1 / 15

Progress in sequencing chromosome 6

Progress in sequencing chromosome 6. - State of the art - Selection of minimal overlapping Extension BACs from tomato chr.6 using BAC-end sequences. René Klein Lankhorst Plant Research International Wageningen, NL. Sept 2005. CBSG Tomato Sequencing Program State of the Art for chromosome 6:.

emelda
Download Presentation

Progress in sequencing chromosome 6

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Progress in sequencing chromosome 6 - State of the art - Selection of minimal overlapping Extension BACs from tomato chr.6 using BAC-end sequences • René Klein Lankhorst • Plant Research International • Wageningen, NL Sept 2005

  2. CBSG Tomato Sequencing ProgramState of the Art for chromosome 6: • Total number of finished BACs: 35 (33 in Phase 1/2 and 2 in Phase 3) • Number of BACs in current sequencing pipeline: 2 • Number of BACs in current FISH pipeline:8 • Sequences of 16 BACs have been uploaded to SGN for public release

  3. +0/+0 AFLP fingerprint BAC binning High-Resolution local physical map Seed BAC BlastN BAC-ends Assembly of selected BAC-ends Pre-selected BACs Largest extending, minimal overlapping BAC Selection of Extension BACs by BAC-end screening

  4. AFLP AFLP AFLP AFLP AFLP AFLP AFLP AFLP Finished seed BAC Blast homology search and assembly of BAC ends +0/+0 fingerprinting, binning and physical mapping Selection for minimal overlap and largest extension Sequencing, assembly, walking Selection of Extension BACs by BAC-end screening

  5. Mi-2 hsp’s Hyp. protein transposon Candidate Extension BAC Selection of Extension BACs (I) BlastN analysis of finished seed BAC P250I21

  6. Selection of BACs (III) P073H07 assembled BAC end on P250I21

  7. Selection of Extension BACs (II) Assembly of BAC ends on P250I21

  8. Selection of Extension BACs (III) Repetitive BAC end sequences on P308F14 Perfect Blast score • High quality base discrepancy • Repetitive

  9. AFLP fingerprints from chromosome 6 tomato BACs

  10. Binning and high resolution physical map Mi Contig 3,9 kb overlap

  11. Results: • 24 Seed BACs were screened with 350.000 BAC ends • 188.000 HindIII, 112.000 MboI, 49.000 EcoRI • On average 16 overlapping BAC-ends per seed BAC were found • Almost all ‘seeds’ have candidates from the 3 BAC libraries: • SL_MboI008H07 overlaps 3.6kb with LE_HBa125P18 • SL_MboI027B07 overlaps 880bp with LE_HBa153O03 • SL_EcoRI113K10 overlaps 4,2 kb with LE_HBa106K23 • LE_HBa103N18 overlaps 2.1kb with LE_HBa046G10 • LE_HBa073H07 overlaps 3.9kb with LE_HBa250I21 • 3 Extension BACs have been sequenced • 100% sequence overlap with Seed BACs was confirmed • BlastN-analysis has been fully automated

  12. Conclusions • Proof of concept for the “BAC-end strategy” • Works for both fully closed BACs as for Phase 1 – 2 BACs • No need to fingerprint an entire BAC library • Blast, Assembly and AFLP work complementary • Accurate selection of BACs having minimal overlap and largest extending insert • Protocol will be published: • TOPAAS, a Tomato and Potato Assembly Assistance System for selection and finishing of BACs. Peters et al., Plant Physiology, accepted for publication

  13. Taco Jesse Kim Jansen Dennis Woltinge Jan van Haarst Thamara Hesselink Marjo van Staveren Marleen Henkens Paul Mooijman Sander Peters Roeland van Ham René Klein Lankhorst

  14. High resolution physical mapping (II) Contig P103

  15. Candidate BAC Selection of BACs (II) BlastN analysis for draft assembled seed BAC P046G10

More Related