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Maize Production Sequencing

Maize Production Sequencing. lfulton@watson.wustl.edu. Maize Production Goals. BAC End Sequencing of 220,000 Clones Fosmid End Sequencing of 500,000 Clones Shotgun of 16,000 BAC Clones. Maize BAC End Sequences. 580,000 reads processed 567 average read length 60% success.

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Maize Production Sequencing

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  1. Maize Production Sequencing lfulton@watson.wustl.edu

  2. Maize Production Goals • BAC End Sequencing of 220,000 Clones • Fosmid End Sequencing of 500,000 Clones • Shotgun of 16,000 BAC Clones

  3. Maize BAC End Sequences • 580,000 reads processed • 567 average read length • 60% success

  4. Maize Fosmid End Sequences • 850,000 processed • 79% success • 543 average read length • Completed today

  5. Library Construction Pipeline • Receipt of sheared DNA from AGI • Size selection of insert DNA • Ligation into pSMART vector

  6. Constructed 17,034 Libraries as of August 31st

  7. Average Fail Rate for Library Construction was less than 5%

  8. Shotgun Criteria • 3.5X coverage • Clone size verification • 50% paired ends • BES agreement • 25% of clones failed • 22% need more data • 3% BES disagreement

  9. Shotgun Complete for 12,211 Clones as of August 31st

  10. Final Production Work • 660 Clones Need Library Construction • 2100 Clones In Production Pipeline • Expected Completion Date December 2007

  11. Sequence Improvement Bob Fulton Dick McCombie Rod Wing

  12. Sequence Improvement Pipeline • Shotgun_done triggers the prefinishing pipeline • Initial identification of “do finish” regions • Manual sorting and use of autoedit(Gordon) to break apart misassembly. • Autofinish(Gordon) used to choose directed reactions for all gaps and regions of low quality in “do finish” regions • Reassembly and 2nd iteration of prefinishing pipeline • Final identification of “do finish” regions and handoff to finishing pipeline

  13. Clone Improvement through the Prefinishing Pipeline

  14. Assembly View-Entire Clone Coverage (green) Spanning Plasmids End

  15. Assembly View-Do Finish Region EST sequence GSS sequence Do Finish Repeat Tags

  16. Alignment with cDNA read pairs Alignment with End Sequences

  17. Actual Projected

  18. Maize GenBank Submissions Joanne Nelson

  19. HTGS_FULLTOPHTGS_PREFINHTGS_ACTIVEFINHTGS_IMPROVED Submission Landmarks

  20. Improved Sequence “Non-repetitve portions of the sequence have had sequence improvement (directed attempts) and have been labeled as ‘improved.’ Improved regions are double stranded, sequenced with an alternate chemistry or covered by high quality data (i.e. phred quality greater than or equal to 30 or approval by an experienced finisher), unless otherwise noted. Regions of low sequence complexity (such as dinucleotide repeats and small unit tandem repeats) in the improved regions have not been resolved to previously established finishing standards. BAC end sequence, cot and methyl filtered genome survey sequence and data from overlapping projects of strain B73 may have been included in this project. Where possible, contigs have been ordered and oriented based on read pairing. These regions are designated as scaffolds. Additional order and orientation will be provided upon completion of detailed analysis of the complete finished tiling path.”

  21. FEATURES Location/Qualifiers source 1..184604 /organism="Zea mays" /mol_type="genomic DNA" /db_xref="taxon:4577" /chromosome="1" /clone="CH201-132J17; ZMMBBc0132J17" misc_feature 1..69252 /note="scaffold_name:Scaffold1" misc_feature 1..34245 /note="assembly_name:Contig28 vector_side:SP6" misc_feature 32401..34245 /note="Improved sequence." unsure 34230..34245 /note="Non-repetitive but unresolved region" gap 34246..34345 /estimated_length=unknown misc_feature 34346..68071 /note="assembly_name:Contig27" misc_feature 34346..36695 /note="Improved sequence." unsure 34346..34356 /note="Non-repetitive but unresolved region" misc_feature 38146..46795 /note="Improved sequence." gap 68072..68171 /estimated_length=unknown misc_feature 68172..69252 /note="assembly_name:Contig14" gap 69253..69352 /estimated_length=unknown misc_feature 69353..132243 /note="scaffold_name:Scaffold2” Improved Sequence

  22. HTGS_FULLTOP 3342HTGS_PREFIN 2014HTGS_ACTIVEFIN 4151HTGS_IMPROVED 2660 TOTAL 12167 Submission Totals

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