1. Tomato Chromosome 4: A Mapping & Sequencing Update 28 th September 2005 Christine Nicholson Mapping Core Group Welcome Trust Sanger Institute, UK

2. FPC Database Mapping strategy = to develop the physical map in order to select minimal tiling paths across the chromosome to sequence, in conjunction with BES data and markers.Mapping strategy = to develop the physical map in order to select minimal tiling paths across the chromosome to sequence, in conjunction with BES data and markers. FPC database worked on in-house at SangerFPC database worked on in-house at Sanger Obtained from Arizona Genomics InstituteObtained from Arizona Genomics Institute The FPC Database:The FPC Database: –LE_HBa library fingerprints –88,584 fingerprinted BACs (68% of total library) –Possibly due to contamination of library Therefore, Therefore, Have ~ 10 X coverage in the fingerprint map.

3. Libraries * Heinz1706 **Based on genome size of 950Mb. No. of clones Average insert size **No. of genome equivalents Coverage in fingerprints BESavailable Mated Pairs % LE_HBa*129,024 117 kb 15 X 10 X 152,81968 SL_MboI52,992 135 kb ~ 7 X -101,75570 SL_EcoRI72,264 95-100 kb ~ 7 X -99,45565

4. Chromosome 4 Contigs Examined seed BACs/contigs for each chromosome using overgo probes. (Conducted by Cornell)Examined seed BACs/contigs for each chromosome using overgo probes. (Conducted by Cornell)ftp://ftp.sgn.cornell.edu/tomato_genome/seedbacs/20050112_chr4_long_short.xls 54 markers on chromosome 4 in the current FPC build54 markers on chromosome 4 in the current FPC build Each potential chromosome 4 contig assessed in silico in FPC: - structure based on fingerprints - marker content – assigned to how many contigs? - possible merges to other contigs?Each potential chromosome 4 contig assessed in silico in FPC: - structure based on fingerprints - marker content – assigned to how many contigs? - possible merges to other contigs? September 2005: 58 contigs currently on chr 4

5. Pilot Sequencing Select 5 BACs to form “pilot sequencing”.Select 5 BACs to form “pilot sequencing”. In two different regions of the chromosomeIn two different regions of the chromosome Tomato BACs are being processed through our sequencing pipeline.Tomato BACs are being processed through our sequencing pipeline. Apply our Finishing programmes to tomato clones & examine sequence features e.g. repeats.Apply our Finishing programmes to tomato clones & examine sequence features e.g. repeats. Sequencing Pipeline at WTSI…… Sequencing Pipeline at WTSI……

6. Sequencing Pipeline MappingSubcloning Production (Shotgun sequencing) FinishingQC At Wellcome Trust Sanger Institute: ANNOTATION Imperial College manual annotation MIPS (automated) Single colonies Clone verification (PCR using BES probes) Further digest check

7. Wellcome Trust Sanger Institute Sequencing Packard Minitrak: Sequencing reactions set-up MJs: Cycle sequencing ABI 3730s: Sequence data generated & analysed

8. Sequencing Pipeline MappingSubcloning Production (Shotgun sequencing) FinishingQC At Wellcome Trust Sanger Institute: ANNOTATION MIPS (automated) Single colonies Clone verification (PCR using BES probes) Further digest check Imperial College manual annotation

9. Selected Chromosome 4 BACs Located in contig 270Located in contig 270 Marker C2_At5g37360Marker C2_At5g37360 LE_HBa clones: LE_HBa clones: 198L24 (contains marker) 31HO5Stage No. of BACs Selected for Sequencing 3 Finishing2

10. Sequence data available for 2 BACs CloneAccession No. of contigs No. of gaps Approx. size 13H05CT02587732 (All spanned) 168kb 198L24CT02587376 (5 spanned) 165.5kb ftp://ftp.sanger.ac.uk/pub/sequences/tomato/unfinished_sequence/

11. Finishing - Analysis in GAP 4 PHRED – calls basesPHRED – calls bases PHRAP – assemblyPHRAP – assembly GAP4 – view & editGAP4 – view & edit

12. FISH Analysis BAC 198L24 underwent metaphase FISH.BAC 198L24 underwent metaphase FISH. Confirmed chr 4.Confirmed chr 4. Reported to be in heterochromatic region.Reported to be in heterochromatic region. However, no major issues (repeats etc.) in FinishingHowever, no major issues (repeats etc.) in Finishing Image courtesy of S. B. Chang, Prof S. Stack’s Laboratory, University of Colorado, USA.

13. Genes in Sequenced BACs? Used WUBLASTX  align proteins against the tomato sequence.Used WUBLASTX  align proteins against the tomato sequence. Partial gene highlighted in each BACPartial gene highlighted in each BAC 31H05  putative carboxyl-terminal peptidase 198L24  AMT1.2 Ammonium transporter 1 member2

14. Mapping Strategy * AIM = Reduce the current contig number * * AIM = Reduce the current contig number *WHY? Select longer minimal tilepaths across the chromosomeSelect longer minimal tilepaths across the chromosome Smaller overlaps of selected sequence BACs (aim for 15- 20kb)Smaller overlaps of selected sequence BACs (aim for 15- 20kb) More efficient sequencingMore efficient sequencingHOW? Work on FPC database to improve continuityWork on FPC database to improve continuity Walk off sequenced clones (once available) using BES hitsWalk off sequenced clones (once available) using BES hits Incorporate further BES/fingerprint data as generatedIncorporate further BES/fingerprint data as generated Possible walk from contig ends by hybridization.Possible walk from contig ends by hybridization.

15. Further Map Development & Fingerprinting? Have been able to replicate the fingerprinting technique from AGI.Have been able to replicate the fingerprinting technique from AGI. 31H05 and 198L24 fingerprints confirmed.31H05 and 198L24 fingerprints confirmed. Will use this for future clone verification.Will use this for future clone verification. Currently have 10 X coverage in fingerprints.Currently have 10 X coverage in fingerprints. Investigating the practicalities and possibilities of augmenting the fingerprint database → SL_MboI library?Investigating the practicalities and possibilities of augmenting the fingerprint database → SL_MboI library?

16. Further BAC Selection Strategy Intend to select minimal tilepaths to sequence  ideally over reduced number of contigs on chromosome 4.Intend to select minimal tilepaths to sequence  ideally over reduced number of contigs on chromosome 4. Will FISH more BACs across the chromosome  obtain confirmation of chromosome location of BACs & contigs they are contained within.Will FISH more BACs across the chromosome  obtain confirmation of chromosome location of BACs & contigs they are contained within.

17. Acknowledgements Wellcome Trust Sanger Institute: Jane Rogers Sean Humphray Carol Scott Karen Barlow Helen Beasley Sarah Sims Jennifer Harrow Carol Carder Paul Hunt Mark Maddison Imperial College London: Gerard Bishop University of Warwick: Graham Seymour Cornell University: Lukas Mueller Arizona Genomics Institute: Rod Wing Seunghee Lee Colorado State University: Stephen Stack Song-Bin Chang Scottish Crop Research Institute: Glenn Bryan FUNDING