Present: David Denning (Univ of Manchester): Chair; Stephen Oliver (UMIST); Celia Calcott (Wellcome Trust); Bart Barrell (Beowulf Genomics); Philippe Glaser (Institut Pasteur); Andy Brass (Univ. of Manchester); Michael Anderson (Univ. of Manchester): secretary.
Apologies for absence: Jayne Brookman (Univ. of Manchester)

Introductions

P. Glaser: applied to EU for A. nidulans sequencing project (1 chromosome): not funded; considering with J-P Latgé sequencing random shotgun genomic clones of A. fumigatus to create an expression array.
Celia Calcott: involved in the funding of projects for pathogen genome sequencing.
Bart Barrell: pathogen genome sequencing at the Sanger centre; 25-30 Mb shotgun/year. Current projects include S. pombe as part of an EU consortium and a pilot project for Candida albicans.

Introduction

D. Denning: outlined reasons for sequencing A. fumigatus because it is the main cause of Aspergillus disease in humans (85 %) and because as demonstrated by one autopsy study (1992) there has been a proportional increase (17 to 60 %) in the number of cases so that by 1992 4 % of patients were infected with Aspergillus.  There is also only a 35 to 45 % response rate to available antifungals.  In contrast, 1 % of infections are caused by A. nidulans.  D. Denning also stated that A. fumigatus is asexual, haploid and an opportunistic pathogen.  S. Oliver enquired about the phylogeny of the Aspergilli and C. Calcott about the level of genetic similarity.  I have subsequently checked these out: the Aspergilli are a closely related group on a branch that includes Penicillium species with  2 % sequence difference (18 S rRNA) and there is 72-74 % DNA identity between A. fumigatus and A. nidulans catalase homologues.

A. nidulans

S. Oliver: wanted to know why not sequence A. nidulans as its sequence will be of more general use?
C. Calcott stated that the Infection and Immunity panel had discussed which pathogens it considered important enough to sequence.  A. fumigatus has been chosen because it is an increasing clinical problem and also to an extent a 'Cinderella' organism.  The Wellcome Trust would consider the arguments for sequencing a model organism, but prefer concentrating on pathogens.  There is a need though to find out what is being done with A. nidulans and D. Denning will find out from Geoff Turner (Sheffield).  S. Oliver suggested linking the sequencing of A. fumigatus with the available information on A. nidulans such as using its genetic map to decide on an interesting region to sequence in the pilot project.  Again D. Denning is to discuss this with G. Turner.

Genomic versus cDNA sequencing

Both C. Calcott and S. Oliver voiced strong opposition to any cDNA sequencing.  The Wellcome Trust has a principle of going for complete information and for doing it properly and S. Oliver stated the example of the Arabidopsis situation where valuable time has been lost with cDNA sequencing rather than just getting on with the genomic sequence.  D. Denning stated that cDNA clones might be useful for expression arrays.

Pilot Project

B. Barrell suggested that the whole genome shotgun approach was not applicable even though it is being done for A. fumigatus (Incyte) and Candida (Scherer).  The Sanger centre prefers a more methodical approach using physical mapping to reduce the number of gaps between contigs.  There is an argument for making both cosmid and BAC libraries and using both types for physical mapping.  Two genetically closely linked genes from A. nidulans could be used as a starting point for isolating clones.  B. Barrell suggested a similar pilot project to that developed for Candida involving the sequencing of 10 overlapping cosmids.  This pilot project would see if there are potential problems with the map and to work out sequencing costs.  P. Glaser suggested the use of BAC clones with the additional task of sequencing many ends which would act as an aid to future mapping.  B. Barrell suggested that there might be problems with recombination within BACs because of their size.  M. Anderson worried about the size of the task involved in generating a complete physical map using cosmids.

Chromosomal analysis

As it was decided that the chromosomes of A. fumigatus are probably too large to isolate and shotgun clone, it was nevertheless proposed that further effort be made to work out an electrophoretic karyotype as this will aid the mapping.

International element

As the Wellcome Trust would not be funding the complete genome sequence without first approving a pilot project, it was felt that an international element both in terms of funding and sequencing would be applicable for the whole genome.  The establishment of a good cosmid and/or BAC bank with associated mapping during the pilot would be a suitable resource for fielding out variously sized projects.  It was stressed that the setting up of an International consortium should start now and D. Denning mentioned his ESF workshop in the autumn as a suitable forum.

Which isolate?

The isolate to be sequenced should be scientifically the most appropriate.  It was agreed that Incyte would not be releasing their A. fumigatus sequence data and so there was no reason not to use the same isolate that they used (AF10 ATCC 90240).  J-P Latgé and D. Denning will discuss this at their June meeting.

Pilot proposal

Construction and mapping of library: to be done in most appropriate UK lab (will be peer-reviewed) with another library to be constructed by an European lab (e.g. J-P Latgé's).
Sequencing (and possibly also the mapping) to be done at the Sanger centre.
Exploratory work: CHEF analysis.

To do

C. Calcott: will investigate the most appropriate funding path and whether one or two pilot projects should be considered.
D. Denning: to discuss A. nidulans with G. Turner; to discuss libraries and isolate with J-P Latgé and to set up international consortium.

 

M. Anderson
20 May 1998

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