Sequencing the MTP of Ae . tauschii

1. Sequencing the MTP of Ae. tauschii

2. Shotgun sequencing of Ae. tauschii genome using Roche 454 FLX In collaboration with the NSF-funded wheat Radiation Hybrid mapping project, we have generated 28 Roche 454 FLX runs of Ae. tauschii genomic sequences, representing 3.1 X genome coverage. The average read length is about 377 bp

3. Sequence complexity of wheat genome Ay Ax Wheat Glu-1region  2 3a 4a 3b 4b 5 III II I Fatimah-1 Sabrina-2 Ames-1 Eos-1 Deimos-1 Gujog-1 Solo-3s Apiip-1 Sabrina-3 Jorge-1 Nusif-1 Solo-2s WHAM-2 Lahuwi-1 Hawi-1 WHAM-1 Fatimah-2 Hawi-1 Sabrina-1 Wilma-1s Solo-4s Solo-1s Wilma-2 Sabrina-4p Pivu-1 62,445 bp 241,895 bp Ay HMW-glutenin Wilma, LTR Ax HMW-glutenin

4. Why choose the GS FLX+ System ?

5. 16 x 16 two-dimensional pooling strategy • Single BAC clones from the MTPs of 256 different contigs will be selected and arranged into a grid of 16 x 16 clones. • Cell cultures of 16 clones in a row or a column of the grid will be pooled, generating a total of 32 pools; DNA will be isolated from each pool of 16 clones. • Indexed fragment library will be prepared from DNA of each pool, 32 libraries will be combined and sequenced to a depth of 10X genome equivalent with Roche 454 FLX+ system. • Reads of each pool will be individually assembled into sequence contigs. • Each sequence contig will be assigned to a BAC clone on the basis of sharing sequence information by a row pool and a column pool. • Sequence contigs will be ordered and oriented along each BAC clone using the global pair-end Illumina sequences of AL8/78 generated in CSHL (Richard McCombie, PI), overlaps of neighboring BAC clones in the BAC contigs, and BES. • Sequence scaffolds and pseudomolecules will be constructed on the basis of the location of BACs within BAC contigs and BAC contigs along the physical maps of chromosomes.

6. 16X16 two dimensional pooling and BAC DNA purification A1 A2 A3 A16 Row A O.D. reading BAC DNA purification (Qiagen Kit) 16 x 16 = 256 BAC clones 16 Row pools (Row A to Row P) and 16 Column pools (Col 1 to Col 16) = 32 DNA pools

7. 454 FLX+ run of indexed BAC libraries Rapid Library Construction Pool indexed Row libraries Row A MID1 Row B MID2 em-PCR Bead wash and load Row P MID16 Col 1 MID16 Col 2 MID17 454 FLX+ Run Pool indexed Col libraries em-PCR Bead wash and load Col 16 MID32

8. Sequence assembly and deconvolution of sequence contigs Col Col Col Col Col Col Col Col Col Col Col Col Col Col Col Col Row Row Row Row Row Row Row Row Row Row Row Row Row Row Row Row

9. Sequence assembly using col pool data

10. Sequence assembly using row pool data

11. Dotplot analysis of 454 assembled contigs against reference BACs control1 control2 control3 control4

12. Deconvolution of Row and Col pool assembled contigs to specific BAC clones based on two-dimensional pooling design 2DPool Contigs Assembled Contigs Reference 2 Reference 2 2DPool Contigs Assembled Contigs Reference 13 Reference 13

13. Genic regions covered by 454 sequence contigsin control BACs

14. Singletons in each Row and Col pool assembly

15. Dotplot analysis of assembled BACs against reference BACs Assemble scaffold Assemble scaffold Reference 2 Reference 1 Assemble scaffold Assemble scaffold Reference 3 Reference 4

16. Sequence coverage of column and row pool

17. Number of scaffolds of the assembled control BACs