Скачать презентацию Progress in sequencing chromosome 6 — State of Скачать презентацию Progress in sequencing chromosome 6 — State of

b0651ecafabd01c1390bac9f9883e6b1.ppt

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Progress in sequencing chromosome 6 - State of the art - Selection of minimal 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: ► Total number CBSG Tomato Sequencing Program State 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

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

Selection of Extension BACs by BAC-end screening AFLP AFLP Finished seed BAC Blast homology Selection of Extension BACs by BAC-end screening 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 (I) Blast. N analysis of finished seed BAC P 250 Selection of Extension BACs (I) Blast. N analysis of finished seed BAC P 250 I 21 Mi-2 hsp’s Hyp. protein transposon Candidate Extension BAC

Selection of BACs (III) P 073 H 07 assembled BAC end on P 250 Selection of BACs (III) P 073 H 07 assembled BAC end on P 250 I 21

Selection of Extension BACs (II) Assembly of BAC ends on P 250 I 21 Selection of Extension BACs (II) Assembly of BAC ends on P 250 I 21

Selection of Extension BACs (III) Repetitive BAC end sequences on P 308 F 14 Selection of Extension BACs (III) Repetitive BAC end sequences on P 308 F 14 Perfect Blast score ► High quality base discrepancy ► Repetitive

AFLP fingerprints from chromosome 6 tomato BACs Panel I, lanes 2 to 6 contains AFLP fingerprints from chromosome 6 tomato BACs Panel I, lanes 2 to 6 contains fingerprints for BACs from the Mi contig. Panel II, lanes 9 to 16 contains fingerprints for BACs from contig P 103. All fingerprinted BACs originate from a Hind. III BAC library except for lane 12 which was pulled from an Mbo. I library. For all fingerprints Eco. RI/Mse. I +0/+0 primer combinations have been used where +0 indicates the absence of selective nucleotides. Lane 1, 8, and 18 contain a 10 bp size marker. The molecular weight size range of the fingerprints is between 50 and 500 nts an indicated at the right size. BACs used for fingerprints are indicated below. 1 2 3 4 5 6 7 8 9 = = = = = 10_BL P 073 H 07 P 096 H 22 P 092 A 17 P 111 A 08 P 250 I 21 P 112 G 05 10_BL P 046 G 10 10= P 118 F 20 11= M 054 C 01 12= P 200 L 01 13= P 061 I 06 14= P 008 K 02 15= P 103 N 18 16= P 188 J 12 17= P 197 N 20 18= 10 -BL

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

Results: ► 24 Seed BACs were screened with 350. 000 BAC ends | 188. Results: ► 24 Seed BACs were screened with 350. 000 BAC ends | 188. 000 Hind. III, 112. 000 Mbo. I, 49. 000 Eco. RI ► On average 16 overlapping BAC-ends per seed BAC were found ► Almost all ‘seeds’ have candidates from the 3 BAC libraries: | | | SL_Mbo. I 008 H 07 overlaps 3. 6 kb with LE_HBa 125 P 18 SL_Mbo. I 027 B 07 overlaps 880 bp with LE_HBa 153 O 03 SL_Eco. RI 113 K 10 overlaps 4, 2 kb with LE_HBa 106 K 23 LE_HBa 103 N 18 overlaps 2. 1 kb with LE_HBa 046 G 10 LE_HBa 073 H 07 overlaps 3. 9 kb with LE_HBa 250 I 21 ► 3 Extension BACs have been sequenced | 100% sequence overlap with Seed BACs was confirmed ► Blast. N-analysis has been fully automated

Conclusions ► Proof of concept for the “BAC-end strategy” | Works for both fully 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

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

High resolution physical mapping (II) Contig P 103 High resolution physical mapping (II) Contig P 103

Selection of BACs (II) Blast. N analysis for draft assembled seed BAC P 046 Selection of BACs (II) Blast. N analysis for draft assembled seed BAC P 046 G 10 Candidate BAC