Chromosome 12Chromosome 12

M. Pietrella1, G. Falcone1, E. Fantini1, A. Fiore1, C. Perla1, M.R. Ercolano2, A. Barone2, M.L. Chiusano2, S. Grandillo3, N. D’Agostino2, S. Melito2, S. Torre2, A. Traini2, L. Frusciante2, A. Vezzi4, S. Todesco4, M. D'Angelo4, R. Schiavon4, D. Campagna4, A. Zambon4, S. Pescarolo4, F. Levorin4, G. Valle4, G. Giuliano1

1Italian Agency for New technologies, Energy and the Environment (ENEA), Roma, Italy

2Department of Soil, Plant, Environmental and Animal Production Sciences, Univ. of Naples "Federico II", Portici, Italy

3CNR, Institute for Plant Genetics-Portici, Portici, Italy

4CRIBI Biotechnology Centre and Department of Biology, Univ. of Padova, Padova, Italy

IL mapping of BACs: workflowAn optimized workflow has been developed for the mapping of BACs using esculentum/pennellii Introgression Lines

IL mapping of BACs: resultsThe procedure has been used to confirm the map position of BACs previously mapped on Chrom 12 at Cornell and at Syngenta, or, as a service to the community, to “de novo” map novel seed BACs on the entire genome. The data are avalable on SGN.

Cornell Syngenta De novo

Confimed on Chr 12

29

(24 in pipeline)

56

(10 in pipeline)

5

(2 in pipeline)

Map on other Chr

22 16 56

Total 52 72 61

IL mapping of “orphan” sequenced BACsSome BACs sequenced by a country have been found “a posteriori” not to map on that country’s chromosome. As a service to the community, those BACs are being assigned on a novel chromosome. The data are available on SGN.

BAC extensionA tool for extending seed BACs (PABS, Todesco et al., 2008) has been developed and is available at http://tomato.cribi.unipd.it/index.html.

A)

B)

Genome annotationIn order to contribute to the genome annotation, we developed ISOL@ (Chiusano et al, 2008), a bioinformatics resource for Solanaceae genomics. Based on EST analysis, ISOL@ allows a cross-link from the BAC sequences with other databases (transcriptome-EST and proteome-UNIPROT) to obtain a functional annotation.

Fosmid end sequencingPaired end sequences have been obtained for approx. 50,000 fosmids. Approx. 10% of the sequences match chloroplast DNA. The data are being uploaded on SGN.

N° of bacterial plates 135

N° of sequencing plates produced 270

N° of sequenced ends 103410

(270 * 383)

N° of available fosmid clone paired-ends

95698

(92.5%)

% of clones with both ends failed 4.8%

Chromosome 12 sequencing statusCurrently, there are 71 BACs in various steps of the sequencing process, i.e. around 62% of the projected gene-rich region. A total of 5.1 Mb non redundant sequence have been submitted (up from 1.2 Mb last year). A comparative genetic and cytogenetc map is under construction, in collaboration with H. De Jong and D. Szinay.

What does the genome look likeDistribution of repeat and EST sequences in sequenced BACs

Chiusano et al

454 sequencing of 24 BACs

Shotgun sequencingShotgun sequencing(using MIDs)(using MIDs)

Preparation of single libraries, one

per BAC, using adaptors with MIDs.

There’re 12 different MIDs, therefore

the 24 BACs were sorted in 2

groups:

S1S1: BACs 1 to 12 (MIDs 1-12)

S2S2: BACs 13 to 24 (MIDs 1-12)

Sequenced in

distinct regions of

the PTP.

Input DNA: 24 BACs

Nebulization

BAC fragments, 24 samples

DNA fragments with MID adaptors, 24 samples

ssDNA fragments linkedto beads, 2 samples

MID adaptorsligation

Pooling of libraries and Binding to beadsfor emPCR

Slide kindly provided by BMR-genomics, spin-off of CRIBI

Long Paired EndLong Paired End

The same two groups of

pooled BACs were

hydrosheared to obtain two

pools of fragments of about 3-

4 kbases.

The purified fragments were

used to produce 2 long paired

end libraries.

In this case BACs were not tagged!

Slide kindly provided by BMR-genomics, spin-off of CRIBI

454 sequencing of 24 BACs

After a shotgun sequencing run usually we obtain a series of contig of aligned sequences, but….

we don’t know:- how these contigs are oriented- how they are ordered- the distance between two consecutive contigsUsing the Long Paired End approach, we can obtain scaffolds of ordered and oriented contigs.

~ 3kb~ 3kb

~ 3kb

The Long Paired End Strategy

Sequence Assembly

Obtained reads were:

automatically sorted in different folders according to the MID sequence (found at the beginning of each read and then trimmed).

24 folders BAC specific

assembly of the shotgun reads contigs construction and, after, addition of the long paired end reads scaffolds production

LPE reads (not tagged!) were assigned to the proper BAC by performing a series of BLAST against the shotgun (tagged) sequences.