available, in which all of these topics could be addressed at greater length.  A possible time for the next meeting is the autumn of 2001.

Michael Anderson
Joan Bennett

*refs: Dear PH Cook PR. 1993. Happy mapping: linkage mapping using a physical analogue of meiosis. Nucleic Acids Res 21:13-20

Piper MB, Bankier AT Dear PH. 1998. A HAPPY map of Cryptosporidium parvum. Genome Res 8:1299-1307

Williams JG Firtel RA. 2000. HAPPY days for the Dictyostelium genome project. Genome Res 10:1658-1659

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