Tomato Satellite Meeting

16:40-17:50 Session overview: Lukas Mueller

16:50-17:00 Chromosome 1, 10, 11, BAC end sequencing update: Lukas Mueller

17:00-17:15 Chromosome 2: Dr. Sanghyeob Lee “Current status and problems that we met during sequencing tomato chromosome No. 2”

17:15-17:30 Chromosome 3: Eileen Wang. “Progress on chromosome 3”

17:30-17:45 Chromosome 4: Christine Nicholson "Tomato Chromosome 4: A Mapping & Sequencing Update"

17:45-18:00 Chromosome 5: Jitendra P. Khurana “Progress on sequencing tomato chromosome 5”

18:00-18:15 Chromosome 6: Rene Klein Lankhorst

18:15-18:30 Chromosome 8: Satoshi Tabata

18:30-18:45 Chromosome 12: Mara Ercolano

18:45 - 19:00 Dr. Cheol-Goo Hur, Experiences and suggestions for annotation of tomato BAC clones.

1900-19:15 Maria Luisa Chiusano: Comparison of gene prediction programs on tomato BAC clones

19:15-19:25 Heiko Schoof: Getting databases talking

19:25-19:35 Remy Bruggmann: First annotation results for tomato BACs

19:15-19:40 Round table discussion on Sequencing/Annotation

Morning Session

Overview of Phenotypic databases

8:30-8:40 Session overview

8:40-10:00 Annotation and sequencing project discussion

10:00-10:10 Yaniv Semel: The In silico QTL Phenotypic database and future directions

10:10-10:20 Jim Giovannoni: The TOMET database

10:20-10:30 Nic Schauer: Overview of activities at MPI Golm.

10:30-10:40 Daisuke Shibata: "KaPPA-View: A web-based tool for Integration of transcript and metabolite data on plant metabolic pathway maps".

10:40-11:15 Round table discussion: Phenotypic databases

11:15-11:30 Planning of next meeting, wrapup

Overview

Sequencing Strategy

BAC registry

Sequence data upload

BAC naming conventions

BAC end sequences

Full BAC sequences

Sequencing Strategy

Sequencing the gene containing euchromatin (250MBases) of the tomato genome (900MBases)

Ordered BAC approach

Sequence and gene order

High quality sequence

“Seed BACs” are identified using the genetic map. Overgo probes identify sets of BACs that are linked with the genetic marker.

“Extension BACs” for walking through the gaps

anchored bacs

Obtaining the Tiling Path

A

B

C

genetic map

overgos

“seed BAC”

“seed BAC”

Seed BAC identification & verification

Identification

A list of recommended seed BACs has been identified and is available from SGN for each chromosome(ftp://ftp.sgn.cornell.edu/tomato_genome/seedbac/).

Verification

For each of the two recommended BACs per anchor point, take T7 or/and SP6 reads from the end

Design internal primer, take sequence read

Design primer such that it lies within the marker sequence (test for intron using the intron finder)

Check if marker sequence can be retrieved

Extension BAC identification and verification

Identification

Through BLAST of full BAC sequence against BAC end data

Screen the obtained BAC ends against the repeat database

Verification

Take a T7 or/and SP6 read and verify with information in the SGN database

In case of doubt: FISH

BAC registry

Database that keeps track of who sequences what and stage of sequencing

Please register the BACs that you intend to sequence

Email BAC ids to sgn-feedback@sgn.cornell.edu

Login to SGN and adjust the BAC status as appropriate

Uploading BAC data to SGN

Special UNIX upload account has been created for each project

Programs to use for upload:

UNIX: scp, sftp

Windows: WinSCP

Use file naming consistent with the conventions in the guidelines doc

Mailing list for newly uploaded BACs:

sgn-bac-announce@sgn.cornell.edu

BAC nomenclature

C01HBa0001A01

Chromosome Library Plate Well

C01HBa0001A01/

seq_dir/C01HBa0001A01.01A01.R.ab1

seq_dir/....

edit_dir/C01HBa0001A01.ace

phd_dir/...

C01HBa0001A01.seq

C01HBa0001A01.xml

C01HBa0001A01.tar.gz

C01HBa0001A01.tar.gz.md5

http://sgn.cornell.edu/solanaceae-project/

US tomato sequencing project

Steven Tanksley, Cornell

Jim Giovannoni, Boyce Thompson Institute

Joyce van Eck, Boyce Thompson Institute

Stephen Stack, Colorado State U

Lukas Mueller, Cornell

2 year project

400,000 BAC end reads / sheared library

12 full BACs

FISH

BAC End Sequencing

SeqWright Inc, Houston, TX

Libraries

Read statistics

HindIII MboI EcoRI sheared total

BAC End Annotation

Repeat Data

Annotated Repeat Dataset

Recon dataset derived from BAC end data

Full BAC sequencing – Anchor points

Sequenced 6 BACs

Full BACs sequenced

Finished:

C10HBa0111D09	(SeqWright Inc.)

C11HBa0034I10	“

C11HBa0119D16	“

C01HBa0088L02	“

C01HBa0252G05	“

C01HBa216G16	“

In Progress:

C01Hba0163B20	(Agencourt Inc.)

C11HBa0064J13	“

C10HBa0041K23	“

3 in pipeline	(Agencourt, Inc.)

3 in preparation	(SYMBIO, Inc)

FISH

9 experiments in SGN

Sequenced BACs will be FISHed

Interactive FISH map

Comparison to genetic map in mapviewer

BAC End sequences and full BAC sequences: availability

SGN BAC search

SGN BLAST page

SGN FTP site

BAC ends: basecalled, vector and quality trimmed sequences, chromatograms

BACs: assembly files, chromatograms

Submitted to Genbank

Summary

Sequenced 350,000 BAC ends

Sequenced full BACs with 6 more in progress and 3 more in preparation

Established a simple, extensible annotation pipeline

Derived extensive repeat datasets

Outlook

☞ Grant needs to be renewed this fall

Full BAC sequences of 600 BACs of chromosome 1, 10 & 11.

Generate additional genomic library: small insert fosmid & 100,000 clones

Annotation in the framework of the SOL project

Discussion Points

Sequence quality standards

Annotation

Decide a list of gene prediction programs for calibration to tomato

What datasets to align against

2005-09-21T13:58:00