16 th April 2007 Christine Nicholson, Mapping Core Group Wellcome Trust Sanger Institute Tomato Chromosome 4 Mapping & Use of FPC Copyright Wellcome Trust Medical Photographic Library

Tomato Genome Sequencing Strategy BAC by BAC sequencing Seed BACs selected for sequencing using mapped markers BAC selection from the physical map: constructed using restriction fingerprint digests & mapped marker information → orders & orientates contigs

Tomato Genome Sequencing Strategy Shotgun subclone & sequence Annotation : MIPs Imperial College London Overlapping BACs anchored by mapped markers Minimal tiling path “Finishing” Gap closure Problem solving Contiguous < 1 error in 10,000 …….. TAGCTGTGTACGATGATC ………. ~15 contigs per clone EMBL GenBank DDBJ Markers EMBL GenBank DDBJ Computer assembly – paired plasmid reads

WTSI Sequencing Pipeline - Tomato FinishingQC Mapping Sub- cloning Production Shotgun sequencing AB 3730 Auto Pre- Finishing

BAC Library & Map Resources LibraryNo. of clones Average Insert Genome equivalents Coverage in fingerprints End Sequenced ? LE_HBa129, kb15 X10 XYes SL_MboI52, kb7 X-Yes SL_EcoI72, kb7 X-Yes FPC Database Agarose gel fingerprints ~ 10 X fingerprint coverage BAC End Sequences Available for all three libraries

Fingerprinting – SL_MboI Library 1. Increase map coverage 2. Fingerprint data from > 1 library 3. Make clones visible in FPC - Assess potential deletions? 4. Additional resource for community Why fingerprint additional BACs ? How the fingerprinting was conducted……….

Agarose Gel Fingerprinting Digitised on: Amersham Typhoon 8600 Scanner Why agarose fingerprinting? Same protocol as AGI Same marker Integrate datasets

Processing Fingerprint Data Digitised agarose gel From the SL_MboI library: > 43,000 fingerprints generated Marker every 5 th well across gel

Image Gel Images processed using Image: ( Marker Locked (Standard - from AGI) Band called Lane tracked

WTSI Prefixes – Clone Names Library PrefixWTSI Internal Prefix LE_HBabTH SL_MboIbTM SL_EcoRIbTE EMBL/GenBank accessions released with International Prefix

Confirmation of Fingerprint Placement Re-built FPC with fingerprints from both libraries Fingerprint placements verified and confirmed by BAC End Sequence

Accessing SL_MboI Fingerprint Data Fingerprint data can also be accessed from SGN ftp://ftp.sanger.ac.uk/pub/tomato/map/ Assembled database available at: (sizes, bands, gels files available) General FPC info:

BAC Library & Map Resources – April ’07 LibraryNo. of clones Average Insert Genome equivalents Coverage in fingerprints End Sequenced ? LE_HBa129, kb15 X10 XYes SL_MboI52, kb7 X6 XYes SL_EcoI72, kb7 X-Yes Increased map coverage – fingerprints Fingerprint data – multiple libraries Visible – fingerprint assessed for sequence BAC selection Public

Anchoring Contigs to Chromosome 4 Tomato EXPEN-2000 map FPC Contig Analysis In silico assessment contigs with chromosome 4 markers Contigs merged with supporting evidence → longer minimal tile paths Use of BES and fingerprint data for BAC selection Overgo analysis at Cornell Some examples of chromosome 4 contigs……

Assessing & Editing Chromosome 4 Contigs Sequence BACs highlighted Chr 4 marker

Marker has several overgo hits in contig

Assessing & Editing Chromosome 4 Contigs Chr 4 marker Tilepath selection from anchored BAC

A further Chromosome 4 Contig

Marker has ONE hit in contig bTH53M2 FISHed to long arm, chromosome 4 → same region as marker All FPC contigs with chromosome 4 markers examined & assessed individually

Selection of Minimal Tiling Paths – Using BES Tile Path selected from initial chromosome 4 confirmed clone Design probes from BES Confirm overlaps by colony PCR Selecting minimal tiling paths across chromosome 4 contigs

Additional Uses of Fingerprints FinishingQC Mapping Sub- cloning Production Shotgun sequencing Auto Pre- Finishing Fingerprint confirmation Digest confirmed with original FP

Digest Check Prior to Sequencing Confirm HindIII digested DNA against original map fingerprint

Additional Uses of Fingerprints FinishingQC Mapping Sub- cloning Production Shotgun sequencing Auto Pre- Finishing Fingerprint confirmation Digest confirmed with original FP Digest information utilised

BAC Registration at SGN

Identifying Further Chromosome Coverage Screen unanchored markers Walk from contig ends by hybridization Grid libraries to generate filters CHROMOSOME VERIFICATION : Confirmation of Overlaps by PCR using BES Genetic Mapping FISH

FISH Analysis 36C23 20F17 114C15 198L24 & 119A16 308B7 6E18 78E4 132O11 53M2 106F7 36C23 114C15 308B7 6E18 198L24 & 119A16 20F17 78E4 132O11 53M2 106F7 Tomato-EXPEN 2000 FISH OBSERVATIONS: Euchromatin confirmation Slight marker order variation around centromere/heterochromatin Islands of heterochromatin within euchromatin Gene space requires further definition

Potential Use of Fosmid End Sequences Fosmids Smaller than BACs Sheared Known insert sizes Contig Extension & Gap Closure Assembly Confirmation

Acknowledgements Wellcome Trust Sanger Institute: Jane Rogers Sean Humphray Paul Hunt Mark Maddison Richard Clark Kate Fraser Rebecca Hannah Subcloning Shotgun Sequencing Finishing Imperial College London: Gerard Bishop Daniel Buchan James Abbott Sarah Butcher University of Nottingham: Graham Seymour Scottish Crop Research Institute: Glenn Bryan Cornell University: Lukas Mueller Jim Giovannoni Steve Tanksley Yimin Xu Colorado State University: Stephen Stack Suzanne Royer Song-Bin Chang Arizona Genomics Institute: Rod Wing Seunghee Lee MIPS/IBI Institute for Bioinformatics: Klaus Mayer Remy Bruggmann FUNDING Mapping Core Group