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Solanum lycopersicum Chromosome 4 Sequencing Update

Wellcome Trust Medical Photographic Library. Solanum lycopersicum Chromosome 4 Sequencing Update. UK-SOL– Dec 2008. Overview. Summary of Project from WTSI Transfer of BAC selection/contig building QC-checking has moved to Imperial College London Call for your help in annotation.

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Solanum lycopersicum Chromosome 4 Sequencing Update

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  1. Wellcome Trust Medical Photographic Library Solanum lycopersicum Chromosome 4 Sequencing Update UK-SOL– Dec 2008

  2. Overview • Summary of Project from WTSI • Transfer of BAC selection/contig building QC-checking has moved to Imperial College London • Call for your help in annotation

  3. Mapped Markers Overlapping Clones anchored by mapped markers BAC Library Minimal tiling path Subcloning & Shotgun sequencing Computer assembly – paired plasmid reads ……..TAGCTGTGTACGATGATC………. “Finishing” Order contigs Gap closure Sequence Quality Clone by Clone Sequencing Strategy Contiguous Sequence < 1 error in 10,000

  4. Overview of Clone Pipeline • Clone Selection and Verification • Clones entered into pipeline Mapping BACs assigned to chr4 sequencing project on SGN BAC registry • Clone DNA Prep • Digest Confirmation • Library Construction (plasmid) Subcloning • Plasmid Prep • Sequencing & Processing Sequence Contigs >2Kb available on Sanger FTP site and Public Databases “Sequencing in Progress” Shotgun Sequencing HTGS Phase 1 • Sequence Improvement • Contig Orientation and Gap Closure • Confirmation of Assemby (QC) Finishing HTGS Phase 2 • Sequences Uploaded • to SGN • BAC Registry Updated • Ready for Annotation Finished Sequence Final EMBL submission “Complete Sequence” HTGS Phase 3

  5. BAC Library & Map Resources Tomato EXPEN-2000 map • - 2585 mapped markers across genome • - 242 Chr4 mapped markers • Overgo analysis at Cornell BAC Libraries FPC Map Construction 1st FPC map build of HindIII Library by Arizona Genomics Institute 2nd FPC map build incorporated MboI Library mid 2006 at WTSI

  6. Fosmid Library End Sequencing • 150 plates (1-150) End Sequenced at WTSI December 2007 • Approx. ~57600 fosmids (115200 FES) • Fosmid Analysis (January 2008) • 107681 reads with total bp count of 70900576 giving average length = 658.4bp (after quality and vector clipping) (60.3% bases repeat masked) • Hits within existing chromosome 4 BACs: • 380 fosmids with good read pair alignments (within expected size range) • 31 fosmids with bad read pair alignments • Hits to single end • 48 single ends - from fosmids with only 1 end sequenced • 1027 single ends - from fosmids where only 1 end is found

  7. Selection of Minimum Tile Path Markers Overlaps identified by fpc and BES alignment Fingerprinted BACs Seed BAC Anchored by marker (Cornell) Verify overlaps by colony pcr Framework Markers in FPC Anchor further BACs by hybridisation to marker sequences and FISH

  8. Increasing Map Coverage using PseudoGoldenPath (PGP) Analysis Bridging clones identified from BES alignments to sequence Sequenced clones MAP GAP

  9. FISH Map for Chr 4 on SGN • FISH is used: • to confirm BAC assignment to chr 4 • to confirm contig order along chr 4 Steve Stack Dora Szinay, Hans DeJong

  10. FISH Map for Chr 4 on SGN • FISH is used: • to confirm BAC assignment to chr 4 • to confirm contig order along chr 4

  11. WTSI Tomato Clone Pipeline 2006-2008 HTGS: Phase 1 Phase 2 Phase 3

  12. Chr 4 Map and Sequence Update • Chromosome 4 estimate : 19 Mb of euchromatin • 80 contigs with sequence

  13. Distribution of Contigs 227 markers mapped to Chr_4 Centromere {62 markers} {41 markers} {124 markers} 29 contigs 11 contigs 36 contigs Unordered: 4 contigs (5 BACs) 55 BACs (27 markers) 59 BACs (16 markers) 63 BACs (61 markers) = Heterochromatin Average Contig Length = 250Kb Average BACs/Contig = 2.3 Largest Contigs = ~450-500Kb = Euchromatin

  14. Some facts and figures • ~81 contigs (80 contigs with sequence available). • Average contig length is just under 250 kb. • The average number of BACs per contig is 2.3. • The largest sequence contigs are in the range of 450kb-500kb with 5 or 6 BACs.

  15. Summary of Progress on Chromosome 4 • 81 map contigs have been built • 119 BACs/44 contigs definitely on chr4 in FISH/ IL mapped • 57 BACs under confirmation of Chr4 location (28 on SGN 29 to be placed after confirmed location) • ~60 Markers for which BACs have not been identified. • ~13 BACs have been sequenced to HTGS3 and placed on chr0, definitely not on chr4 (others initiated, in same contig etc but stopped in pipeline). • 22 Missing markers missing sequence?

  16. Summary of what we will do next 1) Confirm chr4 location of BACs that lack chr4 marker sequence and or have conflicting map location. IL mapping. 2) Use missing marker sequences to identify further BACs (3D pools) and confirm chr4 location using IL mapping. 3) Use 3D BAC pools to identify BACs to extend current contigs. 4) Analyse output from GS-FLX and GA-Illumina sequencing runs on cDNA from chr4 IL and parental lines to identify SNPs and further chr4 markers. 5) Use any markers from (4) to isolate further BACs for sequencing.

  17. Analysis of cDNA sequence to identify Chr4 specific sequences

  18. Call for your help Need your help in checking and verifying the automated annotation. Please respond to e-mails in 2009 calling for help in annotation your favourite genes.

  19. Acknowledgements Cornell University: Lukas Mueller Jim Giovannoni MIPS/IBI Institute for Bioinformatics: Klaus Mayer Remy Bruggmann FISH Resources Stephen Stack Group (Colorado) Hans de Jong (Wageningen) Dora Szinay (Wageningen) • Wellcome Trust Sanger Institute: • Carol Churcher • Jane Rogers • Sean Humphray • Clare Riddle and Mapping Core Group • Karen McLaren and Finishing Team 46 • Stuart McLaren and Pre-finishing Team 58 • Christine Lloyd and QC Team 57 • Karen Oliver • Matt Jones • Carol Scott • Imperial College London: • Gerard Bishop • Daniel Buchan • James Abbott • Sarah Butcher • Rosa Lopez-Cobollo • University of Nottingham: • Graham Seymour • Scottish Crop Research Institute: • Glenn Bryan FUNDING

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