1. WGP Tomato EU-SOL meeting July 15, 2009 Antoine Janssen

2. overview Whole Genome Profiling  Whole Genome Profiling: the concept  POP in Arabidopsis  WGP melon  Combining WGP and WGS  WGP Tomato

3. Whole Genome Profiling: Sequence-based physical mapping BAC clones using Illumina Genome Analyzer (Solexa)‏

4. Next-generation sequencing technologies have accelerated whole genome re-sequencing approaches and reduced their costs dramatically but, de novo construction of genomes in complex organisms is still costly therefore, An improved de novo draft genome sequencing strategy is needed taking full advantage of the power of next-generation sequencing The challenge Whole Genome Profiling

5. BAC libraries - BACs 125 kb average insert size, covering 5-20 times the genome (GE)‏ Chromosome BAC1 BAC3 BAC5 BAC4 BAC2 Whole Genome Profiling

6. TTAA……ACTTAGTTAGCTTGGACTAACGAATTCGTAGGCATAGTGACTAGCATTG…..……TTAA EcoRIMseI Restriction fragments Whole Genome Profiling

7.  Arabidopsis Genome – 125 Mbp  6144 BACs (5 GE) in 384 well plates  Each Illumina GA lane: 768 BACs ~ 3 M reads  Total 8 lanes  Individual BAC target preparation is too time consuming/costly  Therefore: BAC 2D pooling  Each pool identified by unique sample identification tag Pooling BAC clones R1 - CTACT R2 - CAGGT R3 - GCATC R4 - TGCAG R5 - TACTA R6 - CCTAG TCTGT - AGACT - GAGTC - GTGCA - ATCAC - GTATC - 384 wells plate = 384 BACs column pools row pools 19 20 21 22 23 24 Whole Genome Profiling

8. Illumina Genome Analyzer GTTGACAATTCAGTATTACTAGAGCTGCGTA TAACTCAATTCTCATCAGAAAAAATATATGA TACGCCAATTCCAAAAGATTGTAGAGTATTT GCCATCAATTCCAGTTTTGGGTTCTTCACCA GAAGTCAATTCAATTCCTTCAATCATCACAC GGTCGCAATTCAGTGTAAAAAGAAATTTCTA GCGGTCAATTCATGGTATGATGATAATAAGT TAACTCAATTCGAGTTCAGTTTGTTACTACT GCGGTCAATTCGGCAGAAGACACCAGTAACA TAAGGCAATTCATAGGTATAGGAAGAAGCCC TAACTCAATTCGTTCATACTTACACATGCAT TATTACAATTCTATATGATTTGTTACAGGTT GAATACAATTCAAAACCCTAATCACACGCAC TGAACCAATTCCACAATCGATGCTTATGAAA TATTACAATTCCAGTGGTCTAACAAGTAAAC GTTGACAATTCTTCATTCGAGTATTTCATCA GAAGTCAATTCGATTAGAAGCTGAAGAAGAA GTCGCCAATTCACAAAAAGCAAAACCCTTTT GTACTCAATTCTAATGGTGATTGAAAGACAA GTTATCAATTCTCCTCAGAGTTGGCGATGGA GCCATCAATTCTCTCGGGGATGTGTTGGGGG GCGCGCAATTCGCTTATCAAATCATTACCAG GTCAGCAATTCTATGGAGATAATTCGTGGGA TAAGGCAATTCTCATAGAAACAGAGAATGGA TAACTCAATTCTAAACTCAACCACCTAAAAC GAACGCAATTCGTTCTCCCACATGATGTTGC GCGACCAATTCCGTGTTTGTTTCTTGCAGTA GCCATCAATTCGTCTTACATGACTTCCATAG TCAGACAATTCGGGGCATTCCAAAAACTTGG GTTGACAATTCATCAATTCATAATATAATAG TGACGCAATTCCATTTTCCTTCCTCATTCAT TAAGGCAATTCCAGAGATTTGTAGACTCTAG GTCTTCAATTCAGCTCACATTTCTTCGTTTT TAAAACAATTCAGTAAAAAAATACAAGTAGT GTCAGCAATTCTCTTCCATAAGAAGTTGTGA GGTTACAATTCTGGGAACTATAAAAATCAGA TGACGCAATTCTTTTGTACATGGGATAAACA GTTCCCAATTCTACAATGGAGCTCTTATATG GTTCCCAATTCAACTAAAGATAAAGGAGTTC GCGCGCAATTCTAAAACCAATCAATTAAATA GAACGCAATTCCTACATAGATGGTGGGACCC GTCTTCAATTCAAATAAGCTAATACCAAGTA GTACTCAATTCCTCTCTATAAACCATTACAA GGTGTCAATTCTGATGCTCGTCCTCGTCATA GGTACCAATTCCAAATCAAATAAAATCCTAT TATTACAATTCTTGAACGTGTCGTCTCACAC GTCTTCAATTCGTGCGTTTCTATCAAAACTG TAACTCAATTCAGACTCTGAATAAAGTTTAC TATGTCAATTCGGATTCACCTATAGCTTGAT GAATACAATTCAGAACCTTCTCTTCCTCAAA GTCTTCAATTCATGGAGACTTCAAATTTGAA GTTCCCAATTCTGGTTTTGTGTTTACAGTTA GTTGACAATTCAGTATTACTAGAGCTGCGTA TAACTCAATTCTCATCAGAAAAAATATATGA TACGCCAATTCCAAAAGATTGTAGAGTATTT GCCATCAATTCCAGTTTTGGGTTCTTCACCA GAAGTCAATTCAATTCCTTCAATCATCACAC GGTCGCAATTCAGTGTAAAAAGAAATTTCTA GCGGTCAATTCATGGTATGATGATAATAAGT TAACTCAATTCGAGTTCAGTTTGTTACTACT GCGGTCAATTCGGCAGAAGACACCAGTAACA TAAGGCAATTCATAGGTATAGGAAGAAGCCC TAACTCAATTCGTTCATACTTACACATGCAT TATTACAATTCTATATGATTTGTTACAGGTT GAATACAATTCAAAACCCTAATCACACGCAC TGAACCAATTCCACAATCGATGCTTATGAAA TATTACAATTCCAGTGGTCTAACAAGTAAAC GTTGACAATTCTTCATTCGAGTATTTCATCA GAAGTCAATTCGATTAGAAGCTGAAGAAGAA GTCGCCAATTCACAAAAAGCAAAACCCTTTT GTACTCAATTCTAATGGTGATTGAAAGACAA GTTATCAATTCTCCTCAGAGTTGGCGATGGA GCCATCAATTCTCTCGGGGATGTGTTGGGGG GCGCGCAATTCGCTTATCAAATCATTACCAG GTCAGCAATTCTATGGAGATAATTCGTGGGA TAAGGCAATTCTCATAGAAACAGAGAATGGA TAACTCAATTCTAAACTCAACCACCTAAAAC GTCGCCAATTCTACAGCCAGGTTTTGGATCT TACCACAATTCCCGGGGGCAAATTACGTTGA GGTACCAATTCCGTAGGCGACTTGAGTGCGG GTTATCAATTCAACAGTCATTTCATTGGACC GGTACCAATTCATTATTTTCATATAAATTTT GTTATCAATTCTTCGCTTTCGCCACTTGGTC TGACGCAATTCTGATCCATTGATTGCTCTTG GTTCCCAATTCTTGTTTAGGCAGTTCATACC GTTGACAATTCTCAGTATGCTAGGTGGTTGA GGTACCAATTCAGTTCAAGAGCCCAAGGACT GGTACCAATTCATCGTGAGAGAATGAGTAAA GCCATCAATTCGGTTCAGTATTTCCTTTCGG GGTGTCAATTCGCAAGGATTTGTAGGCCGGA TATTACAATTCTGGGTTTTTCCTTCTGGTGA GAATACAATTCTGAAGTCCTACGAAATATAG TATGTCAATTCTCCCAAAATGTGAGAGGTCC GTCAGCAATTCATTTTCATTCTGACCGAACC TATGTCAATTCCATATTCGAAGTTGCGATCA GAATACAATTCAAAGTTGTAAGTAATATCTC TATTACAATTCCAATAGAGAAAAGAGTCGTA GTCAGCAATTCGCCCTATAGTGAGTCGTATT GTCAGCAATTCCATTTCCGGCGTGATGATGC GAACGCAATTCGTTCTCCCACATGATGTTGC GCGACCAATTCCGTGTTTGTTTCTTGCAGTA GCCATCAATTCGTCTTACATGACTTCCATAG TCAGACAATTCGGGGCATTCCAAAAACTTGG GTTGACAATTCATCAATTCATAATATAATAG TGACGCAATTCCATTTTCCTTCCTCATTCAT TAAGGCAATTCCAGAGATTTGTAGACTCTAG GTCTTCAATTCAGCTCACATTTCTTCGTTTT TAAAACAATTCAGTAAAAAAATACAAGTAGT GTCAGCAATTCTCTTCCATAAGAAGTTGTGA GGTTACAATTCTGGGAACTATAAAAATCAGA TGACGCAATTCTTTTGTACATGGGATAAACA GTTCCCAATTCTACAATGGAGCTCTTATATG GTTCCCAATTCAACTAAAGATAAAGGAGTTC GCGCGCAATTCTAAAACCAATCAATTAAATA GAACGCAATTCCTACATAGATGGTGGGACCC GTCTTCAATTCAAATAAGCTAATACCAAGTA GTACTCAATTCCTCTCTATAAACCATTACAA GGTGTCAATTCTGATGCTCGTCCTCGTCATA GGTACCAATTCCAAATCAAATAAAATCCTAT TATTACAATTCTTGAACGTGTCGTCTCACAC GTCTTCAATTCGTGCGTTTCTATCAAAACTG TAACTCAATTCAGACTCTGAATAAAGTTTAC TATGTCAATTCGGATTCACCTATAGCTTGAT GAATACAATTCAGAACCTTCTCTTCCTCAAA GTCTTCAATTCATGGAGACTTCAAATTTGAA GTTCCCAATTCTGGTTTTGTGTTTACAGTTA GTTGACAATTCAGTATTACTAGAGCTGCGTA TAACTCAATTCTCATCAGAAAAAATATATGA TACGCCAATTCCAAAAGATTGTAGAGTATTT GCCATCAATTCCAGTTTTGGGTTCTTCACCA GAAGTCAATTCAATTCCTTCAATCATCACAC GGTCGCAATTCAGTGTAAAAAGAAATTTCTA GCGGTCAATTCATGGTATGATGATAATAAGT TAACTCAATTCGAGTTCAGTTTGTTACTACT GCGGTCAATTCGGCAGAAGACACCAGTAACA TAAGGCAATTCATAGGTATAGGAAGAAGCCC TAACTCAATTCGTTCATACTTACACATGCAT TATTACAATTCTATATGATTTGTTACAGGTT GAATACAATTCAAAACCCTAATCACACGCAC TGAACCAATTCCACAATCGATGCTTATGAAA TATTACAATTCCAGTGGTCTAACAAGTAAAC GTTGACAATTCTTCATTCGAGTATTTCATCA GAAGTCAATTCGATTAGAAGCTGAAGAAGAA GTCGCCAATTCACAAAAAGCAAAACCCTTTT GTACTCAATTCTAATGGTGATTGAAAGACAA GTTATCAATTCTCCTCAGAGTTGGCGATGGA GCCATCAATTCTCTCGGGGATGTGTTGGGGG GCGCGCAATTCGCTTATCAAATCATTACCAG GTCAGCAATTCTATGGAGATAATTCGTGGGA TAAGGCAATTCTCATAGAAACAGAGAATGGA TAACTCAATTCTAAACTCAACCACCTAAAAC GTCGCCAATTCTACAGCCAGGTTTTGGATCT TACCACAATTCCCGGGGGCAAATTACGTTGA GGTACCAATTCCGTAGGCGACTTGAGTGCGG GTTATCAATTCAACAGTCATTTCATTGGACC GGTACCAATTCATTATTTTCATATAAATTTT GTTATCAATTCTTCGCTTTCGCCACTTGGTC TGACGCAATTCTGATCCATTGATTGCTCTTG GTTCCCAATTCTTGTTTAGGCAGTTCATACC GTTGACAATTCTCAGTATGCTAGGTGGTTGA GGTACCAATTCAGTTCAAGAGCCCAAGGACT GGTACCAATTCATCGTGAGAGAATGAGTAAA GCCATCAATTCGGTTCAGTATTTCCTTTCGG GGTGTCAATTCGCAAGGATTTGTAGGCCGGA TATTACAATTCTGGGTTTTTCCTTCTGGTGA GAATACAATTCTGAAGTCCTACGAAATATAG TATGTCAATTCTCCCAAAATGTGAGAGGTCC GTCAGCAATTCATTTTCATTCTGACCGAACC TATGTCAATTCCATATTCGAAGTTGCGATCA GAATACAATTCAAAGTTGTAAGTAATATCTC TATTACAATTCCAATAGAGAAAAGAGTCGTA GTCAGCAATTCGCCCTATAGTGAGTCGTATT GTCAGCAATTCCATTTCCGGCGTGATGATGC GAACGCAATTCGTTCTCCCACATGATGTTGC GCGACCAATTCCGTGTTTGTTTCTTGCAGTA GCCATCAATTCGTCTTACATGACTTCCATAG TCAGACAATTCGGGGCATTCCAAAAACTTGG GTTGACAATTCATCAATTCATAATATAATAG TGACGCAATTCCATTTTCCTTCCTCATTCAT TAAGGCAATTCCAGAGATTTGTAGACTCTAG GTCTTCAATTCAGCTCACATTTCTTCGTTTT TAAAACAATTCAGTAAAAAAATACAAGTAGT GTCAGCAATTCTCTTCCATAAGAAGTTGTGA GGTTACAATTCTGGGAACTATAAAAATCAGA TGACGCAATTCTTTTGTACATGGGATAAACA GTTCCCAATTCTACAATGGAGCTCTTATATG GTTCCCAATTCAACTAAAGATAAAGGAGTTC GCGCGCAATTCTAAAACCAATCAATTAAATA GAACGCAATTCCTACATAGATGGTGGGACCC GTCTTCAATTCAAATAAGCTAATACCAAGTA GTACTCAATTCCTCTCTATAAACCATTACAA GGTGTCAATTCTGATGCTCGTCCTCGTCATA GGTACCAATTCCAAATCAAATAAAATCCTAT TATTACAATTCTTGAACGTGTCGTCTCACAC GTCTTCAATTCGTGCGTTTCTATCAAAACTG TAACTCAATTCAGACTCTGAATAAAGTTTAC TATGTCAATTCGGATTCACCTATAGCTTGAT GAATACAATTCAGAACCTTCTCTTCCTCAAA GTCTTCAATTCATGGAGACTTCAAATTTGAA GTTCCCAATTCTGGTTTTGTGTTTACAGTTA GTTGACAATTCAGTATTACTAGAGCTGCGTA TAACTCAATTCTCATCAGAAAAAATATATGA TACGCCAATTCCAAAAGATTGTAGAGTATTT GCCATCAATTCCAGTTTTGGGTTCTTCACCA GAAGTCAATTCAATTCCTTCAATCATCACAC GGTCGCAATTCAGTGTAAAAAGAAATTTCTA GCGGTCAATTCATGGTATGATGATAATAAGT TAACTCAATTCGAGTTCAGTTTGTTACTACT GCGGTCAATTCGGCAGAAGACACCAGTAACA TAAGGCAATTCATAGGTATAGGAAGAAGCCC TAACTCAATTCGTTCATACTTACACATGCAT TATTACAATTCTATATGATTTGTTACAGGTT GAATACAATTCAAAACCCTAATCACACGCAC TGAACCAATTCCACAATCGATGCTTATGAAA TATTACAATTCCAGTGGTCTAACAAGTAAAC GTTGACAATTCTTCATTCGAGTATTTCATCA GAAGTCAATTCGATTAGAAGCTGAAGAAGAA GTCGCCAATTCACAAAAAGCAAAACCCTTTT GTACTCAATTCTAATGGTGATTGAAAGACAA GTTATCAATTCTCCTCAGAGTTGGCGATGGA GCCATCAATTCTCTCGGGGATGTGTTGGGGG GCGCGCAATTCGCTTATCAAATCATTACCAG GTCAGCAATTCTATGGAGATAATTCGTGGGA TAAGGCAATTCTCATAGAAACAGAGAATGGA TAACTCAATTCTAAACTCAACCACCTAAAAC GTCGCCAATTCTACAGCCAGGTTTTGGATCT TACCACAATTCCCGGGGGCAAATTACGTTGA GGTACCAATTCCGTAGGCGACTTGAGTGCGG GTTATCAATTCAACAGTCATTTCATTGGACC GGTACCAATTCATTATTTTCATATAAATTTT GTTATCAATTCTTCGCTTTCGCCACTTGGTC TGACGCAATTCTGATCCATTGATTGCTCTTG GTTCCCAATTCTTGTTTAGGCAGTTCATACC GTTGACAATTCTCAGTATGCTAGGTGGTTGA GGTACCAATTCAGTTCAAGAGCCCAAGGACT GGTACCAATTCATCGTGAGAGAATGAGTAAA GCCATCAATTCGGTTCAGTATTTCCTTTCGG GGTGTCAATTCGCAAGGATTTGTAGGCCGGA TATTACAATTCTGGGTTTTTCCTTCTGGTGA GAATACAATTCTGAAGTCCTACGAAATATAG TATGTCAATTCTCCCAAAATGTGAGAGGTCC GTCAGCAATTCATTTTCATTCTGACCGAACC TATGTCAATTCCATATTCGAAGTTGCGATCA GAATACAATTCAAAGTTGTAAGTAATATCTC TATTACAATTCCAATAGAGAAAAGAGTCGTA GTCAGCAATTCGCCCTATAGTGAGTCGTATT GTCAGCAATTCCATTTCCGGCGTGATGATGC GAACGCAATTCGTTCTCCCACATGATGTTGC GCGACCAATTCCGTGTTTGTTTCTTGCAGTA GCCATCAATTCGTCTTACATGACTTCCATAG TCAGACAATTCGGGGCATTCCAAAAACTTGG GTTGACAATTCATCAATTCATAATATAATAG TGACGCAATTCCATTTTCCTTCCTCATTCAT TAAGGCAATTCCAGAGATTTGTAGACTCTAG GTCTTCAATTCAGCTCACATTTCTTCGTTTT TAAAACAATTCAGTAAAAAAATACAAGTAGT GTCAGCAATTCTCTTCCATAAGAAGTTGTGA GGTTACAATTCTGGGAACTATAAAAATCAGA TGACGCAATTCTTTTGTACATGGGATAAACA GTTCCCAATTCTACAATGGAGCTCTTATATG GTTCCCAATTCAACTAAAGATAAAGGAGTTC GCGCGCAATTCTAAAACCAATCAATTAAATA GAACGCAATTCCTACATAGATGGTGGGACCC GTCTTCAATTCAAATAAGCTAATACCAAGTA GTACTCAATTCCTCTCTATAAACCATTACAA GGTGTCAATTCTGATGCTCGTCCTCGTCATA GGTACCAATTCCAAATCAAATAAAATCCTAT TATTACAATTCTTGAACGTGTCGTCTCACAC GTCTTCAATTCGTGCGTTTCTATCAAAACTG TAACTCAATTCAGACTCTGAATAAAGTTTAC TATGTCAATTCGGATTCACCTATAGCTTGAT GAATACAATTCAGAACCTTCTCTTCCTCAAA GTCTTCAATTCATGGAGACTTCAAATTTGAA GTTCCCAATTCTGGTTTTGTGTTTACAGTTA GTTGACAATTCAGTATTACTAGAGCTGCGTA TAACTCAATTCTCATCAGAAAAAATATATGA TACGCCAATTCCAAAAGATTGTAGAGTATTT GCCATCAATTCCAGTTTTGGGTTCTTCACCA GAAGTCAATTCAATTCCTTCAATCATCACAC GGTCGCAATTCAGTGTAAAAAGAAATTTCTA GCGGTCAATTCATGGTATGATGATAATAAGT TAACTCAATTCGAGTTCAGTTTGTTACTACT GCGGTCAATTCGGCAGAAGACACCAGTAACA TAAGGCAATTCATAGGTATAGGAAGAAGCCC TAACTCAATTCGTTCATACTTACACATGCAT TATTACAATTCTATATGATTTGTTACAGGTT GAATACAATTCAAAACCCTAATCACACGCAC TGAACCAATTCCACAATCGATGCTTATGAAA TATTACAATTCCAGTGGTCTAACAAGTAAAC GTTGACAATTCTTCATTCGAGTATTTCATCA GAAGTCAATTCGATTAGAAGCTGAAGAAGAA GTCGCCAATTCACAAAAAGCAAAACCCTTTT GTACTCAATTCTAATGGTGATTGAAAGACAA GTTATCAATTCTCCTCAGAGTTGGCGATGGA GCCATCAATTCTCTCGGGGATGTGTTGGGGG GCGCGCAATTCGCTTATCAAATCATTACCAG GTCAGCAATTCTATGGAGATAATTCGTGGGA TAAGGCAATTCTCATAGAAACAGAGAATGGA TAACTCAATTCTAAACTCAACCACCTAAAAC GTCGCCAATTCTACAGCCAGGTTTTGGATCT TACCACAATTCCCGGGGGCAAATTACGTTGA GGTACCAATTCCGTAGGCGACTTGAGTGCGG GTTATCAATTCAACAGTCATTTCATTGGACC GGTACCAATTCATTATTTTCATATAAATTTT GTTATCAATTCTTCGCTTTCGCCACTTGGTC TGACGCAATTCTGATCCATTGATTGCTCTTG GTTCCCAATTCTTGTTTAGGCAGTTCATACC GTTGACAATTCTCAGTATGCTAGGTGGTTGA GGTACCAATTCAGTTCAAGAGCCCAAGGACT GGTACCAATTCATCGTGAGAGAATGAGTAAA GCCATCAATTCGGTTCAGTATTTCCTTTCGG GGTGTCAATTCGCAAGGATTTGTAGGCCGGA TATTACAATTCTGGGTTTTTCCTTCTGGTGA GAATACAATTCTGAAGTCCTACGAAATATAG TATGTCAATTCTCCCAAAATGTGAGAGGTCC GTCAGCAATTCATTTTCATTCTGACCGAACC TATGTCAATTCCATATTCGAAGTTGCGATCA GAATACAATTCAAAGTTGTAAGTAATATCTC TATTACAATTCCAATAGAGAAAAGAGTCGTA GTCAGCAATTCGCCCTATAGTGAGTCGTATT GTCAGCAATTCCATTTCCGGCGTGATGATGC GAACGCAATTCGTTCTCCCACATGATGTTGC GCGACCAATTCCGTGTTTGTTTCTTGCAGTA GCCATCAATTCGTCTTACATGACTTCCATAG TCAGACAATTCGGGGCATTCCAAAAACTTGG GTTGACAATTCATCAATTCATAATATAATAG TGACGCAATTCCATTTTCCTTCCTCATTCAT TAAGGCAATTCCAGAGATTTGTAGACTCTAG GTCTTCAATTCAGCTCACATTTCTTCGTTTT TAAAACAATTCAGTAAAAAAATACAAGTAGT GTCAGCAATTCTCTTCCATAAGAAGTTGTGA GGTTACAATTCTGGGAACTATAAAAATCAGA TGACGCAATTCTTTTGTACATGGGATAAACA GTTCCCAATTCTACAATGGAGCTCTTATATG GTTCCCAATTCAACTAAAGATAAAGGAGTTC GCGCGCAATTCTAAAACCAATCAATTAAATA GAACGCAATTCCTACATAGATGGTGGGACCC GTCTTCAATTCAAATAAGCTAATACCAAGTA GTACTCAATTCCTCTCTATAAACCATTACAA GGTGTCAATTCTGATGCTCGTCCTCGTCATA GGTACCAATTCCAAATCAAATAAAATCCTAT TATTACAATTCTTGAACGTGTCGTCTCACAC GTCTTCAATTCGTGCGTTTCTATCAAAACTG TAACTCAATTCAGACTCTGAATAAAGTTTAC TATGTCAATTCGGATTCACCTATAGCTTGAT GAATACAATTCAGAACCTTCTCTTCCTCAAA GTCTTCAATTCATGGAGACTTCAAATTTGAA GTTCCCAATTCTGGTTTTGTGTTTACAGTTA GTTGACAATTCAGTATTACTAGAGCTGCGTA TAACTCAATTCTCATCAGAAAAAATATATGA TACGCCAATTCCAAAAGATTGTAGAGTATTT GCCATCAATTCCAGTTTTGGGTTCTTCACCA GAAGTCAATTCAATTCCTTCAATCATCACAC GGTCGCAATTCAGTGTAAAAAGAAATTTCTA GCGGTCAATTCATGGTATGATGATAATAAGT TAACTCAATTCGAGTTCAGTTTGTTACTACT GCGGTCAATTCGGCAGAAGACACCAGTAACA TAAGGCAATTCATAGGTATAGGAAGAAGCCC TAACTCAATTCGTTCATACTTACACATGCAT TATTACAATTCTATATGATTTGTTACAGGTT GAATACAATTCAAAACCCTAATCACACGCAC TGAACCAATTCCACAATCGATGCTTATGAAA TATTACAATTCCAGTGGTCTAACAAGTAAAC GTTGACAATTCTTCATTCGAGTATTTCATCA GAAGTCAATTCGATTAGAAGCTGAAGAAGAA GTCGCCAATTCACAAAAAGCAAAACCCTTTT GTACTCAATTCTAATGGTGATTGAAAGACAA GTTATCAATTCTCCTCAGAGTTGGCGATGGA GCCATCAATTCTCTCGGGGATGTGTTGGGGG GCGCGCAATTCGCTTATCAAATCATTACCAG GTCAGCAATTCTATGGAGATAATTCGTGGGA TAAGGCAATTCTCATAGAAACAGAGAATGGA TAACTCAATTCTAAACTCAACCACCTAAAAC GTCGCCAATTCTACAGCCAGGTTTTGGATCT TACCACAATTCCCGGGGGCAAATTACGTTGA GGTACCAATTCCGTAGGCGACTTGAGTGCGG GTTATCAATTCAACAGTCATTTCATTGGACC GGTACCAATTCATTATTTTCATATAAATTTT GTTATCAATTCTTCGCTTTCGCCACTTGGTC TGACGCAATTCTGATCCATTGATTGCTCTTG GTTCCCAATTCTTGTTTAGGCAGTTCATACC GTTGACAATTCTCAGTATGCTAGGTGGTTGA GGTACCAATTCAGTTCAAGAGCCCAAGGACT GGTACCAATTCATCGTGAGAGAATGAGTAAA GCCATCAATTCGGTTCAGTATTTCCTTTCGG GGTGTCAATTCGCAAGGATTTGTAGGCCGGA TATTACAATTCTGGGTTTTTCCTTCTGGTGA GAATACAATTCTGAAGTCCTACGAAATATAG TATGTCAATTCTCCCAAAATGTGAGAGGTCC GTCAGCAATTCATTTTCATTCTGACCGAACC TATGTCAATTCCATATTCGAAGTTGCGATCA GAATACAATTCAAAGTTGTAAGTAATATCTC TATTACAATTCCAATAGAGAAAAGAGTCGTA GTCAGCAATTCGCCCTATAGTGAGTCGTATT GTCAGCAATTCCATTTCCGGCGTGATGATGC GAACGCAATTCGTTCTCCCACATGATGTTGC GCGACCAATTCCGTGTTTGTTTCTTGCAGTA GCCATCAATTCGTCTTACATGACTTCCATAG TCAGACAATTCGGGGCATTCCAAAAACTTGG GTTGACAATTCATCAATTCATAATATAATAG TGACGCAATTCCATTTTCCTTCCTCATTCAT TAAGGCAATTCCAGAGATTTGTAGACTCTAG GTCTTCAATTCAGCTCACATTTCTTCGTTTT TAAAACAATTCAGTAAAAAAATACAAGTAGT GTCAGCAATTCTCTTCCATAAGAAGTTGTGA GGTTACAATTCTGGGAACTATAAAAATCAGA TGACGCAATTCTTTTGTACATGGGATAAACA GTTCCCAATTCTACAATGGAGCTCTTATATG GTTCCCAATTCAACTAAAGATAAAGGAGTTC GCGCGCAATTCTAAAACCAATCAATTAAATA GAACGCAATTCCTACATAGATGGTGGGACCC GTCTTCAATTCAAATAAGCTAATACCAAGTA GTACTCAATTCCTCTCTATAAACCATTACAA GGTGTCAATTCTGATGCTCGTCCTCGTCATA GGTACCAATTCCAAATCAAATAAAATCCTAT TATTACAATTCTTGAACGTGTCGTCTCACAC GTCTTCAATTCGTGCGTTTCTATCAAAACTG TAACTCAATTCAGACTCTGAATAAAGTTTAC TATGTCAATTCGGATTCACCTATAGCTTGAT GAATACAATTCAGAACCTTCTCTTCCTCAAA GTCTTCAATTCATGGAGACTTCAAATTTGAA GTTCCCAATTCTGGTTTTGTGTTTACAGTTA GTTGACAATTCAGTATTACTAGAGCTGCGTA TAACTCAATTCTCATCAGAAAAAATATATGA TACGCCAATTCCAAAAGATTGTAGAGTATTT GCCATCAATTCCAGTTTTGGGTTCTTCACCA GAAGTCAATTCAATTCCTTCAATCATCACAC GGTCGCAATTCAGTGTAAAAAGAAATTTCTA GCGGTCAATTCATGGTATGATGATAATAAGT TAACTCAATTCGAGTTCAGTTTGTTACTACT GCGGTCAATTCGGCAGAAGACACCAGTAACA TAAGGCAATTCATAGGTATAGGAAGAAGCCC TAACTCAATTCGTTCATACTTACACATGCAT TATTACAATTCTATATGATTTGTTACAGGTT GAATACAATTCAAAACCCTAATCACACGCAC TGAACCAATTCCACAATCGATGCTTATGAAA TATTACAATTCCAGTGGTCTAACAAGTAAAC GTTGACAATTCTTCATTCGAGTATTTCATCA GAAGTCAATTCGATTAGAAGCTGAAGAAGAA GTCGCCAATTCACAAAAAGCAAAACCCTTTT GTACTCAATTCTAATGGTGATTGAAAGACAA GTTATCAATTCTCCTCAGAGTTGGCGATGGA GCCATCAATTCTCTCGGGGATGTGTTGGGGG GCGCGCAATTCGCTTATCAAATCATTACCAG GTCAGCAATTCTATGGAGATAATTCGTGGGA TAAGGCAATTCTCATAGAAACAGAGAATGGA TAACTCAATTCTAAACTCAACCACCTAAAAC GTCGCCAATTCTACAGCCAGGTTTTGGATCT TACCACAATTCCCGGGGGCAAATTACGTTGA GGTACCAATTCCGTAGGCGACTTGAGTGCGG GTTATCAATTCAACAGTCATTTCATTGGACC GGTACCAATTCATTATTTTCATATAAATTTT GTTATCAATTCTTCGCTTTCGCCACTTGGTC TGACGCAATTCTGATCCATTGATTGCTCTTG GTTCCCAATTCTTGTTTAGGCAGTTCATACC GTTGACAATTCTCAGTATGCTAGGTGGTTGA GGTACCAATTCAGTTCAAGAGCCCAAGGACT GGTACCAATTCATCGTGAGAGAATGAGTAAA GCCATCAATTCGGTTCAGTATTTCCTTTCGG GGTGTCAATTCGCAAGGATTTGTAGGCCGGA TATTACAATTCTGGGTTTTTCCTTCTGGTGA GAATACAATTCTGAAGTCCTACGAAATATAG TATGTCAATTCTCCCAAAATGTGAGAGGTCC GTCAGCAATTCATTTTCATTCTGACCGAACC TATGTCAATTCCATATTCGAAGTTGCGATCA GAATACAATTCAAAGTTGTAAGTAATATCTC TATTACAATTCCAATAGAGAAAAGAGTCGTA GTCAGCAATTCGCCCTATAGTGAGTCGTATT GTCAGCAATTCCATTTCCGGCGTGATGATGC GAACGCAATTCGTTCTCCCACATGATGTTGC GCGACCAATTCCGTGTTTGTTTCTTGCAGTA GCCATCAATTCGTCTTACATGACTTCCATAG TCAGACAATTCGGGGCATTCCAAAAACTTGG GTTGACAATTCATCAATTCATAATATAATAG TGACGCAATTCCATTTTCCTTCCTCATTCAT TAAGGCAATTCCAGAGATTTGTAGACTCTAG GTCTTCAATTCAGCTCACATTTCTTCGTTTT TAAAACAATTCAGTAAAAAAATACAAGTAGT GTCAGCAATTCTCTTCCATAAGAAGTTGTGA GGTTACAATTCTGGGAACTATAAAAATCAGA TGACGCAATTCTTTTGTACATGGGATAAACA GTTCCCAATTCTACAATGGAGCTCTTATATG GTTCCCAATTCAACTAAAGATAAAGGAGTTC GCGCGCAATTCTAAAACCAATCAATTAAATA GAACGCAATTCCTACATAGATGGTGGGACCC GTCTTCAATTCAAATAAGCTAATACCAAGTA GTACTCAATTCCTCTCTATAAACCATTACAA GGTGTCAATTCTGATGCTCGTCCTCGTCATA GGTACCAATTCCAAATCAAATAAAATCCTAT TATTACAATTCTTGAACGTGTCGTCTCACAC GTCTTCAATTCGTGCGTTTCTATCAAAACTG TAACTCAATTCAGACTCTGAATAAAGTTTAC TATGTCAATTCGGATTCACCTATAGCTTGAT GAATACAATTCAGAACCTTCTCTTCCTCAAA GTCTTCAATTCATGGAGACTTCAAATTTGAA GTTCCCAATTCTGGTTTTGTGTTTACAGTTA GTTGACAATTCAGTATTACTAGAGCTGCGTA TAACTCAATTCTCATCAGAAAAAATATATGA TACGCCAATTCCAAAAGATTGTAGAGTATTT GCCATCAATTCCAGTTTTGGGTTCTTCACCA GAAGTCAATTCAATTCCTTCAATCATCACAC GGTCGCAATTCAGTGTAAAAAGAAATTTCTA GCGGTCAATTCATGGTATGATGATAATAAGT TAACTCAATTCGAGTTCAGTTTGTTACTACT GCGGTCAATTCGGCAGAAGACACCAGTAACA TAAGGCAATTCATAGGTATAGGAAGAAGCCC TAACTCAATTCGTTCATACTTACACATGCAT TATTACAATTCTATATGATTTGTTACAGGTT GAATACAATTCAAAACCCTAATCACACGCAC TGAACCAATTCCACAATCGATGCTTATGAAA TATTACAATTCCAGTGGTCTAACAAGTAAAC GTTGACAATTCTTCATTCGAGTATTTCATCA GAAGTCAATTCGATTAGAAGCTGAAGAAGAA GTCGCCAATTCACAAAAAGCAAAACCCTTTT GTACTCAATTCTAATGGTGATTGAAAGACAA GTTATCAATTCTCCTCAGAGTTGGCGATGGA GCCATCAATTCTCTCGGGGATGTGTTGGGGG GCGCGCAATTCGCTTATCAAATCATTACCAG GTCAGCAATTCTATGGAGATAATTCGTGGGA TAAGGCAATTCTCATAGAAACAGAGAATGGA TAACTCAATTCTAAACTCAACCACCTAAAAC GTCGCCAATTCTACAGCCAGGTTTTGGATCT TACCACAATTCCCGGGGGCAAATTACGTTGA GGTACCAATTCCGTAGGCGACTTGAGTGCGG GTTATCAATTCAACAGTCATTTCATTGGACC GGTACCAATTCATTATTTTCATATAAATTTT GTTATCAATTCTTCGCTTTCGCCACTTGGTC TGACGCAATTCTGATCCATTGATTGCTCTTG GTTCCCAATTCTTGTTTAGGCAGTTCATACC GTTGACAATTCTCAGTATGCTAGGTGGTTGA GGTACCAATTCAGTTCAAGAGCCCAAGGACT GGTACCAATTCATCGTGAGAGAATGAGTAAA GCCATCAATTCGGTTCAGTATTTCCTTTCGG GGTGTCAATTCGCAAGGATTTGTAGGCCGGA TATTACAATTCTGGGTTTTTCCTTCTGGTGA GAATACAATTCTGAAGTCCTACGAAATATAG TATGTCAATTCTCCCAAAATGTGAGAGGTCC GTCAGCAATTCATTTTCATTCTGACCGAACC TATGTCAATTCCATATTCGAAGTTGCGATCA GAATACAATTCAAAGTTGTAAGTAATATCTC TATTACAATTCCAATAGAGAAAAGAGTCGTA GTCAGCAATTCGCCCTATAGTGAGTCGTATT GTCAGCAATTCCATTTCCGGCGTGATGATGC Whole Genome Profiling

9.  Illumina sequence reads: TCTGT CAATTC TAGTACCAAGCTTGCCATGA TAAGG CAATTC GTTCCCGGGCCTTGTACACA GTCGC CAATTC CATCCAATAAATAGCTCTAT GCATC CAATTC TAGTACCAAGCTTGCCATGA TATTA CAATTC AATTAGAAGAAATGATATTC Whole Genome Profiling Sequence Tags sample identification tag (“barcode”)‏ Restriction site part of the primer 20 base genome sequence tag flanking RE site = pool R3 = pool C19

10.  70% of sequence 20-mer tags are unique in rice; > 85% in Arabidopsis Impact sequence tag length Whole Genome Profiling

11. FingerPrinted Contigs (FPC) assembly BAC1 BAC2 Assembly physical BAC map using adapted FPC Whole Genome Profiling

12. Whole Genome Profiling Whole Genome Arabidopsis  Arabidopsis Genome – 125 Mbp  6144 BACs (6 GE) in 384 well plates  Each Illumina GA lane: 768 BACs ~ 3 M reads  Total 8 lanes

13. Whole Genome Profiling Results 6 GE Arabidopsis 4599 BACs 65,000 tags 234 contigs (2 – 125 BACs)‏ 541 singletons 85% coverage FPC BAC1 BAC2

14.  WGP Arabidopsis thaliana ecotype Colombia  6144 BACs (5 GE);  WGP using one Illumina GA classic run  65,000 sequence tags  assembly 4599 BACs (75%): 234 contigs (2 – 125 BACs/contig)‏  Validation on genome sequence by BLAST analysis WGP sequence tags:  52,000 tags 100% hits, covering 99% of genome; max. gap 125 kbp  50,000 unique hits; average 2,355 bp between tags  86% of all EcoRI sites represented PoP Arabidopsis thaliana Whole Genome Profiling

15. PoP Arabidopsis thaliana Whole Genome Profiling

16.  450 Mbp estimated genome size  47,000 BACs (EcoRI and HindIII libraries) ~ 13 GE in total  Available for contig building: - 5 GA runs - 300 M reads - 196,000 unique sequence tags - 40,000 BACs (85%) uniquely tagged, average 33 tags/BAC WGP melon Whole Genome Profiling

17. WGP melon: results  549 contigs, 6416 singleton BACs  Median 21 BACs / contig  78% genome coverage Whole Genome Profiling

18. Combining WGP and WGS Roche GS FLX Titanium and Illumina Genome Analyzer II Whole Genome Profiling and Whole Genome Sequencing

19.  GS FLX Titanium sequencing (15 X):  10 GS FLX Titanium random shotgun runs  3 3-kb and 4 long jump p.e. GS FLX Titanium runs  Illumina GA II paired-end sequencing (30 X):  500 bp, 2 kb and 10 kb  Status:  GS and GA sequencing completed  GS assembly completed  GA assembly in progress WGS melon genome Whole Genome Profiling and Whole Genome Sequencing

20. Combining WGP and WGS Whole Genome Profiling and Whole Genome Sequencing EcoRI WGP BAC contigs EcoRI WGP sequence tag  2 - 3 kb distance → WGP sequence tag 400 nt Titanium (Paired-end) WGS contigs 36 nt GA II

21. Combining WGP and WGS Advantages:  WGP provides sequence-based anchor points for WGS  Use WGP to create high-resolution sequence-based physical BAC map, eg. 10 X BAC library coverage  Use WGS to generate (deep) coverage whole genome sequence  Superior assembly: WGP map contains far less contigs (549) than genomes sequenced by conventional random shotgun WGS strategies (tens of thousands) and produces more accurate maps than fingerprint based PM  Cost reduction: no Sanger sequencing required  Direct access to BAC clones in regions of interest Whole Genome Profiling and Whole Genome Sequencing

22. Status WGP Tomato 4 types of BAC libraries:  HindIII15360 clones120Kbp insert  EcoRI15360 clones120Kbp insert  MboI15360 clones120Kbp insert  20 pools  total 5.5 GBp / 950 Mbp = 5.7 x  Random sheared (Lucigen)50688 clones90 kb insert  16 pools  Total 4.6 Gbp / 950 Mbp = 4.8 x Total nr of clones: 96786 of which 92160 are analyzed (95%)‏  Approximately 85% RE bacs deconvolutable  Approximately 60% of sheared bacs deconvolutable

23. Comparison WGP results WGP Tomato 515771average nr reads/tag 403140average nr tags/BAC 74%84%75% tagged BACs (FPC ready)‏ 67,74239,9354,599nr tagged BACs (FPC ready)‏ 336,258181,25465,734nr unique tags 42%50%43% deconvolutable reads 136.796.012.1nr deconvolutable reads (M)‏ 326.919128.2nr OK reads generated (M)‏ E/M enzyme combination 12.113.25.9genome equivalents BACs tested 92,16047,6166,144nr BACs tested 26 tag length (incl. restriction site)‏ 950450130genome size (Mbp)‏ TomatoMelonArabidopsis

24. What next WGP Tomato  Finish last 5% (planned for next run)‏  Contiging with FPC  Deliver data  EU-SOL: Integrate with WGS data

25. Amplicon Express: Robert Bogden Keith Stormo Quanzhou Tao 454 Life Sciences / Roche Applied Science: Jason Affourtit Brian Desany Hans Lunstroo University of Udine: Michele Morgante CBSG / EU SOL: Willem Stiekema Roeland van Ham René Klein Lankhorst BioSeeds companies: Rijk Zwaan Enza Zaden Vilmorin & Cie Takii & Co Keygene N.V.: Upstream ResearchApplied Research Marcel PrinsRené Hofstede Marjo de RuiterAnker Sørensen Hein van der PoelRichard Feron Marjolijn KelderMartin Zevenbergen Anita BonnéLinda de Leeuw Nathalie van OrsouwAlberto Maurer Esther VerstegeMarco van Schriek Taco JesseJeroen Rombout Bio-informatics ICT Jan van Oeveren Kornelis Stol Antoine Janssen Harold VerstegenContact: Hanne Volpinmichiel.van-eijk@keygene.com Jifeng Tangherco.van-liere@keygene.com Business Development Jon Wittendorp Herco van Liere Mark van Haaren Keygene N.V. owns patents and patent applications covering its Whole Genome technologies Thanks to: Whole Genome Profiling and Whole Genome Sequencing