Chromosome 12Chromosome 12
M. Pietrella1, G. Falcone1, E. Fantini1, A. Fiore1, M.R. Ercolano2, A. Barone2, M.L. Chiusano2, S. Grandillo3, N. D’Agostino2, A. Traini2, L. Frusciante2, A. Vezzi4, S. Todesco4, 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: workflow
An optimized workflow has been developed for the mapping of BACs using esculentum/pennellii Introgression Lines
IL mapping of BACs: results
The 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
39(33 in pipeline)
56(10 in pipeline)
7(4 in pipeline)
Map on other Chr
25 16 64
Total 64 72 71
IL mapping of “orphan” sequenced BACs
Some 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.
Mapping methodMapped BACs n°
Mapped BACs on Chr 12
CAPS 3 -
pennellii-specific PCR 9 1
Total 12 1
BAC extension
PABS, a tool for extending seed BACs (Todesco et al., 2008) has been developed and is available at http://tomato.cribi.unipd.it/index.html.
A)
B)
Genome annotation
ISOL@ (Chiusano et al, 2008), a bioinformatics resource for Solanaceae genomics, allows a cross-link from the BAC sequences with other databases (transcriptome-EST and proteome-UNIPROT) to obtain a functional annotation. The new update of the EST collections (October 2008) contains EST (dbEST) and mRNAs (GenBank).
Paired end sequences have been obtained for approx. 50,000 fosmids. Approx. 10% of the sequences match chloroplast DNA. The data have beeen 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%
Fosmid end sequencing
Currently, there are 80 BACs in various steps of the sequencing process, i.e. around 70% of the projected gene-rich region. A total of 5.3 Mb non redundant sequence have been submitted (up from 1.2 Mb last year). A comparative genetic and cytogenetic map has been built, in collaboration with H. De Jong and D. Szinay. New markers (SSR and non-overgo SGN markers) have been sought for filling gaps.
SSR: markers identified and IL-mapped in the gaps
Chromosome 12 sequencing status
What does the genome look like
Distribution of repeat and EST sequences in sequenced BACs
Chiusano et alProjected heterochromatin
ESTs Repeats
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
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.