Current Sequencing Effort of Tomato Chromosome 2

Sunghwan Jo, KRIBB

• Identical segment duplication/ Chimera BAC clone?

• What is best way to speed up sequencing progress?

Increase seed BACs!?

query query_len strandquery_startquery_endsbjct sbjct_start sbjct_end identityC02HBa0011A02 94 15261 Plus 34741 50001 C02HBa0142D08 89.3 10260 25520 100C02HBa0011A02 94 82527 Minus 40984 1E+05 C02SLe0123B22 89.3 203018 120496 99C02HBa0013N18 82 8359 Plus 41285 49640 C02HBa0177F12 142 2710 11062 99C02HBa0016A12 92 15168 Minus 16016 31183 C02SLm0014P22 96 15167 1 99C02HBa0016A12 92 58370 Minus 17790 76159 C02HBa0236E02 96 131788 73419 100C02HBa0016A12 92 34270 Minus 80320 1E+05 C02HBa0236E02 96 67914 33645 100C02HBa0044O16 75.1 51370 Plus 1 51370 C02SLm0049G16 75 36495 87863 99C02HBa0044O16 75.1 44154 Plus 7217 51370 C02HBa0075D08 74.8 1 44154 99C02HBa0044O16 75.1 8553 Plus 71134 79686 C02HBa0075D08 74.8 57840 66392 100C02HBa0059M17 67 9101 Plus 16907 26007 C02SLm0108P14 ? 25190 34290 100C02HBa0075D08 74.8 57839 Plus 1 57839 C02SLm0049G16 75 43711 101548 99C02HBa0075D08 74.8 8553 Minus 57840 66392 C02SLm0073G04 75.3 72905 64353 100C02HBa0134G09 89.3 62072 Plus 1 62072 C02HBa0011O23 90 97803 159872 99C02HBa0155D20 130 77420 Plus 38185 1E+05 C04HBa0203P08 1 77419 99C02HBa0161P02 69.5 4494 Plus 88189 92676 C02HBa0323A14 72.5 101015 105496 98C02HBa0161P02 69.5 6723 Minus 132056 1E+05 C02SLm0114O11 70 147037 140315 100C02HBa0190P16 92 43491 Minus 1 43491 C02HBa0236E02 96 43490 1 99C02HBa0204D01 73 28476 Plus 85540 1E+05 C02SLm0008E03 71 63 28537 99C02HBa0215M12 89 92934 Plus 66834 2E+05 C02SLm0128E12 89 1 92933 99C02HBa0323A14 72.5 590 Plus 1 590 C02SLe0075O22 67 93921 94510 100C02HBa0323A14 72.5 25190 Minus 40147 65331 C02SLm0108P14 ? 25189 1 99C02SLe0022J22 118 10019 Plus 115275 1E+05 C02HBa0012A12 118 1 10019 100C02SLm0008E03 71 28476 Plus 63 28537 C02HBa0204D01 73 85540 114014 99C02SLm0014P22 96 15168 Minus 1 15167 C02HBa0016A12 92 31183 16016 99C02SLm0014P22 96 16471 Plus 15168 31638 C02SLm0065M14 92 80689 97159 99C02SLm0049G16 75 35695 Minus 1 35695 C02HBa0165K22 76 35695 1 100C02SLm0065M14 92 3538 Plus 1 3538 C02SLm0014P22 96 11631 15167 99C02SLm0065M14 92 16471 Plus 80689 97159 C02SLm0014P22 96 15168 31638 99C02SLm0108P14 ? 25190 Minus 1 25189 C02HBa0323A14 72.5 65331 40147 99

Redundant sequence in chromosome 2

67.0

70

71

72

72.5

73

74.5

Mbo108P14(34290bp)

HBa323A14 72.5cM (128329bp)

Hba059M17, 67cM (115036bp)

65331bp

1

40147bp 1

16907bp26007bp

9.1kb25.2kb

Mbo008E0371cM(46,886bp)

HBa204D0173cM(179,870bp)

1

1

~28Kb

Case 1

4.9kb

(RC) M108P14

H323A14

M108P14-confirm (455bp)

M108P14 H323A14

M108P14-confirm (455bp)

CATTTTATGCTGGCGAAACC

LEFT PRIMER

ACCCTTCCTCTTGCATCCTT

RIGHT PRIMER

M82

Mbo108P14 (34,290bp)

HBa323A14 (128,329bp)

65,331bp

1

40,147bp 1

9.1kb25.2kb

34,290

H323A14-M108P14-confirm (462bp)

TGTCCGAGTGGATCTCCTTC

LEFT PRIMER

CATTTTATGCTGGCGAAACC

RIGHT PRIMER

111111

H323A14-M108P14-confirm (462bp)

M108P14H323A14 M82H059M17

Mbo108P14 (34,290bp)

HBa323A14 72.5cM (128,329bp)

H059M17, 67cM (115,036bp)

65331bp

1

40147bp

1

16907bp 26007bp

9.1kb25.2kb

4.9kb

Mbo108P14 (34,290bp)

HBa323A14 72.5cM (128,329bp)

H059M17, 67cM (115,036bp)

65,331bp

1

40,147bp

1

16,907bp 26,007bp

9.1kb25.2kb

4.9kb

H059M17-confirm (421bp)

GTGGTGGGATCAACCTGTCT

LEFT PRIMER

TGCATGGCAATTTTGTATCC

RIGHT PRIMER

H059M17-confirm (421bp)

M108P14 M82H059M17

67.0

70

71

72

72.5

73

No segmental duplication but chimera clone

Mbo108P14 (34,290bp)

HBa323A14 72.5cM (128,329bp)

H059M17, 67cM (115,036bp)

65,331bp

1

40,147bp

1

16,907bp 26,007bp

9.1kb25.2kb

4.9kb

72

72.5

73

74.5

70

71

67.0

0008E03 0204D01 G

No segmental duplication but chimera cloneCase 2

Mbo008E0371cM(46,886bp)

HBa204D0173cM(179,870bp)

1

1

~28Kb

Hba75D08_sp6(74.8cM)

Mbo049G16_sp6 (75.0cM)

T0702(76.0cM)

H044O16_sp6 (75.1cM)

MboI73G04_T7 (75.3cM)

2-I

2-H2-H

2-I

2-I

Tomato-EXPEN 2000 IL mapBAC contig

Case 3

H075D08

M020N15TO-M073G04

H044O16

1 218853

or

M020N15TO-M073G04

M021D12

1 214253

H165K22TO-H228I09

TO-M049G16M021D12

1 288675

Hba75D08_sp6(74.8cM)

Contig 1

Contig 2

Contig 3

All contigs are well assembled!!

H165K22TO-H228I09

TO-M049G16M021D12

TO-M073G04M020N15

1 412497

H165K22TO-H228I09

TO-M049G16M021D12

1 288675

M020N15TO-M073G04

M021D12

1 214253

Contig 1

H075D08 H044O16

Contig 3

H165K22TO-H228I09

TO-M049G16M021D12

1 288675

M020N15TO-M073G04

H044O16

1 218853

H165K22TO-H228I09

TO-M049G16M021D12

1 288675

M020N15TO-M073G04

H044O16

1 218853

H075D08

Contig 1

Contig 2

H075D08

H044O16

H075D08

H075D08- H044O16 -confirm (397bp)

H044O16 confirm (427bp)

GGCCGTTCAACTTGCTCTTA

LEFT PRIMER

GGCACAAACATGTCAAATGC

RIGHT PRIMER

GGCCGTTCAACTTGCTCTTA

LEFT PRIMER

CTGAATTCGCGAACCAATCT

RIGHT PRIMER

Hba0075D08(74.8cM)

Mbo0049G16 (75.0cM)

T0702(76.0cM)

Hba044O16 (75.1cM)

MboI73G04 (75.3cM)

M020N15TO-M073G04

Hba0044O16

Hba0075D08

H165K22

TO-H228I09

Mbo0049G16M021D12

1 288675

75D08 44O16 G 75D08 44O16 G

75D08 49G16 G 75D08 49G16 G

H165K22TO-H228I09

TO-M049G16M021D12

TO-M073G04M020N15

1 412497

H165K22TO-H228I09

TO-M049G16M021D12

1 288675

M020N15TO-M073G04

M021D12

1 214253

Contig 1

H075D08 H044O16

Contig 3

89.3

92

94

96

TG426

TG48

TG147H236E02

M014P22

E128J14

1 257525

H190P16H016A12

M065M14H189G15

1 406359

E123B22

H142D08H180C15

H134G09

E010E18_112044_bp

1 524515 TG426(89.3cM)

TG48(92cM)

TG147(96cM)

H011A02 TG373(94cM)

Case 4

80kb

H125L12 (12,174bp)

1bp

E011K05 (91,579bp)671bp 6,462bp

5,798bp

H125L12 (12,174bp)

T0493 (48.0 cM)Expect

11,466bp E011K05 (91,579bp)

H125L12

E01

1K05

Sequence result

Dot-Matrix result Two sequence alignment result

2_G

Case 5

Summary

Name Genome Enzyme site

Name Genome

Enzyme site

Mbo108P14

X O HBa075D08

X X

Mbo008E03

X O Mbo049G16

O X

HBa323A14

O X HBa044O16

X X

HBa059M17

O X

10

20

30

40

50

60

70

80

90

100

110

140

120

130

0

142

13

Probably, chimeric BAC clonesWere selected as “Next BAC “ clones during extension process

1. Chimeric BAC clone itself exist in libraries but not in genome.2. Not all strange BAC clones have enzyme site.

SL_EcoRI0057O21 LE_HBa0155E05 SL_MboI0065K08

no BAC DQ672601

no BAC

LE_HBa0303I24

ing

LE_HBa0044L14 LE_HBa0163K16

LE_Hba0190N21 LE_HBa0025N15 LE_HBa0280E02

SP6 LE_HBa0146O12

T7

SL_EcoRI0042O06 LE_HBa0025A22 LE_HBa0209K17 LE_HBa0101G09

LE_HBa0320M09 SL_EcoRI0002K14

SL_MboI0045L06 LE_HBa0168N10 LE_HBa0155C04 LE_HBa0162I09 LE_HBa0027B01

SL_EcoRI0018B07 SL_MboI0064E19 LE_HBa0026M05

LE_HBa0160F05

no BAC LE_HBa0091J18 LE_HBa0122F06 SL_EcoRI0104N06 LE_HBa0185P07

no BAC

no BAC SL_EcoRI0016P15 SL_MboI0100O21 LE_HBa0072A04 LE_HBa0066C13

no BAC LE_HBa0125L12

no BAC

no BAC SL_EcoRI0011K05

no BAC LE_HBa00209I20

LE_HBa0122E16no BAC

no BAC SL_MboI108P14

no BAC

3' SL_EcoRI0075O22

LE_HBa0059M17

LE_HBa0031A21

no BAC LE_HBa0161P02 LE_HBa0329G05 SL_MboI0019I01 LE_HBa0090O01

SL_MboI0008E03 LE_HBa0167J21

LE_HBa0323A14

LE_HBa0006L05

LE_HBa0204D01

LE_HBa0320D04

LE_HBa0075D08

SL_MboI0049G16 LE_HBa0228I09

LE_HBa0165K22

LE_HBa0044O16

SL_MboI0073G04 SL_MboI0020N15

SL_EcoRI0026H18 LE_HBa0291P19 SL_EcoRI0033D19

LE_HBa0238L13

LE_HBa0013N18 LE_HBA0060J03 LE_HBa0159F19 LE_HBa0030D08 LE_HBa0009K06 LE_HBa0056D15

LE_HBa0130B04 SL_MboI0057H03

LE_HBa0164H08 SL_MboI0097L01 SL_MboI0128E12 LE_HBa0116F10 LE_HBa00106H06 (BAC19,AF27333) LE_HBa0023N04 LE_HBa0215M12 LE_HBa0074A14 LE_HBa0030A21 LE_HBa0213A01

SL_EcoRI0123B22 LE_HBa0142D08 LE_HBa0180C05 LE_HBa0134G09 SL_EcoRI0010E18 LE_HBa0011O23

LE_HBa0189G15 SL_MboI0065M14 LE_HBa0016A12 LE_HBa0190P16

ing LE_HBa0011A02

no BAC

LE_HBa0236E02 SL_MboI0014P22 SL_EcoRI0128J14

SL_EcoRI0010H16

SL_MboI0025E22 LE_HBa0172G12 SL_MboI0042B19

LE_HBa0124N09

SL_EcoRI0092M23 LE_HBa0046M08 LE_HBa0214B22

SL_EcoRI0096G02 LE_Hba00108N03 LE_HBa0111M10 SL_Mbo0066K03 LE_HBa0226F10

5' FW2.2

What is best way to speed up sequencing progress?

Increase seed BAC

• 64 Seed BACs• 74 Extended BACs• 28 contigs• 16 single BAC+ increase extending chance- Increase overlap length

Seed BACs BAC extension Total reading length

Non-overlapped

reading length

64 73 15,016kb

10,888kb

Contig single Average overlap

% done

(22Mb)

28 16 30kb 49.5%

BAC Sequencing SummaryBAC Sequencing Summary

Difference between the order of Genetic marker and

sequence result• cosII

Increase overlap length or lost BAC sequence