wageningen, april 24-25, 2008 ii tomato finishing workshop chromosome 12 update enea, rome...
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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
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
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
-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.
*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
*Strategies:• sequencing of BAC DNA and/or plasmid clones withcustom 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
Examples of problematic clonesC12HBa0062P09: 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 ContigsI) 312 reads, 39741 bpsII) 392 reads, 32205 bps
Examples of problematic clonesC12HBa077H15
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
Examples of problematic clonesLE_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?
-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
BAC N° IL Mapping method
16 Sequencing
26 S.pennellii-specific PCR
7 CAPS method
TOT: 49
Chr. 1 Chr. 2 Chr. 3 Chr. 4 Chr. 5 Chr. 6 Chr. 7 Chr. 8 Chr. 9 Chr. 10 Chr. 11 Chr. 12
9 4 4 3 1 5 4 3 6 5 1 4
Mapped BACs on
Result Summary of BAC IL mapping methods
Wageningen, April 24-25, 2008
Thanks for the attention!