Mo17 shotgun project

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Mo17 shotgun project

• Goal sequence Mo17 gene space with inexpensive
  new technologies
• Datasets in progress
• Four-phases of 454-FLX sequencing to max of 12X
• Include 3kb paired-end sequencing (for
  short-range structural variation)
• Ultra-short-read Solexa or ABI-SOLID (for
  polishing)
• Preparation of methyl-spanning linkers to augment
  IBM map integration, detect rearrangements
  (Sanger end-sequence)
• (Ideally would add Mo17 BAC-ends from DuPont, if
  available)

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Shotgun

• Independent of tiling path
• -Can detect non-repetitive gene space even
  within otherwise complex regions that may not be
  in tiling path
• Disadvantages of short-reads
• -Cant expect to recover repetitive sequences

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Four Phases of Sequencing Complete in 2007

• Sequencing contract established with 454/Roche.
  Four Phases, including collaborative runs at no
  cost in P2-4.
• Phase I underway (30 FLX runs.) Library QC and
  initial assessment of data quality (30 FLX runs).
  
• 10 FLX runs totaling 1 Gb (0.4X)
• 20 FLX pair runs spanning 12 Gb (5X span in 3kb
  inserts)
• Assess quality, coverage, contamination,
  chimerism, accuracy
• Phase II. (80 runs plus 30 runs from Roche, total
  110 runs). Rough draft stage.
• 40 FLX-pair runs spanning 36 Gb (total 48 Gb10X
  span)
• 70 FLX runs for 7 Gb (total 8Gb 3.5X sequence)
• Assess rough draft assembly (3 methods), compare
  B73, sorghum

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Phases III and IV

• Phase III (50 runs 20 contributed)
• 20 FLX-pair runs (total spanning cover 20X)
• 50 FLX runs (total 13 Gb sequence 5.5X)
• Draft assembly. Rough annnotation. Assessment
  of structural
• variation based on 20X clone cover.
  Assessment complete by
• end of 2007.
• Phase IV (60 runs 30 contributed)
• 90 FLX runs (to reach total 22 Gb 10X)
• Data collection complete by end of 2007.
• Early 08. Final assembly. Integration with MSSL
  ends and IBM
• map. Proceed to annotation and full analysis.
• Note Later phases may use next FLX release with
  longer
• read lengths. To be conservative, sequence from
  FLX-pair
• reads not included in sequence coverage
  estimates.
• Total sequencing cost for Phase I-IV 1.6M

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454-FLX reads are typically either mostly masked,
or mostly clean
29 of reads have lt quarter of positions masked
58 of reads have gt 2/3 of positions masked
0 0.5
1.0 Percent masked by over-repd
16mers
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Mo17 454 unique full length alignments vs. B73
MAGIs show high quality of unique alignments
Residual repeats in MAGIs with multiple hits in
454 data
Unique full alignments
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SNPs and indels of 454 reads relative to MAGIs
consistent with few variation of Mo17/B73
(combines variation with sequencing errors)
SNPs or indels per base
Frequency of reads
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Multiple assembly alternate plans

• Divide and conquer
• Reduce 100 million reads to 50K unique gene
• spaces of thousands of reads each (10kb) by
• clustering based on various comparisons
• Plan A De novo clustering of masked reads
• Plan B map to B73, assemble (de novo for
  remainder)
• Plan C sorghum-assisted
• Use various assemblers to lay-out and produce
• consensus for each cluster (454 assembly team
  engaged)
• Polish sequence with Solexa or SOLID for
• accuracy
• Link with MSSL pairs, integrate with map

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Backup analyses vs. B73 reference

• SNP/variation detection by alignment to B73
  sequence
• -454/Solexa/Solid (various successful models in
  other species at JGI, elsewhere)
• Structural variation detection via paired-end
  placements
• -Needs to be tolerant of chimerism rate
• -Model of successful human structural analysis
  done with 454 (unpublished)

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Timeline

• Phase I in progress, complete by end of month.
  Analysis to OK phase II 10 days.
• Phase II October
• Phase III November
• Phase IV December
• 454 sequencing complete by end of year

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58 of each BAC is masked by over-represented
16-mers
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Outreach Dick McCombie
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Types of Outreach

• Public presentations
• Collaborations
• CSHL DNA Learning Center

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Public Presentations
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Collaborations

• The Maize Genetics and Genomics Database.
  --Letter
• for Carolyn Lawrence-MaizeGDB
• MaizeGDB-web site text, links to data
• Gramene
• EBI Ensembl
• Affymetrix Maize Pilot Expression Array Project
• Optical map
• TWINSCAN
• Vmatch
• Full-Length cDNA Project

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CSHL DNA Learning Center
http//www.dnalc.org/maize/maize.html