Tomato Tomato Chromosome 4:Chromosome 4:

A Mapping & A Mapping & Sequencing Sequencing

UpdateUpdate

2828th th September 2005September 2005

Christine NicholsonChristine NicholsonMapping Core GroupMapping Core Group

Welcome Trust Sanger Institute, UKWelcome Trust Sanger Institute, UK

FPC DatabaseFPC Database• Mapping strategy = to develop the physical map in order to Mapping strategy = to develop the physical map in order to

select minimal tiling paths across the chromosome to select minimal tiling paths across the chromosome to sequence, in conjunction with BES data and markers.sequence, in conjunction with BES data and markers.

• FPC database worked on in-house at Sanger FPC 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 fingerprintsLE_HBa library fingerprints– 88,584 fingerprinted BACs (68% of total library)88,584 fingerprinted BACs (68% of total library)– Possibly due to contamination of libraryPossibly due to contamination of library

Therefore, Therefore, Have ~ 10 X coverage in the fingerprint map.Have ~ 10 X coverage in the fingerprint map.

Libraries Libraries

** Heinz1706Heinz1706

**Based on genome size of 950Mb.**Based on genome size of 950Mb.

No. of No. of clonesclones

Average Average insert insert sizesize

**No. of **No. of genome genome

equivalentsequivalents

Coverage in Coverage in fingerprintsfingerprints

BESBES

availableavailable

Mated Pairs Mated Pairs %%

LE_HBa*LE_HBa* 129,024129,024 117 kb117 kb 15 X15 X 10 X10 X 152,819152,819 6868

SL_MboISL_MboI 52,99252,992 135 kb135 kb ~ 7 X~ 7 X -- 101,755101,755 7070

SL_EcoRISL_EcoRI 72,26472,264 95-100 95-100 kbkb

~ 7 X~ 7 X -- 99,45599,455 6565

Chromosome 4 ContigsChromosome 4 Contigs• Examined seed BACs/contigs for each chromosome Examined seed BACs/contigs for each chromosome

using overgo probes. (Conducted by Cornell)using overgo probes. (Conducted by Cornell)

ftp://ftp.sgn.cornell.edu/tomato_genome/seedbacs/20050112_chr4_long_short.xlsftp://ftp.sgn.cornell.edu/tomato_genome/seedbacs/20050112_chr4_long_short.xls

• 54 markers on chromosome 4 in the current FPC 54 markers on chromosome 4 in the current FPC buildbuild

• Each potential chromosome 4 contig assessed Each potential chromosome 4 contig assessed in in silicosilico in FPC: in FPC:- structure based on fingerprints- structure based on fingerprints- marker content – assigned to how many contigs?- marker content – assigned to how many contigs?- possible merges to other contigs?- possible merges to other contigs?

September 2005: 58 contigs currently on chr 4

Pilot SequencingPilot Sequencing

• Select 5 BACs to form “pilot sequencing”.Select 5 BACs to form “pilot sequencing”.• In two different regions of the chromosome In two different regions of the chromosome • Tomato BACs are being processed through our Tomato BACs are being processed through our

sequencing pipeline.sequencing pipeline.• Apply our Finishing programmes to tomato Apply our Finishing programmes to tomato

clones & examine sequence features clones & examine sequence features e.g. repeats.e.g. repeats.

Sequencing Pipeline at WTSI……Sequencing Pipeline at WTSI……

Sequencing PipelineSequencing Pipeline

Mapping SubcloningProduction

(Shotgun sequencing)

Finishing QC

At Wellcome Trust Sanger Institute:At Wellcome Trust Sanger Institute:

ANNOTATIONImperial College manual annotation

MIPS (automated)

•Single colonies

•Clone verification(PCR using BES probes)

Further digest check

Wellcome Trust Sanger Institute SequencingWellcome Trust Sanger Institute Sequencing

Packard Minitrak: Sequencing reactions set-up

MJs:Cycle sequencing

ABI 3730s:Sequence data generated & analysed

Sequencing PipelineSequencing Pipeline

Mapping SubcloningProduction

(Shotgun sequencing)

Finishing QC

At Wellcome Trust Sanger Institute:At Wellcome Trust Sanger Institute:

ANNOTATION

MIPS (automated)

•Single colonies

•Clone verification(PCR using BES probes)

Further digest check

Imperial College manual annotation

Selected Chromosome 4 BACsSelected Chromosome 4 BACs

•Located in contig 270Located in contig 270•Marker C2_At5g37360Marker C2_At5g37360• LE_HBa clones:LE_HBa clones:

198L24 (contains marker)198L24 (contains marker)

31HO531HO5

StageStage No. of No. of BACsBACs

Selected for Selected for Sequencing Sequencing

33

Finishing Finishing 22

Sequence data available for 2 BACsSequence data available for 2 BACs

CloneClone AccessionAccession No. of No. of contigscontigs

No. of gapsNo. of gaps Approx. Approx. sizesize

13H0513H05 CT025877CT025877 33 22

(All spanned)(All spanned)

168kb168kb

198L24198L24 CT025873CT025873 77 66

(5 spanned)(5 spanned)

165.5kb165.5kb

ftp://ftp.sanger.ac.uk/pub/sequences/tomato/unfinished_sequence/

Finishing - Analysis in GAP 4Finishing - Analysis in GAP 4

• PHRED – calls basesPHRED – calls bases• PHRAP – assemblyPHRAP – assembly• GAP4 – view & editGAP4 – view & edit

FISH AnalysisFISH Analysis

• BAC 198L24 underwent BAC 198L24 underwent metaphase FISH.metaphase FISH.

• Confirmed chr 4.Confirmed chr 4.• Reported to be in Reported to be in

heterochromatic region.heterochromatic region.• However, no major However, no major

issues (repeats etc.) in issues (repeats etc.) in FinishingFinishing

Image courtesy of S. B. Chang, Prof S. Stack’s Laboratory, University of Colorado, USA.

Genes in Sequenced BACs?Genes in Sequenced BACs?

• Used WUBLASTX Used WUBLASTX align proteins against the align proteins against the tomato sequence.tomato sequence.

• Partial gene highlighted in each BACPartial gene highlighted in each BAC

31H05 31H05 putative carboxyl-terminal peptidase putative carboxyl-terminal peptidase

198L24 198L24 AMT1.2 Ammonium transporter 1 member2 AMT1.2 Ammonium transporter 1 member2

Mapping StrategyMapping Strategy * AIM = Reduce the current contig number ** AIM = Reduce the current contig number *

WHY?WHY?• Select longer minimal tilepaths across the chromosomeSelect longer minimal tilepaths across the chromosome• Smaller overlaps of selected sequence BACs (aim for 15-Smaller overlaps of selected sequence BACs (aim for 15-

20kb)20kb)• More efficient sequencingMore efficient sequencing

HOW? HOW? • 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.

Further Map Development & Further Map Development & Fingerprinting?Fingerprinting?

• Have been able to replicate the fingerprinting Have been able to replicate the fingerprinting technique from AGI.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 Investigating the practicalities and possibilities of

augmenting the fingerprint database augmenting the fingerprint database → SL_MboI library?→ SL_MboI library?

Further BAC Selection StrategyFurther BAC Selection Strategy

• Intend to select minimal tilepaths to sequence Intend to select minimal tilepaths to sequence ideally over reduced number of contigs on ideally over reduced number of contigs on chromosome 4.chromosome 4.

• Will FISH more BACs across the chromosomeWill FISH more BACs across the chromosome obtain confirmation of chromosome location obtain confirmation of chromosome location of BACs & contigs they are contained within.of BACs & contigs they are contained within.

AcknowledgementsAcknowledgementsWellcome Trust Sanger Institute:Jane RogersSean HumphrayCarol ScottKaren BarlowHelen BeasleySarah SimsJennifer HarrowCarol CarderPaul HuntMark Maddison

Imperial College London:Gerard Bishop

University of Warwick:Graham Seymour

Cornell University:

Lukas Mueller

Arizona Genomics Institute:Rod WingSeunghee Lee

Colorado State University:Stephen StackSong-Bin Chang

Scottish Crop Research Institute:Glenn Bryan

FUNDING