Tomato Project Group

1
Second Tomato Finishing Workshop Chromosome 4

• Tomato Project Group
• Wellcome Trust Sanger Institute
• 25th April 2008

2
Chromosome 4 Introduction

• Data Flow at WTSI
• Sequencing Method Used
• Finishing Strategies
• Use of Overlapping Data
• Chr4 Sequence Update
• Discussion points for Workshop
• Unmapped BACs
• Examples of Problem Clones
• Dealing with Large Repeats

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UK - Chromosome 4

• Gene space estimate for Chromosome 4 is 19Mb
• Mapping, sequencing and finishing at Wellcome
  Trust Sanger Institute (WTSI)
• BAC by BAC sequencing approach
• Approximately 200 BACs
• Funding at WTSI ends October 31st 2008

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Overview of WTSI Clone Pipeline

• Clone Selection and Verification
• Clones entered into pipeline

Mapping
BACs assigned to chr4 sequencing project on SGN
BAC registry

• Clone DNA Prep
• Digest Confirmation
• Library Construction (plasmid)

Subcloning

• Plasmid Prep
• Sequencing Processing

Sequence Contigs gt2Kb available on Sanger FTP
site and Public Databases Sequencing in
Progress
Shotgun Sequencing
HTGS Phase 1

• Sequence Improvement
• Contig Orientation and Gap Closure
• Confirmation of Assemby (QC)

Finishing
HTGS Phase 2

• Sequences Uploaded
• to SGN
• BAC Registry Updated

Finished Sequence Final EMBL submission Complete
Sequence HTGS Phase 3
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Clones Selection and Verification

• BACs selected primarily from the
• HindIII (LE-HBa-) and MboI (SL_MboI) libraries
• Using Seed BACs from SGN,
• end sequence alignment and FPC analysis
• New BACs selected from in house overgoes for
  markers
• Selected 5 clones from the fosmid library
• based on end sequence alignments and
  fingerprints

6
Plasmid Prep and Shotgun Sequencing

• Optimised for 384 well prep and sequencing
• Capillary Sequencing
• AB3730s with AB Big Dye Terminator
• pUC118 Double Stranded Sequencing Vector
• 4-6Kb inserts, double end sequenced

BACs Aim for 6x-8x Coverage Average Insert
100-150Kb (LE_HBa- and SL_MBol- Libraries) 2x
or 3x 384 plates per BAC 750 paired end
reads 1500 reads in total Average 10-15 contigs

Fosmids Average Insert 35Kb 1x 384 plates
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Clone Finishing
Gap4 (Staden) used to view and manipulate
sequence data

• Sequence Improvement

Manual Finishing
QC Checking
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Manual Finishing of BACsBACs viewed in relation
to map

• BACs are viewed in relation to the mapped minimal
  tile path
• Use in house tpf visualisation tool e.g. ctg503

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Use of Overlapping Sequences

• From Minimal Tile Path the region finished in
  each clone depends on the order the clones enter
  finishing
• Finish unique sequence with a 2000bp overlap
  between clones

BAC1
BAC4 gap closure
BAC2
BAC3
total BAC insert
finished region
Final order and orientation of finished BACs are
given in the AGP file e.g. BAC1-BAC2-BAC4-BAC3
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Summary of Clone Gap Closure Strategies

• Make use of paired ends to order and orientate
  contigs
• Identify whether gaps are spanned or unspanned
  orchid example
• Identify any repeats associated with gaps
  dotter example
• Estimate gap sizes using restriction digest data
• This will determine appropriate strategy for gap
  closure e.g.
• primer/oligo walking into regions of low quality
  or gaps spanned by paired end reads
• PCR and direct walking on BAC DNA into regions of
  low quality and unspanned gaps (also attempted on
  unresolved spanned gaps)
• Use of alternative chemistries where appropriate
• structural problems, mono- di-nuclotide runs

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OrchidRead pair Visualisation Tool
Contiguous sequence with good read pair coverage
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Visualising Repeats associated with gaps
Inverted Repeat
Direct Repeat

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Restriction Digests

• Minimum of three restriction enzymes used to
  confirm the assembly
• Selection depends on organism and the nature of
  the sequence
• S. lycopersicum BACs are digested with
• BamHI
• EcoRI
• HindIII
• Comparison of real and virtual digest of entire
  BAC sequence

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ConfirmWTSI In-house digest visualisation tool
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In-house digest visualisation tool
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Clone Gap Closure Strategies

• Make use of paired ends to order and orientate
  contigs
• Identify whether gaps are spanned or unspanned
  orchid
• Identify any repeats associated with gaps
  dotter
• Estimate gap sizes using restriction digest
• This will determine appropriate strategy for gap
  closure e.g.
• primer/oligo walking into regions of low quality
  or gaps spanned by paired end reads
• PCR and direct walking on BAC DNA into regions of
  low quality and unspanned gaps (also attempted on
  unresolved spanned gaps)
• Use of alternative chemistries where appropriate
• structural problems, mono- di-nuclotide runs

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Sequencing Chemistries and Additives used in
Finishing

• 41 mix ratio of AB Big Dye Terminator AB dGTP
  Terminator
• used for general finishing reactions, not
  problem specific
• AB dGTP Terminator
• used for di-nucleotide runs and inverted repeats
• Additive A (SequenceRx Enhancer Solution A -
  Invitrogen)
• Dimethyl sulfoxide (DMSO)
• Additive ADMS0dGTP
• used for mono-nucloetide runs, inverted repeats
• Sequence Finishing Kit (SFK) (TempliPhi -
  Amersham)
• used to increase DNA yield
• useful for structural problems caused by
  inverted repeats

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Alternative Gap Closure Strategies

• Specialist Subcloning
• Small Insert Libraries (SIL)
• Double Stranded pUC or Single Stranded M13
• Large Insert Libraries (LIL)
• Transposon Libraries (TIL)
• Restriction Fragment SIL (RFSIL)
• Alternative Strategies for dealing with large
  repeats
• - points for further discussion on Tuesday
• - what repeats have other chromosomes found?

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Clone Gap Closure Strategies

• Make use of paired ends to order and orientate
  contigs
• Identify whether gaps are spanned or unspanned
  orchid
• Identify any repeats associated with gaps
  dotter
• Estimate gap sizes using restriction digest
• This will determine appropriate strategy for gap
  closure e.g.
• primer/oligo walking into regions of low quality
  or gaps spanned by paired end reads
• PCR and direct walking on BAC DNA into regions of
  low quality and unspanned gaps (also attempted on
  unresolved spanned gaps)
• Use of alternative chemistries where appropriate
• structural problems, mono- di-nuclotide runs

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Use of Misc_Feature Tags in EMBL/GenBank/DDBJ

• Used regularly on finished sequence to identify
  regions of
• uni-directional chemistry when dGTP only
• single subclone regions
• including SIL and TIL only regions
• pcr only
• Single reads from direct walks on BAC DNA
• data only from overlapping BACs
• E.coli Transposon insertion sites
• tag sp6 and t7 ends of overlaps (tomato)
• gap sizes of force joins in tandem repeats

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Misc_Feature Tag Example Clone End Tags
Accession
Length of sequence
Whole Clone Finished
Both ends of clone cited
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Misc_Feature Tag Example
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QC Check of Clone Assembly

• Before submission to public databases as HTGS
  phase 3 complete, all assembled BACs undergo
  several QC checks
• all reasonable chemistry attempts have been made
  for any specific problem types
• all bases are above phred30
• orientation of paired end reads checked across
  assembly
• assembly is confirmed by restriction digest data
• correct misc_feature tags have been used to
  identify any regions where appropriate

Ensures on high quality contiguous sequence with
low error rate
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Chromosome 4 Clone Pipeline
Additional 15 BACs finished - not on chromosome 4
from FISH
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Unmapped BACs moved from chr4
bTH82D4 (LE_HBa082D04) moved to chr7 (on FISH
map) bTH91D14 (LE_HBa091D14) moved to chr5 (on
FISH map)
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Points for Discussion at Workshop

• What problematic sequence have other groups
  encountered?
• Strategies for finishing repeats used by other
  chromosome groups?
• Unmapped BACs any from other chromosomes?

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Acknowledgements
Cornell University Lukas Mueller Robert
Buels Jim Giovannoni Steve Tanksley 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 Wageningen University Rene Klein
Lankhorst Hans de Jong Dora Szinay

• Wellcome Trust Sanger Institute
• Karen McLaren
• Clare Riddle
• Sean Humphray
• Christine Nicholson
• Carol Scott
• Stuart McLaren
• Matt Jones
• Christine Lloyd
• Sarah Sims
• Karen Oliver
• Jane Rogers
• Imperial College London
• Gerard Bishop
• Daniel Buchan
• James Abbott

FUNDING