NETWORK MEETING

AND GENOMICS WORKSHOP

Smith College, Northampton, MA

31st March - 6th April 1997

the 1997 Filarial Genome Meeting: dinner in the Green Street Cafe, Northampton
back row: Martin Aslett, Reda Ramzy, Gary Weil, David Guiliano, Sara Lustigman, Steve Williams, Kaliraj, Narayanan
front row: Ibrahim Kamal, Sandra Laney, Barton Slatko, Mark Blaxter, Tom Nutman, Alan Scott, Jen Daub

The third Filarial Genome Project co-ordination meeting and training workshop was held at Smith College, MA, in March-April 1997. The meeting, organised and co-ordinated by Prof. Steve Williams, had three goals:

¥ to review progress over the year to April 1997

¥ to plan for the coming years

¥ to share and train each other in new genomics techniques.

Participants

Ibrahim H. Kamal	Ain Shams, Cairo	                FGP/WHO
Reda M. Ramzy	   Ain Shams, Cairo	                FGP/WHO
Ravi Varadharajulu   Centre for Biotechnology, Madras	FGP/WHO
R.B. Narayanan	  Centre for Biotechnology, Madras	FGP/WHO
Alan Scott	      Johns Hopkins University, MD USA	FGP/WHO
Steven Williams	 Smith College, MA USA                FGP/WHO
Michelle Lizotte
        -Waniewski   Smith College, MA USA	     FGP/WHO
David Guiliano	  Smith College, MA USA                FGP/WHO
Mark Blaxter	    ICAPB, Edinburgh	                FGP/MRC
Jennifer Daub	   ICAPB, Edinburgh	                FGP/MRC
Martin Aslett	   EBI, Hinxton, Cambridge              Parasite Genomes Centre/WHO
Barton Slatko	   New England Biolabs, MA USA          FGP/WHO&NEB
Thomas Nutman	   Lab for Parasitic Diseases, NIH USA  scientific advisory group
Gary Weil	       Jewish Hospital of St. Louis, MO USA scientific advisory group
Sara Lustigman	  NY Blood centre, NY USA              scientific advisory group
Boris Dobrokhotov    WHO, Geneva	                     WHO observer

1 Library construction

a cDNA libraries

To add to the five libraries already available to the filarial community (microfilaria, L3 (spliced leader), L3 (conventional), L4 (spliced leader) and adult male) two new stages were obtained:

L2 a library made from L2 larvae day six after infection of the mosquito by Al Scott's group. This library was constructed using the SL-oligodT method, as it is not possible to isolate L2 in the quantities needed for conventional library construction.

AF a library constructed from fecund adult females by Steve Williams' group. The library was made using the conventional methodology.

b genomic libraries

Two genomic libraries were announced.

cosmids a 18,000 clone cosmid library in the vector SuperCos was constructed by Steve Williams' lab. The library was very new and was partially characterised during the genomics workshop.

BACs a 5000 clone bacterial artificial chromosome library was constructed in the vector pBeloBac by Steve Williams' lab. The library was new and uncharacterised.

a cDNA libraries

Three cDNA libraries have been gridded. 18,000 clones from each were picked into microtitre plates and these plates used to print filter mats for screening. Thus far only a limited amount of screening has been carried out, but the grids and libraries are available.

b genomic libraries

The cosmid and BAC libraries were being picked to microtitre plates (indeed 2400 were picked during the workshop) and filtermat imprints of the grids will be distributed shortly.

The FGP had submitted just under 8000 ESTs to dbEST at the time of the meeting. These came from six life cycle stages and represented around 3500 different genes. One important advance presented was the development of a clustering algorithm (D. Guiliano) which permits the derivation of "clusters" of related sequences by identity, and thus the definition of the number of "genes" thus far identified.

The FilGenNet WWW site continues to develop: there are now over 6.5 Mb of data on the WWW site and it is accessed at the rate of 5-10 logins per day. The WWW site also houses the Bibliography on Filariasis (BiblioFil) developed with the aid of the Clark Foundation.

The parasite-genomes computing resource centre at the EBI has provided a very useful filarial-specific BLAST server.

The new Filarial Genome Project database, FilDB, was also premiered. This ACeDB-based database includes over 20,000 references (from BiblioFil), 8000 sequences and has extensive cross referencing and analysis options.

The 1997 Genomics Workshop (see annexe 2) was deemed to have been successful. The participants went through the process of plating out clones, picking them to microtitre plates, imprinting filter mats with colonies and processing them for hybridisation. The workshop mapped the first genes to cosmids (ribosomal proteins) and performed cross screening of cDNA library filters to eliminate previously sequenced clones.

The project discussed plans for the future. These can be divided into three segments:

cDNA libraries and EST generation; genomic DNA libraries and mapping; bioinformatics and networking.

1 cDNA Libraries and EST sequencing

The FGP will generate additional cDNA libraries from early mammalian stage larvae. Day 6 L3 larvae have been collected and will be used for the construction of a pre-moult L3-L4 transition library. The Blaxter lab will attempt to get day 9 post infection (late L3-L4 transition) and L4 larvae for the construction of additional conventional libraries.

EST sequencing will continue. The project aims to get a sizeable dataset from each library/stage. A tentative schedule of EST sequencing was agreed

Library          Number of ESTs   Sequencing laboratory
                 current  1988-9
microfilaria     1400	600     SAW/BS
L2                550     450     MLB
L3 (C & SL)      2200       -
L4 (SL)           850     650     KJ/MLB
Adult female     1400    1600     MLB
Adult male       1300     700     RR/SAW
new libraries			
L3 day 6            -     500     MLB
L3-4 day 9          -     500     MLB
L4 conventional     -     500     MLB

2 Genomic libraries and genome mapping

(see here for a rationale for mapping the genome presented at the workshop and here for an outline of the procedures chosen)

This is to be the main thrust of the new work of the project. The Williams lab has constructed a representative cosmid library (insert size ~35 kb) and a BAC library (insert size ~75 kb). These libraries have been gridded and will be distributed to the genome labs.

The cosmid library has >18,000 recombinants and thus has ~6 fold coverage of the 100 Mbase genome. The BAC library currently has 5000 clones and thus 3.75 fold coverage. The BAC library will be supplemented by additional clones.

The BAC grids will be the substrate for the generation of a physical map of the genome of Brugia. We will use two methods to construct the map. The first will be the hybridisation of cDNA clones to the BAC grids. The genome project labs will each attempt to hybridise 200 ESTs to the grids in the year. This will give us 1200 sequenced and mapped markers. The ESTs for hybridisation will be chosen from the datasets produced by the labs, and to ensure that there is not significant overlap between EST sets Al Scott will co-ordinate clone choice, with reference to the clustered EST dataset (see informatics below).

The second method used will be based on a "sample without replacement" model, carried out in Edinburgh. A BAC clone will be selected, end probes synthesised and used to identify overlapping BACs on the grids. A second BAC will then be selected which has not been identified by hybridisation, and used to generate probes. This process will be repeated until all the clones have been identified.

These two datasets (EST and end-probe) will then be combined to produce a first generation physical map. The map will be refined through by making of end-probes from outlying clones in the larger contigs: these will be hybridised to the grids to identify neighbouring contigs. We expect the first generation map to consist of between 1000 and 1500 contigs, and the second generation one to have 300-500 contigs.

The project identified future goals for genome mapping research. The first is the identification of the repetitive DNAs of Brugia. It is known that the repetitive DNA content is approximately 17% and that a single repeat, the Hha I repeat, makes up ~10% of the genome, but the sequence and structure of other repeats has not been analysed. The project will sponsor the investigation of the non-Hha I repeats of Brugia. These repeats will be used to aid the genome mapping element, by identifying possibly confounding repeat-containing clones.

The second was the generation of long-range restriction maps of the genome. The chromosomes of Brugia (n = 5) are too large to separate by pulsed field electrophoresis. A long range restriction map generated using "rare cutters" might allow both the linking of contigs made by hybridisation and thus the construction of whole chromosomes. To generate the map, a "linking library" (of clones whose inserts contain a rare-cutter site will have to be made and hybridised to restriction digests analysed on pulsed field gels.

Thirdly, the telomeres of Brugia may be of interest from a biological (in that they may contain genes with exceptional expression patterns), genomics (in that they define chromosome ends) and diagnostic (in that they may harbour repeats useful for PCR diagnosis) standpoint. Methods for telomere cloning will be investigated.

3 Informatics and networking

1 Informatics

The clustering algorithms developed by David Guiliano will be used to define a reduced redundancy set of Brugia "genes". These clusters will be used to generate consensus sequences for each gene and this database of genes then used for further analysis. The gene database will be analysed for database similarities and thus putative function, sequence features (such as signal peptides, transmembrane regions, polyaminoacid tracts) and stage specific expression patterns. A world wide web based server, using the SRS system of the European Bioinformatics Institute, will be developed.

The genome database FilDB will be further refined and made public. Tools for input of genome map data and other features will be developed. The database will be installed in all project labs.

2 Networking

The project will continue to use the filarial-genome email network and the world wide web site FilGenNet to publicise and communicate results. The full protocols for the genomics workshop will be made available on the www.

The project will hold the next network meeting and genomics workshop in Ain Shams University, Egypt, in the spring of 1998.

All project members will continue to promote the project through presentations at meetings, seminars and publications.

Thanks to the Smith Lab for organising the meeting, especially the food and the snow!

Mark Blaxter

Network Secretary

Edinburgh, 20 May 1997