ATTENDANCE Karen Barlow - Sanger Christine Nicholson - Sanger Eileen Wang - China Lorietta Lukas Mueller Giorgi Valle - Italy Giovanni Giuliano - Italy Remy Bruggmann Carl M. Jones Naama Menda Rene Klein-Lankhorst Steven Stack Roeland van Hamm Jim Giovannoni - U.S. Alessandro Vezzi - Italy Erwin Datema MINUTES Called to order at 1:12pm. 1:16 - Lukas presents agenda. Chromosomes 2,_,8, and 9 are not represented. LUKAS: All projects have started except Spain. Tomato chloroplast is finished (by Plastomics), but sequence is not available. All BAC end sequencing is finished (400,000 reads, 333,808 good reads, 85% good). Success rate was pretty much exactly 85%. The sequencing was performed by a company named Seqwright in Texas. On the US end, 11 BACs have been fully sequenced. NSF is interested in seeing sequences for BACs at the junction between euchromatin and heterochromatin. We tried to choose BACs from these junctions, but most of them turned out to be heterochromatic. We have also done some work on deriving some repeat data sets from the BAC ends. There needs to be a serious effort to generate a repeat dataset for tomato, I think. We are in the process of annotating some repeat families we obtained using Recon (we got 137 families with >100 members). Also, not everyone is aware of the intron finder tool at SGN. This helps avoid designing primers from cDNA transcript sequence that turn out to overlap with an intron in the genomic sequence, and thus don't work. The intron finder aligns your tomato cDNA sequence with Arabidopsis and predicts where the introns are in your sequence based on where they are in Arabidopsis. These introns are usually very well conserved. ___: As of Dec. 2005, the tobacco genome genomic sequence is available. LUKAS: I didn't know that. We should work that into the intron finder. Valle: But there's not much annotation on that yet. LUKAS: Also, it's important that we all use the BAC registry on SGN to avoid duplicating effort. LUKAS: Also, on SGN, you now have the ability to add a BAC to your project yourself. CHROMOSOME 2 - Korea 39 seed BACs, 34 extended, 40 in sequencing pipeline found gene density of 4.6kB/gene CHROMOSOME 5 - India CHROMOSOME 8 - Japan 14 BACs completed, currently assembling 6 BACs, and another 10 BACs further back in the pipeline. CHROMOSOME 3 - China Ying Wang 2 institutes involved in sequencing - Wuhan Botanical garden and Institute for Developmental Biology Now doing 2 things - sequencing euchromatic regions and dynamic development of a fine physical map. -we have confirmed about 50 BACs through FISH -20 BACs have been confirmed by FISH on chromosome 3 -18 on euchromatin, 2 in heterochromatin -5 have been finished -15 have sequences at stage 1 and 2 developing fine physical map for the tomato genome -manual editing of FPC map, reduced the contig number from 6794 to 3000 -PCR screening of positive BACs containing the genetic markers -dynamically integrate the genetic map and the FPC -manually edit the overgo and FPC results -in silico digest of sequenced BACs, integrate and compare with FPC -integrating known sequences (BAC ends, EST, GGS, markers) -PCR screening results -genetically mapping the unanchored contigs -manual FPC current status -contigs: 3000 -anchored contigs: 465 -genome coverage of anchored contigs: 200.5 Mb -coverage of FPC: 788 Mb (75%) -Newly anchored markers by PCR screening: 27 CHROMOSOME4 - Sanger Christine Nicholson PLUS: progress on fingerprinting MboI library strategy for mapping chromosome 4: -fully develop physical map in order to select min. tiling paths -significant comp. analysis prioer to clone selection -AIM: reduce number of contigs and gaps fpc analysis -54 chromosome 4 markers in original fpc build -examined chromosome 4 marker-containing contigs -as of Jan 2006, 61 contigs 2 finished HindIII clones - 31H5, 198L24 WTSI Sequencing pipeline -pipeline status 4 in mapping 3 in subcloning 1 in shotgun 2 finished strategies for reducing number of contigs -continued FPC development of chromosome 4 contigs -walk off sequenced clones using bac end sequence hits -incorporate further bac end sequences location confirmation - FISH -198L24 confirmed on chromosome 4, in heterochromatin -strangely, there were no problems finishing this BAC, which is unusual for one from heterochrom. -next, 15 more BACs being verified by Stack via FISH Fingerprinting an Additional Library -LE_HBa done at Arizona Genomics Institute -agarose gels -marker: hi-lo with lambda-hindiii, lambda-sall, lambda-stul -we replicated this technique at Sanger -used for clone verification -summary: SL_MboI library -52,922 clones on 144 plates, 135kb average insert size -7X genomic coverage why do additional fingerprints? -increase map coverage, depth gel process -gel images are processed using Image (www.sanger.ac.uk/Software/Image) progress -fingerprinted 103 plates so far (about 40,000 clones, 72% of lib) -scheduled to complete in April 2006 further BAc selection and timeline -incorporate MboI fingerprints by April -tilepath selections in April and May in FPC -further sequence gen Summer 2006 -80% sequence in pre-finish or better by Dec 2006 -additional bridging clones selections as required -projected to be sequencing 193 BACs Giovanni Giuliano: We put through a proposal for fingerprinting EcoRI, but we did not get the money. So we probably can't do it. CHROMOSOME 6 - Rene Klein-Lankhorst -state of the art -selection of minimal overlapping extension BACs from tomato chr.6 using BAC end sequences CBSG tomato sequencing program status -have finished 35 BACs, 33 in phase 1/2 and 2 in phase 3 -2 BACs currently in seq pipeline -8 currently in FISH pipeline -16 BAC sequences uploaded to SGN selection of extension BACs by BAC-end screening seed BAC blastN versus bac ends assembly of selected BAC ends preselected BACs +0/+0 AFLP fingerprint to reconfirm the selection of BAC BAC binning High-res local physical map select largest-extending, minimal-overlapping BAC have sequenced 3 extension BACs, confirmed 100% sequence overlap in each case -this proves the concept of the BAC-end strategy -no need to fingerprint an entire BAC library -blast, assembly, and AFLP work complementarily -accurate selection of BACs having minimal overlap and largest extending insert if you don't confirm with AFLP, you might jump to another chromosome and start sequencing that one! EU-SOL Project Overview - I will be the managing director of the program office - EU-SOL is geared toward characterizing tomatoes and potatoes. - 56 partners, 16 countries, 70 groups - budget of 31 million euros for 5 years CHROMOSOME 12 - Giovanni Giuliano and Giorgio Valle funding comes from 3 projects - Agronanotech (ministry of agriculture), - FIRB (Italian Ministry of Research) - EU-SOL (European Commission) validation at UniNA -single colony picking -medium-scale prep -restriction analysis to check E. coli contamination -BAC-end sequencing (check end sequences) -PCR markers development based on marker seqs -amplicon sequencing and sequence alignment -mapping on ILs There are large gaps in anchored BACs on chromosome 12. As much as 17 cM large. In physical distance, these could be as large as 20MB or more! -10+3 seed BACs slected -2 BACs finished and ready to submit -1 BAC pre-finished, ready to submit -3 BACs in progress, should be ready within 2-3 weeks Need more seed BACs and need to work out a fast way to obtain contiguous BACs. TALK - Jim Giovannoni BAC Verification TALK - Remy Bruggman Training Gene Finders for Tomato GenomeThreader - identified 164 genes on available BACs (48 finished bacs) - ~3.4 genes/BAC - these genes can be used to train gene finders - these may be biased toward short genes or genes with few introns Which programs can be trained for tomato? - EuGene? - performed well for arabidopsis and medicago - combines the output of many other programs TALK - Steven Stack Tomato FISH Functions of FISH -confirm location of BACs -demonstrating the order of BACs -determining the distance between BACs -assembling BACs into contigs We always use pachytene chromosomes for FISH. 2 main ways to prepare - fix in 1-3 acetic ethanol, and squash cell with some other means - burst cell 1-3 acetic shows difference between euchromatin and heterochromatin disadvantage is there is some stretching in the chromosome with squashing we prefer bursting doesn't stretch the chromosomes, and they tend to fall out in a nice distribution. if you have a BAC that has a lot of repeats, you can get hybridization to places you don't want. to alleviate that, put a bunch of unlabeled repeated sequences into the preparation to bind to the repeats and effectively remove them from the equation. 45 BACs have been FISHed so far. TALK - Naama Menda Ontologies for Describing Solanaceae Phenotypes DISCUSSION SESSION