26 th July 2006 Christine Nicholson, Mapping Core Group Karen McLaren, Finishing Group Leader Wellcome Trust Sanger Institute Sequencing the Gene Space of Tomato Chromosome 4

A Summary of… Chromosome 4 sequence FISH data Fingerprint generation Map analysis Sequence content Euchromatin & heterochromatin Data deposition Points for discussion

Sequence Generated Chromosome 4 Sequence2,233,136 bp Unique2,182,043 bp 124,063 bp / clone

WTSI Pipeline – Tomato Clones * BAC verification processes Phase 3 Phase 1 Phase 2 HTGS: Pipeline StageNo. of BACs Selected for Sequencing40 Subcloning2 Shotgun8 Assembly Start3 Auto-prefinishing7 Finishing3 QC Checking1 Completed4 193 BACs to sequence (projected) Aim : 80 % at prefinish (phase 1-2) by end of 2006

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-EXPEN2000 FISH OBSERVATIONS: 1. Slight variation of marker order around centromere / heterochromatin 2. Euchromatic regions within heterochromatin

Fingerprinting – SL_MboI Library Why Fingerprint ? Increase map coverage & facilitate contiguity Fingerprint data from > 1 library Make clones visible in FPC Additional resource for community > 43,000 fingerprints generated Assessment of Fingerprints: Replicate existing LE_HBa fingerprints BES matches to neighbouring clones in contigs –BLAST –Colony PCR verification sizes, bands, gels files

bTH = LE_HBa bTM = SL_MboI SL_MboI clones with BES match by BLAST to sequence of LE_HBa- 31H5 ftp://ftp.sanger.ac.uk/pub/tomato/map/ Sizes/Bands and Gel files of new fingerprints as well as a assembled database available at;

Map Coverage – Chromosome 4 Contigs with chromosome 4 markers assessed Map information – SGN Chromosome 4: 57 markers in FPC 42 FPC contigs 9 markers anchored to singletons in FPC

FPC Contig Merging Relatively low marker coverage in FPC (57 markers) Screen MboI Library ** Ideally more chr 4 confirmation of contigs ** Genetic Mapping Colony PCR with BES Further FISH

Finishing Solanum lycopersicum Chromosome 4 2,233,136 bp of sequence HTGS Phase 3 – 461,727bp Features of the sequence observed so far Outline data deposition

Sequence Content Assess sequence early project to identify patterns Clone set selected from different regions > 2Mb of sequence at various stages

Tomato Chromosome 4 FISH Clone Locations Centromere Chromomere Euchromatic region Heterochromatic region -- Clone FISHed LE_HBa-78E4 euchromatin LE_HBa-78E4

LE_HBa-78E4 Euchromatin Phase 3 – Finished Features include: 6Kb tandem 10Kb direct repeat 10 known repeats - TIGR

Tomato Chromosome 4 FISH Clone Locations Centromere Chromomere Euchromatic region Heterochromatic region -- Clone FISHed LE_HBa-308B7 heterochromatin/centromere border LE_HBa-308B7 Assess possible heterochromatic features

LE_HBa-308B7 Heterochromatin/Centromere Border HTGS Phase 2 accession No problematic sequence features 10 known repeats - TIGR Currently assessing latest repeat data to compare against euchromatic - SGN

Tomato Chromosome 4 FISH Clone Locations Centromere Chromomere Euchromatic region Heterochromatic region -- Clone FISHed LE_HBa-27G19, LE_HBa-198L24, LE_HBa-119A16 & LE_HBa-31H5 Heterochromatin close to heterochromatic/euchromatic border 4 BAC contig Contig contains marker = C2_At5g37360

Chromosome BAC contig near to Heterochromatin/Euchromatin Border 450Kb of 3 Phase Phase 2 accessions Features include direct repeats inverted repeats Phase 3 region has been annotated – 3 gene objects found. What is the gene density in the gene space?

Finishing Conclusions ~0.5Mb of HTGS Phase 3 Finished sequence Assessment of euchromatin and heterochromatin Annotation feedback for density in gene space

WTSI – Data Deposition ftp://ftp.sanger.ac.uk/pub/sequences/tomato Clones are finished & QC checked → stable Phase 3 accn. no submission of assembly or associated quality value files

Acknowledgements Wellcome Trust Sanger Institute: Jane Rogers Sean Humphray Carol Scott Helen Beasley Sarah Sims MattJones Ratna Shownkeen Stuart McLaren Christine Lloyd Jennifer Harrow Carol Carder Paul Hunt Mark Maddison Richard Clark Kate Fraser Violetta Steeples Thomas Bounford 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 Colorado State University: Stephen Stack Song-Bin Chang Arizona Genomics Institute: Rod Wing Seunghee Lee FUNDING

Discussion Points Harmonise HTGS phases with NCBI How do we determine gene space has been sequenced Methods for reporting clone order and orientation? eg TPF and AGP

TPF File T ile P ath F ormat file – tab delimited flat file GAPtype-3? ? LE_HBa-24G5ctg145 CT LE_HBa-20F17ctg145 GAPtype-3? CT LE_HBa-114C15ctg5716 ? SL_MboI-143K21ctg5716 GAPtype-3? ? LE_HBa-147F16ctg5014 CT LE_HBa-308B7ctg5014 GAPtype-3? CT LE_HBa-27G19ctg15 CT LE_HBa-198L24ctg15 CT LE_HBa-119A16ctg15 CT LE_HBa-31H5ctg15

chr N50000cloneno chr N50000cloneno chr N50000contigno chr N50000cloneno chr FCT chr FCT chr FCT chr N50000contigno chr N50000cloneno chr N50000contigno AGP File Accesioned Golden Path – tab delimited flat file Gaps and unfinished clones are entered as 50,000bp sections to more accurately represent the chromosome in each build Order and alignment of Phase 3 finished accessions