1. Wageningen, April 24-25, 2008 II Tomato Finishing Workshop Chromosome 12 Update ENEA, Rome University of Naples ‘Federico II’ CRIBI and Univ. of Padua Giulia Falcone & Marco Pietrella

2. centromeric region (ext) Extensions length are shown for information purpose only 67 cM 70 cM 50 cM 60 cM 12 Overall BACs in progress (Chr 12) IL map Wageningen, April 24-25, 2008

3. ENEA SEQUENCE PROVIDER: *Sequence type: capillary (AB 3730). *Chemistry type: Big Dye Terminators. *Subcloned library: 2 libraries of different size: 2.5-3.5kb an 3.5-5kb, cloned in the pUC18 vector. The fragments are obtained through hydroshearing. *Sequence gap closure strategies: Finishing standard methods and sequence manual inspections. *Data flow between sequence provider and finishing: data production and finishing done by Cogenics and in case of problems, we provide the finishing by ourselves. Wageningen, April 24-25, 2008

4. -Concern region annotations for the HTGS3 BACs: *single subclone coverage and unsure bases Wageningen, April 24-25, 2008 “Misc_feature” tags “Comment” tags -Additional informations: *Confirmation by restriction enzyme digest of HTGS3 BAC assembled sequences *Confirmation by FISH of seed BAC positions.

5. *So far, all submitted clones have been sequenced by AB3730, with Big Dye terminator chemistry. *17 BAC clones are now in processing by means of 454 pyrosequencing technology but the results are not yet available. Sequence type in UniPD AB3730 BAC clones: Usually one plasmid shotgun library with an insert length of 2-2.5kbp. Average coverage of 8-10X on shotgun phase. For each BAC clone pair-end reads are produced, useful for: assembly check during finishing phase; design of finishing experiments. Phred/phrap/consed software used for assembly and finishing purposes Wageningen, April 24-25, 2008

6. *Strategies: sequencing of BAC DNA and/or plasmid clones with custom primers; sequencing of PCR spanning a single subclone region. *Notes: Long stretches of TA are better resolved with a modification of the sequencing reaction: 2 step cycle:96°C(denaturation) 58°C (annealing/extension) Presence of long repeats requires the synthesis of other plasmid libraries (shotgun, with longer inserts, and/or restriction library) *On HTGS3 BAC clones all the Tomato Finishing Standards are followed, and moreover: at least two independent reads (with phred >30) have to cover each base. the assembly consistency is checked with 2-3 different restriction analyses. Finishing of AB3730 BAC clones: Wageningen, April 24-25, 2008

7. Examples of problematic clones C12HBa0062P09: GAP of poly TA -Estimated length: ~70 Kb (restriction enzyme digestion data) -Position: heterochromatic BAC on Chr.12 long arm (FISH data, Dóra Szinay) 62P09 Band length: ~1000 kb Putative poly-TA length: ~200 bp PCR to evaluate the size of the region: We consider this BAC as a finished one. We have submitted C12HBa0062P09 as HTGS2. To do: annotate the regions of concern and specify the length of TA stretch. Wageningen, April 24-25, 2008 2 Contigs I) 312 reads, 39741 bps II) 392 reads, 32205 bps

8. Examples of problematic clones C12HBa077H15 LE_HBa077H15 was submitted to NCBI and SGN as Chr. 12 BAC: Subsequent IL mapping data have demonstrated that it belongs to Chr. 3. Wageningen, April 24-25, 2008 How to change the submission to SGN? How to change the name? C12HBa077H15-->C03HBa077H15

9. Examples of problematic clones LE_HBa024A16 LE_HBa024A16 is completely sequenced. IL mapping analysis has demonstrated that it maps on Chr. 7. The mistake was caused by the presence of non univocal marker. The marker cLET-5-M3 is present on Chr. 7 and Chr. 12 (see below). Wageningen, April 24-25, 2008 Can we submit this BAC as C07HBa024A16?

10. -Doubt concerning the trace file names: *Is SGN nomenclature suggested and not obligatory..or not? 2 DOUBTS -Doubt concerning the sequencing extension procedures: *Which accuracy grade is requested for the sequencing of overlapping regions during the extension? Wageningen, April 24-25, 2008

11. Result Summary of BAC IL mapping methods Wageningen, April 24-25, 2008

12. Thanks for the attention!