Progress on sequencing tomato chromosome 12

045N22 (Chromosome 12—Telomere P)

Stack Lab--April 8,2005

LE_HBa0045N22

LE_HBa0026C13

Estimated euchromatin 11 MbN. of BACs to be sequenced 113N. of overgo markers 39

120 cMS. pennelli

ILs

IL12

-1 IL12

-1-1

IL12

-2

IL12

-3

IL12

-4

IL12

-4-1

IL12

-3-1

Silvana GrandilloCNR-IGVSOL2006

Tomato chromosome 12

Principal InvestigatorsLuigi Frusciante (Univ.of Naples)Giovanni Giuliano (ENEA, Rome)Giorgio Valle (CRIBI, Univ. of Padua)

Other InvestigatorsAmalia Barone (Univ. of Naples)Maria Luisa Chiusano (Univ. of Naples)Mara Ercolano (Univ. of Naples)Silvana Grandillo (IGV-CNR, Portici,Naples)Alessandro Vezzi (Univ. of Padua)

FundingAgronanotech (Italian Ministry of Agriculture - started October, 2004)FIRB (Italian Ministry of Research - started October, 2005)EU-SOL (European Commission - started May 2006)

14,0 cLPT-6-E912,5 C2_At4g03280

33,0 T002832,0 T1481

24,0 T148721,0 TG6819,0 TG263

36,0 T098939,0 T166741,0 cLET-8-k4

51,0 T104553,0 T121153,5 CT9954,0 T109354,5 C2_At5g4274055,0 T107855,5 TG28356,0 T162257,2 P6257,5 T145157,7 cLET-8-E1557,8 T118558,2 SSR2059,0 CT18960,0 SSR12460,0 SSR4460,0 cLET-8-G15

65,0 T194766,0 TG11168,0 TG39468,5 TG36771,0 T1266

86,0 T1676

96,0 T178496,0 TG29697,0 T0882

101,0 T0770

115,0 T1504

120,0 CD2

Map position of overgo markers used for Chr. 12

Map Position(cM)

Marker

Map Position(cM)

Marker

Near centromere

BAC validation procedure

√Single colony picking

√Medium scale DNA preparative

√BAC-end sequencing

√Fingerprinting

√PCR amplification of genetic marker

√Amplicon sequencing and sequence alignment

√BAC physical location tested by mapping in ILs

lines

PCR marker development and IL mapping

PCR

1 BAC C12HBa0161H10

2,3 parental genotypes

4,5 IL lines

6 negative control

A B C

E

ALLINEAMENTO MULTIPLO DEL BAC (161H10) E PARENTALI CON IL MARCATORE (T1045) Pennelli --------------------------------------------------------AAGG 4 T1045 ATCTTGAAGCTTAAAAAAAACAAAAAAAAAAACAAACGATGTTGGCTATATTTCACAAGG 60 IL8-1 -----------------------------------------------TCTCGTCACAAGG 13 P161H10 ------------------------------------------------CTCGTCACAAGG 12 IL12-2 -------------------------------------------------CTCTCACAAGG 11 M82 --------------------------------TKAGCAGGACTATTAATCTCTCACAAGG 28 **** Pennelli CATTTGCTCATCCTCCTGAAGAATTAAATAGTCCTGCATCAAAAAAATGTTTACTTCCTC 60 T1045 CATTTGCTCATCCTCCTGAAGAATTAAATAGTCCTGCATCAAAAAAATGTTTACTTCCAC 120 IL8-1 CATTTGCTCATCCTCCTGAAGAATTAAATAGTCCTGCATCAAAAAAATGTTTACTTCCAC 70 P161H10 CATTTGCTCATCCTCCTGAAGAATTAAATAGTCCTGCATCAAAAAAATGTTTACTTCCAC 69 IL12-2 CATTTGCTCATCCTCCTGAAGAATTAAATAGTCCTGCATCAAAAAAATGTTTACTTCCTC 67 M82 CATTTGCTCATCCTCCTGAAGAATTAAATAGTCCTGCATCAAAAAAATGTTTACTTCCAC 84 ********************************************************** * Pennelli AACAAACGCTACAAAAATTCGTATCGACTCGTCCATTTGACACCTCTTATGTTACTTTG 116 T1045 AACAAACGCTACAAAAATTCGTATCGACTCGTCCATTTGACACCTCTTATGTTACTTTT 179 IL8-1 AACAAACGCTACAAAAATTCGTATCGACTCGTCCATTTGACACCTCTTATGTTACTTTT 129 P161H10 AACAAACGCTACAAAAATTCGTATCGACTCGTCCATTTGACACCTCTTATGTTACTTTT 123 IL12-2 AACAAACGCTACAAAAATTCGTATCGACTCGTCCATTTGACACCTCTTATGTTACTTTT 121 M82 AACAAACGCTACAAAAATTCGTATCGACTCGTCCATTTGACACCTCTTATGTTACTTTG 139 ***********************************************************

Multiple alignment of :S. pennellii, M82, IL 12-2, BAC P161H10,

EST T1045 and IL 8-1

S. pennelliiT1045IL8-1P161H10IL12-2M82

S. pennelliiT1045IL8-1P161H10IL12-2M82

S. pennelliiT1045IL8-1P161H10IL12-2M82

T1045 Centromericregion

1 2 3 4 5 6

Sequencing and assembly strategy

Sequencing template:

Moving from PCR product to plasmid mini-preparation

Average plasmid insert size: 2000bp

Usually double-barrel strategy *

* (when the overlap between BACs is very high, the choice has been to sequence only

one end of the plasmid clones, and then to sequence from the other end only the

informative clones)

Assembly program:

phred/phrap/consed package

Sequencing standard for each BAC:

< 3% single strand region

< 1% single subclone (possibly none)

8-10X coverage

Sequencing and assembly (CRIBI, Univ of Padua)

To date 18 seed BACs associated to 16 markers (11 mapping on the short arm

and 5 on the long arm of chr. 12) have been selected for validation and

sequencing.

The map position of the clones is being confirmed by means of SNPs identified

on the S. pennellii IL population.

A total of 23 BACs are currently at different phases of the sequencing pipeline.

4 seed BAC sequences (Phase 3) have been submitted to SGN repository.

In order to identify new BACs to move out of the 4 finished seed BACs, as well as

of the Phase 1 or 2 BAC clones, a program complementary to the SGN Online

BLAST Interface has been developed at CRIBI (Univ. of Padua).

A bioinformatic platform has been built at the Univ. of Naples to provide an

Italian resource for supporting the annotation of the tomato genome.

Summary

Sequencing status of selected BAC clones

LE_HBa0021L02 (T1211)LE_HBa0059A05 (SSR124)

LE_HBa0032K07 (T0989)

LE_HBa0026C13 (cLPT-6-E9)

LE_HBa0140M01 (C2_At4g03280)

LE_HBa0161H10 (T1045)

LE_HBa0260C13; LE_HBa0206G16 (T1487)

LE_HBa0075C18 (T1481)

LE_HBa0244C09; LE_HBa0146I19 (T1667)

LE_HBa0163O04 (T0028) (Map position ???)

In pipeline

LE_HBa0183M06 (T0770)

LE_HBa0115G22 (T1676)

LE_HBa0193C03 (T1266)

Validated

HTGS phase 1

Finished & submitted

Finished but problems

LE_HBa0147G13 (T1504 + seq TG350)

LE_HBa0093P12 (T0882)

SL_EcoRI0004H16**LE_HBa0149G24

SL_MboI0126D24 SL_EcoRI0082A18LE_HBa0180O10 (cLPT-8-K4)

Extension in progress

BAC for extension at 3’ in sequencing pipeline

BAC for extension at 5’ in sequencing pipeline

LEGEND

HTGS phase 2

LE_HBa0073O10 LE_HBa0090D09LE_HBa0061F16

(2)

(5)

(6)

(4)

(4)

(4)

25

# of BACs

Sequencing status of selected BAC clones

Sequencing status of selected BAC clones

*

*

Problems

1. No marker-specific amplification by PCR of few selected seed BACs

2. Problems with BAC identity:LE_HBa0075C18: BES verified, but no marker in the consensus sequenceLE_HBa0260C13: no BES available at SGNLE_HBa0244C09: no BES available before sequencing, then identity not confirmed

3. Wrong map position for:LE_HBa0163O04: BES verified, marker enclosed, FISH map on chr. 7, working on assembly for hard repeat region

4. BACs with repeat regions:LE_HBa0059A05: easily resolvedLE_HBa0163O04: hard repeat region ( > plasmid insert size)LE_HBa0147G13: still to work

5. BAC extention:

How to move out of a sequenced BAC?Should we trust a fully automated BLASTN?How to choose the right BAC?

BAC EXTENSION: “BacEnds Extension v 0.1” developed at CRIBI (Univ. of Padua)

This tool has been designed to simplify some of the problems commonly

found during the BAC extension procedure in the Tomato Genome Project.

Repetitive sequences may easily act as a bridge to many BAC ends belonging

to a different part of the genome.

The program is already available to the Solanaceae community:

http://tomato.cribi.unipd.it/USER: tomato

PWD: trial

For further informations please contact:Dott. Davide Campagna

e-mail: davide@cribi.unipd.it CRIBI, University of Padua

BAC EXTENSION: “BacEnds Extension v 0.1” developed at CRIBI (Univ. of Padua)

- This tool displays the alignments of a BAC against the available tomato

BAC ends

- The BAC ends are displayed showing their orientation, thus indicating the

direction where the corresponding BAC is extending

- The electrophoretic profiles can be opened allowing an immediate control

of any discrepancy

- The RAP (Repeat Analysis Program)* and Low Complexity indexes are

shown indicating the “repetitiousness” of each part of the BAC

- Any sequence found in correspondence to a repeat should not be

considered as reliable for BAC extension

* Campagna D. et al. 2005, ‘RAP:a new computer program for de novo identification of

the repeated sequences in whole genomes’ ; Bioinfomatics 21 (5): 582-588

BacEnds Extention v 0.1

Developed at CRIBI, University of Padua,by Dott. Davide Campagna (e-mail: davide@cribi.unipd.it) Available at http://tomato.cribi.unipd.it/USER: tomatoPWD: trial

Sequenced BACcoordinates RAP index

Low complexityindex

BAC endsequences

BAC sequenceBlast against

BACENDS databaseBlast results parsering

Data StructureAnalyse the sequence and Builds RAP index

Partial image of BACENDS alignments, RAP and Linguistic Complexity index

Images of profiles aligned with blast results

RAP database

BacEnds Extention v 0.1

Bioinformatics at Univ. of Naples

-A bioinformatic platform has been built to provide an Italian resource to support

the experimental annotation of the Solanum lycopersicum genome

- Annotated EST database from dbEST (NCBI)* for Tomato and Potato species

(updated May 2006)

- Gbrowse interface for BAC annotation which has been released to the SOL

communityhttp://biosrv.cab.unina.it

-The BACs available at the SGN site were experimentally annotated and are

available through the Generic Genome Browser web interface allowing selection

of reference Gene Models to test predictive approaches.

-The two EST databases and the Gbrowse are cross-referenced and linked to the

Solanaceae Genome Network resources to provide useful data integration.

* D’Agostino, Aversano and Chiusano 2005, “ParPEST: A pipeline for EST data analysis based on parallel computing ”; BMC Bioinformatics, 6 (Suppl. 4):S9

Acknowledgments

University of Naples ‘Federico II’(Dept. DISSPA)Luigi FruscianteAmalia BaroneMara ErcolanoSara MelitoRosa PaparoWalter SanseverinoSara Torre

University of Naples ‘Federico II’(Dept. DSFB)Maria Luisa ChiusanoMario AversanoNunzio D'Agostino Alessandra Traini

CNR-IGV, Portici (Naples)Silvana GrandilloMaria CammareriPasquale Termolino

ENEA, RomeGiovanni GiulianoElio FantiniAlessia FioreGiuseppe Puglia

CRIBI and Univ. of PaduaGiorgio ValleAlessandro VezziDavide CampagnaLaura CollutoMichela D'AngeloFabrizio LevorinGiorgio Mitch MalacridaSilvia PescaroloRiccardo SchiavonSara TodescoAlessandro Zambon