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Tomato Chromosome 4: A Mapping & Sequencing Update

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

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Tomato Chromosome 4: A Mapping & Sequencing Update

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

  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. • FPC database worked on in-house at Sanger • Obtained from Arizona Genomics Institute • The FPC Database: • LE_HBa library fingerprints • 88,584 fingerprinted BACs (68% of total library) • Possibly due to contamination of library Therefore, Have ~ 10 X coverage in the fingerprint map.

  3. Libraries *Heinz1706 **Based on genome size of 950Mb.

  4. Chromosome 4 Contigs • 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 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? September 2005: 58 contigs currently on chr 4

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

  6. Sequencing Pipeline At Wellcome Trust Sanger Institute: Production (Shotgun sequencing) Finishing QC Mapping Subcloning • Single colonies • Clone verification(PCR using BES probes) Further digest check MIPS (automated) ANNOTATION ImperialCollegemanual annotation

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

  8. Sequencing Pipeline At Wellcome Trust Sanger Institute: Production (Shotgun sequencing) Finishing QC Mapping Subcloning • Single colonies • Clone verification(PCR using BES probes) Further digest check MIPS (automated) ANNOTATION ImperialCollegemanual annotation

  9. Selected Chromosome 4 BACs • Located in contig 270 • Marker C2_At5g37360 • LE_HBa clones: • 198L24 (contains marker) • 31HO5

  10. Sequence data available for 2 BACs ftp://ftp.sanger.ac.uk/pub/sequences/tomato/unfinished_sequence/

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

  12. FISH Analysis • BAC 198L24 underwent metaphase FISH. • Confirmed chr 4. • Reported to be in heterochromatic region. • However, 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. • Partial 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 * WHY? • Select longer minimal tilepaths across the chromosome • Smaller overlaps of selected sequence BACs (aim for 15-20kb) • More efficient sequencing HOW? • Work on FPC database to improve continuity • Walk off sequenced clones (once available) using BES hits • Incorporate further BES/fingerprint data as generated • Possible walk from contig ends by hybridization.

  15. Further Map Development & Fingerprinting? • Have been able to replicate the fingerprinting technique from AGI. • 31H05 and 198L24 fingerprints confirmed. • Will use this for future clone verification. • Currently have 10 X coverage in fingerprints. • 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. • Will FISH more BACs across the chromosome obtain confirmation of chromosome location of BACs & contigs they are contained within.

  17. Acknowledgements 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 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 FUNDING

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