Filarial Genome Meeting 14-18 March, 1998
WORKPLAN FOR THE NEXT 3 YEARS
In the workplan the sections are divided into: Questions to be addressed; Discussion and Decisions/Recommendations.
I. EST Sequencing and Gene Discovery
Questions
1. How many ESTs need to be sequenced for the rest of this year and from which libraries?
2. Do we need new libraries for EST sequencing? Which stages are needed? What subtraction libraries should be made?
3. Where should the sequencing be done? Only in Slatko and Williams labs, with mapping in the other labs (Ramzy, Jayaraman, Supali, Scott, Blaxter)?
Discussion
Stages suggested for new library construction were L4 (conventional), new L3-L4 transition, L4-immature adult transition, immature adult, and vector-derived MF/L1. Ted Bianco suggested that L1-L2 or L2-L3 need about 6000 larvae. According to Ted, after the mosquito gets the MF, at 3 days the full repertoire is present; after 1 hour, transcription activity starts. We could pool days 0,5,11,15 due to lack of availability of material (a balance between the number of worms and the effort to get each stage). Al pointed out that this is labor intensive, not difficult. If the goal of the project is to eliminate the disease, we maybe should also look at MF/L1 (blood to peritrophic membrane). A discussion on the biology of the life cycle ensued. Males mature earlier while females do not mature till later. Despite females being inseminated right away they are not mature to have fertilized eggs until later. Then female development continues while males become quiescent and transcriptionally inactive. Day 30 females and males are hard to separate under microscope; 2000-3000 L4 would be needed to make the library.
It was the consensus of the discussion that we have to balance sequencing and mapping. So far, the funds have been used for gene discovery, but we want to go forward with mapping while keeping the sequencing effort up. In the endemic labs (including Indonesia) sequencing is up to speed and they have learned (or know) how to sequence. They need to learn more bioinformatics and mapping technologies. The sequencing cost is also lower in Barton's and Steve's labs while mapping can be done inexpensively in the endemic labs. We will have created technology transfer (sequencing, mapping and informatics) to the endemic labs.
We need to keep up identification of novel gene discovery: subtraction methods are critical. Subtraction approaches work with hybridization onto filters also. Gridding additional libraries and subtraction with L4 abundants on filters might be of interest.
Decisions/Recommendations
Sequencing Effort We are aiming for a total of 20,000 ESTs by end of this year and to identify 8-9000 novel genes. In year 1 of the next grant (1999) we will sequence from L4C, L3-L4, L4-adult, day 30 female, with 2000 ESTs from each to be sequenced. We will need to sequence more due to decreased return, unless subtraction methods are used.
New Libraries It was suggested that we make libraries from L3-L4, L4C, L4-adult, immature adult and vector MF. Due to difficulty of obtaining enough material for stages of L1-L2, we should stockpile material for the library which could be made in upcoming years 2 or 3. We could make a library for these stages or could make an Onchocerca L1-L2 and piggyback to Brugia..
Subtraction Libraries The Williams lab will do more ESTs on each subtracted library to check novel gene discovery since the subtraction method generates internal fragments. We need to see what % of subtracted library is novel and whether some are parts of previous cDNAs not sequenced yet. For continued subtraction, new libraries should be subtracted with the high redundancy ESTs as "drivers". We have some data to suggest what these drivers should be and we will have more information within the next few months.
Gridding New Libraries We should put AF on grids. L4C should also be gridded when ready. Mark will check with the Sanger Center to see if it is less expensive than current costs.
Sequencing Rationalisation The EST sequencing will be done in Slatko and Williams labs (Washington University) with mapping by all the labs. If any (including endemic labs) want to sequence, we can supply templates and materials or let them know what is underway so that there is no duplication. They can, of course, also sequence anything of interest to them. Research strengthening grants (RSGs) can be used to help sequence genes of interest. We will then bring endemic labs up to speed on mapping and bioinformatics.
II. Mapping
Questions
1 Wolbachia DNA in the libraries: what should be done? Can we make Wolbachia-free libraries?
2 Is the BAC library representative? Do we need a YAC library? Why don't all the probes hybridize?
3 How do we make the hybridisation mapping process effective? Is stripping between probings necessary?
4 How do we coordinate mapping? Which and how many clones will each lab map? Should we charge for filters to non-project labs? If so, how much? Should we set a policy for charging for BAC filters? Should we wave the charge if we can get hybridization data form the lab?
Discussion
It is critical to identify Wolbachia in the genomic libraries: Wolbachia contamination will reduce the overall coverage of the library. We will ask if we need to make more BACs or regrid the ones which are not Wolbachia (although this may not be necessary). We also need to make sure there are no chimeras. We hope to have this answer soon before mapping onto the BACs. If there are 3000 BAC clones on the filter grid, the worst case scenario is 100% Wolbachia; 20% Wolbachia might be correct, based upon David Guiliano's and Williams lab's preliminary hybridization data (50% Brugia probes do hybridize; many Wolbachia positives). If the contamination is 20% or less, the filters will still be useful for mapping; we can reuse saved non-radioactive probes for YAC hybridization. End sequencing 50 BACs to see the data (A + T content) will be informative also. We will wait until the data in to decide about the library. DNA isolation without Wolbachia could be made by using sperm to make DNA or one could try tetracycline curing of Wolbachia or use PFG to separate Wolbachia DNA from the rest of the chromosomal DNA. With either method, contamination could be monitored by PCR of Wolbachia sequences. For whatever reason, the cosmids are clearly contaminated with Wolbachia. This indicates Wolbachia DNA clones more efficiently. We will have to check the YAC library as well. We will know fairly soon.
Is the BAC library good enough? If not, a new BAC library will have to be made. The YAC library is being made (Slatko and Blaxter labs). A Mun I digest will also be used to clone into the vector Eco RI site, to get a more random distribution of ends. We want clones of about 200 kb. BAC-end and YAC-end mapping and sequencing will begin in these labs. Contigs may be joined if we use several libraries, as well as use the YAC library to overlap the BACs.
It could be that short probes don't work as well for labeling or that the library is not representative due to the Wolbachia contamination. It is possible that introns may be a problem for probes which may not hit contiguous sequences on BAC filters. We need to correlate probe size with positives. Is there some way to make a heavily biotinylated oligonucleotide which can be splint-ligated or hybridized in some way to a sequence to be made as a probe?
Coordination of the EST hybridisation mapping scheme is essential: this role has been taken on by Al Scott. We need to maintain a current list of positive hybridization onto BACs into a database. Probes which were not positive are also useful data for estimating the coverage of the library.
Decisions/Recommendations
Wolbachia The Williams and Blaxter labs will work on a primary physical map of Wolbachia. This will involve identifying all the Wolbachia DNA containing clones in the library. David Guiliano and the Slatko lab will end sequence 50 BACs to see A + T content. Wolbachia free libraries would be a good thing: the Edinburgh lab will investigate biological and physical methods for generating endosymbiont-free nuclear DNA.
YAC Library The library will be built and tested by Jeremy Foster (Slatko lab) and tested by the Slatko and Blaxter labs. The YAC filters will be prepared by the Sanger Center and filters will be distributed when available.
Hybridisation Probe Selection A list of clusters to hybridise will be prepared by Al Scott in consultation with Edinburgh. We want 50 positives per lab, so that we estimate we will 100 per lab to try; others can be done if one has one particular one of interest by whom-ever. (Slatko and Williams labs will hybridize less due to increased sequencing). Al will assign clones to each lab; one can select other interesting ones if one wants.
Optimising Probe Labeling Slatko and Blaxter labs will co-ordinate the results they have. If probe size is an issue, we will investigate alternative labeling strategies: use a tree'd biotin linker or label with fluorescent and PCR label. Can we PCR biotin label? Will terminal transferase work with tagged nucleotides?
Pooling probe techniques will be useful: We can do 10 probes at a time, can go to the BAC library and pick out positives, respot and rehybridize individuals with the individual probes to see what is what. This will save time and effort. We will start with singles and then make pools from the others. There will be positive controls in each pool. We will investigate whether stripping between probings is necessary. David Guiliano will follow this up.
How many filters If we get 8 reprobings from each filter, each lab will need 15 filters per lab, unless we can extend the lives of the filters.
Hybridisation Database/Coordination All data is to be sent to Al Scott, who will verify it and send it on to Mark Blaxter for inclusion in the project databases. It is also important that lumigraphs be sent to Al for independent verification of clone assignments. A www form for data submission will be made. Autoradiograph verification would also be useful. The hybridization data will be put on the FilGenNet www pages.
III. Informatics and Databases
Data Curation There is a need for hands and eyes to perform curation on the databases, to make them more useful for all. A half-time post in Edinburgh would significantly advance the project.
FilGenNet www site The www site will be updated (by Mark Blaxter) and the mirror Smith site will need to be updated.
FilemakerPro Cluster Database The Blaxter lab will purchase copies of the software for the Jayaraman and Supali labs. The database will be released as soon as is possible.
FilDB Martin Aslett and Mark Blaxter will have a release version available within a month or so. It will be distributed by CD-ROM (the CD will also have the other parasite genome databases and www sites on it) and by ftp from the EBI parasite genome www site.
IV. Grant Issues:
1. RSG grants could be used for informatics. It would be valuable for researchers from the endemic labs to go to Edinburgh for formal training, sequence analysis in FilDB, annotation, datasets, etc. Other labs could come depending on space, although they would have to pay a fee to attend. Boris Dobrokhotov suggested that this workshop might be made available to all the parasite genome projects, but this would necessarily dilute the filarial-specific aspects. It could be held in conjunction with our next meeting. The structure might be 1 week of formal training followed by the meeting, followed by 3-4 weeks of intensive work on the filarial bioinformatics strand, supervised by the Blaxter lab. Would funding provide equipment or software? Mark Blaxter will submit, as soon as possible, a grant for funding. A new proposal for the core WHO Parasite Genomes Computing support facility is also required this year. Mark Blaxter will poll the other networks to coordinate bioinformatics needs and the idea of the informatics workshop.
2. The core Filarial Genome grant renewal is due in July. It will be a three year continuation grant, and will have to include outline plans for the full three years. This grant will include all the decision/ recommendation points above and will be coordinated by Steve Williams and the other principal investigators.
3. Possible initiation of Post-Genomics Research. It was noted that the WHO is keen to start funding (limited) post-genomics research. We can apply for small director's initiative grants any time, and these may be excellent means of funding postgenomics research in endemic labs. These should be submitted independently from the 3-year grant. Interested labs/individuals should submit short pre-proposals to Steve Williams by April 16. The genome project will assist in formulating the final proposal and they will be submitted under the Filarial Genome umbrella. There is a $15,000 maximum.
4. A parasite genomes meeting in India. Kunthala Jayaraman suggested that a research symposium for all the genome projects might be held in Madras. K. J. will submit a grant to WHO for this.
5. The next Filarial Genome Network Meeting It was agreed that a meeting should be held next year, about the same time. Mark Blaxter volunteered to organise it in Edinburgh if it was held at the same time as the informatics workshop.