Download Wellcome Trust Sanger Institute

Tomato
Chromosome 4:
A Mapping &
Sequencing
Update
Christine Nicholson
Mapping Core Group
Welcome Trust Sanger Institute, UK
28th September 2005
FPC Database
• Mapping strategy = to develop the physical map in order to
select minimal tiling paths across the chromosome to
sequence, in conjunction with BES data and markers.
• FPC database worked on in-house at Sanger
• Obtained from Arizona Genomics Institute
• The FPC Database:
– LE_HBa library fingerprints
– 88,584 fingerprinted BACs (68% of total library)
– Possibly due to contamination of library
Therefore,
Have ~ 10 X coverage in the fingerprint map.
Libraries
No. of
clones
Average
insert
size
**No. of
Coverage in
genome
fingerprints
equivalents
BES
available
Mated Pairs
%
LE_HBa*
129,024
117 kb
15 X
10 X
152,819
68
SL_MboI
52,992
135 kb
~7X
-
101,755
70
SL_EcoRI
72,264
95-100
kb
~7X
-
99,455
65
* Heinz1706
**Based on genome size of 950Mb.
Chromosome 4 Contigs
• Examined seed BACs/contigs for each chromosome
using overgo probes. (Conducted by Cornell)
ftp://ftp.sgn.cornell.edu/tomato_genome/seedbacs/20050112_chr4_long_short.xls
• 54 markers on chromosome 4 in the current FPC
build
• Each potential chromosome 4 contig assessed in
silico in FPC:
- structure based on fingerprints
- marker content – assigned to how many contigs?
- possible merges to other contigs?
September 2005: 58 contigs currently on chr 4
Pilot Sequencing
• Select 5 BACs to form “pilot sequencing”.
• In two different regions of the chromosome
• Tomato BACs are being processed through our
sequencing pipeline.
• Apply our Finishing programmes to tomato
clones & examine sequence features
e.g. repeats.
Sequencing Pipeline at WTSI……
Sequencing Pipeline
At Wellcome Trust Sanger Institute:
Production
Mapping
•Single colonies
•Clone verification
(PCR using BES
probes)
Subcloning
(Shotgun
sequencing)
Finishing
QC
Further digest check
MIPS
(automated)
ANNOTATION
Imperial College
manual annotation
Wellcome Trust Sanger Institute Sequencing
MJs:
Cycle
sequencing
Packard Minitrak:
Sequencing
reactions set-up
ABI 3730s:
Sequence data
generated &
analysed
Sequencing Pipeline
At Wellcome Trust Sanger Institute:
Production
Mapping
•Single colonies
•Clone verification
(PCR using BES
probes)
Subcloning
(Shotgun
sequencing)
Finishing
QC
Further digest check
MIPS
(automated)
ANNOTATION
Imperial College
manual annotation
Selected Chromosome 4 BACs
Stage
No. of
BACs
Selected for
Sequencing
3
Finishing
2
•Located in contig 270
•Marker C2_At5g37360
• LE_HBa clones:
198L24 (contains marker)
31HO5
Sequence data available for 2 BACs
Clone
Accession No. of
contigs
No. of gaps
Approx.
size
168kb
13H05 CT025877
3
2
(All spanned)
198L24 CT025873
7
6
165.5kb
(5 spanned)
ftp://ftp.sanger.ac.uk/pub/sequences/tomato/unfinished_sequence/
Finishing - Analysis in GAP 4
• PHRED – calls bases
• PHRAP – assembly
• GAP4 – view & edit
FISH Analysis
• BAC 198L24 underwent
metaphase FISH.
• Confirmed chr 4.
• Reported to be in
heterochromatic region.
• However, no major
issues (repeats etc.) in
Finishing
Image courtesy of S. B. Chang, Prof S. Stack’s Laboratory,
University of Colorado, USA.
Genes in Sequenced BACs?
• Used WUBLASTX  align proteins against the
tomato sequence.
• Partial gene highlighted in each BAC
31H05  putative carboxyl-terminal peptidase
198L24  AMT1.2 Ammonium transporter 1 member2
Mapping Strategy
* AIM = Reduce the current contig number *
WHY?
• Select longer minimal tilepaths across the chromosome
• Smaller overlaps of selected sequence BACs (aim for 1520kb)
• More efficient sequencing
HOW?
• Work on FPC database to improve continuity
• Walk off sequenced clones (once available) using BES hits
• Incorporate further BES/fingerprint data as generated
• Possible walk from contig ends by hybridization.
Further Map Development &
Fingerprinting?
• Have been able to replicate the fingerprinting
technique from AGI.
• 31H05 and 198L24 fingerprints confirmed.
• Will use this for future clone verification.
• Currently have 10 X coverage in fingerprints.
• Investigating the practicalities and possibilities of
augmenting the fingerprint database
→ SL_MboI library?
Further BAC Selection Strategy
• Intend to select minimal tilepaths to sequence
 ideally over reduced number of contigs on
chromosome 4.
• Will FISH more BACs across the chromosome
 obtain confirmation of chromosome location
of BACs & contigs they are contained within.
Acknowledgements
Wellcome Trust Sanger Institute:
Jane Rogers
Sean Humphray
Carol Scott
Karen Barlow
Helen Beasley
Sarah Sims
Jennifer Harrow
Carol Carder
Paul Hunt
Mark Maddison
Imperial College London:
Gerard Bishop
University of Warwick:
Graham Seymour
FUNDING
Cornell University:
Lukas Mueller
Arizona Genomics Institute:
Rod Wing
Seunghee Lee
Colorado State University:
Stephen Stack
Song-Bin Chang
Scottish Crop Research Institute:
Glenn Bryan