Download Method To Verify Re-Arrayed Anchored BAC Clones

Tomato Overgo Project and
Seed BAC Selection
Cornell Team
Ying Eileen Wang, 2005 PAG
TG154
T1566
T347
SSR125
CT9
T147
cLEC7H4
SSR331
SSR580
CT38
SSR50
cLET1I9
T1665
SSR103
SSR5
T697
CT255
T1706
T1117
TG31
SSR57
T1201
T634
cLER17N11
Fw2.2
T1480
T1494
SSR32
SSR26
T562
SSR356
SSR349A
SSR605
SSR96
SSR66
SSR40
SSR586
cLEC7P21
T1616
Objectives for Overgo Project
• anchor tomato BACs/contigs on the highly
saturated genetic map (F2.2000)
• identify the minimum tiling path of BAC
clones for BAC-by-BAC sequencing
overgo --- overlapping oligonucleotide probes
5'
24 mer
3'
(8 bp)
3'
24 mer
5'
Klenow
32P-dATP, 32P-dCTP
*
* * * * * * *
* * * * * *
40 mer
Pools of overgos
Hybridization
Overgo hybridization on tomato BAC filters
BAC fingerprinting
(88,642 clones)
Tomato
BAC library
BAC contigs (7465)
a0037015
a0314C15
a0114G13
a0309K01
(129,024 clones)
TG178
T0244
a0037015
a0314C15
a0114G13+
a0309K01+
40cM
30
P006N16 P004C15
P003A09 P015O20
Tomato
genetic
markers
2
3
4
5
6
7
8
9 10 11 12
A
B
C
E
F
G
(1536)
row 2 positive
clones:
P004C15
P003A09
P010A10
P009P18
D
H
16 plates
10
Anchored BAC contig on
tomato chromosome 6
column 2 positive clones:
1
20
marker 2B
(TG80):
P004C15
P003A09
marker-BAC association
0
Overgo Anchoring Results
• 4857 good marker--BAC associations
• 4857 anchored BACs
• 652 anchor markers are involved in plausible non-conflicted
associations with BACs.
• plausible contigs:
1880 BACs in 705 plausible contigs
• 2166 BAC singletons
• 809 BACs are from 425 implausible contigs
• 3117 implausible marker-BAC associations
• 7235 ambiguous associations
Distribution of Anchor Markers on Chromosomes
92
165
1.8
79
67
143 171
1.8 2.6
62
137
2.2
40
119
3.0
63
101
1.6
51
112
2.2
34
87
2.6
40
116
2.9
41
87
2.1
43
103
2.4
39 # anchors
120 cM chr length
3.1 cM per anchor
Method To Verify BAC-Marker Association
-- sequencing BAC using customized primer
• Select two clones when possible per marker for
sequence verification using the following parameters:
– 1st choice:
– 2cd choice:
– 3rd choice:
Insert size greater than 100 Kb
Only one or two clones for that marker
Insert size > 60 Kb
Insert size unknown, from plates 1-260*
Insert size unknown, from plates >260*
Insert size less than 60 K
* Plates 1 - 260 of the HindIII library are from a ligation yielding larger
insert clones
Clone selection Example
Marker
Chr Offset
TomatoEXPEN
Map
Clone
Size (bp)
Contig
96893
ctg517
cLED-19-B18
2
28
2000
cLED-19-B18
2
28
2000
cLER-1-H17
2
0
2000
181384
cLER-1-H17
2
0
2000
147686 ctg3307
cLER-1-H17
2
0
2000
91460 ctg1334
cLER-1-H17
2
0
2000
39023
cLER-1-H17
2
0
2000
cLER-1-H17
2
0
2000
SSR50
2
143
2000
# BACs Contig Size
in Contig
(bp)
ctg1291
42055 ctg6894
Key
Optimal (between 100-160Kb)
Superoptimal, clone larger than 160Kb
Only one positive clone for that marker
Clone size between > 60 Kb
Clone size unknown & plate number below 260
Clone size unknown & plate number above 260
Clone size less than 60Kb
18
Original
Clone ID
I
Plat
Row Well
e
Sequence
To Verify
236726.35 LE_HBa025A22
1
B
24
Yes
LE_HBa254N13
2
A
1
Yes
LE_HBa007F24
1
A
17
Yes
5
111116.45 LE_HBa209G22
1
N
15
Yes
29
280206.7 LE_HBa332P20
2
F
12
No
LE_HBa271A19
2
B
6
No
376829.7 LE_HBa165A05
1
K
24
No
LE_HBa335D08
2
F
17
No
91791.85 LE_HBa256J01
2
A
3
Yes
25
3
Selection
Ranking
1
1
1
2
2
3
3
Verifying the marker-BAC association by BAC sequencing
• Design customized primer within marker sequence.
customized primer
40bp overgo sequence
marker
SGN predicted intron sites
• Sequencing BAC using the customized primer. One end of the BAC clone
should be sequenced to confirm the quality of BAC DNA. If the BAC end
sequence is good and the sequence from customized primer fails, then another
customized primer should be designed. The SGN tool "intron_finder" could be
used to predict the splice sites.
• Align sequence obtained from customized primer and marker
sequence. If sequences align perfectly or nearly perfectly, then clone is
considered verified.
Ruth White and Jim Giovannoni
Method To Verify BAC-Marker Association
(-- Continued)
-- overgo probe hybridization
Southern hybridization of HindIII digested BACs
-- PCR amplification from BAC DNA
PCR amplification using primers designed based on the
anchor marker sequence. "Inron_finder" should be used to
avoid the intron problem.
Verify the physical location of seed BACs
-- Fluorescence In-situ Hybridization
2D2
1C4
1N16
Dr. Cheng, China
Korean group
Verify the physical location of seed BACs
T1117
IL2-1
SSR586
IL2-2
TG31
SSR57
-- Map BACs in tomato ILs (CAPS)
cLEC7P21
T1616
T1706
SSR40
SSR66
CT255
SSR356
SSR349A
SSR605
IL2-3
T1665
SSR103
SSR5
SSR96
IL2-4
T697
cLET1I9
CT38
SSR50
SSR331
SSR580
IL2-5
T147
CT9
IL2-6
IL2-6-5
2-1
2-2
2-3
2-4
2-5
2-6
T1201
2-6-5
T1566
3-3
T634
M82
Fw2.
2
cLER17N11
S. pennellii
T1480
T347
TG154
• compare sequences and look for enzyme digestion
polymorphism (or search SGN for known information)
• PCR amplification of DNA from chromosome ILs
and mapping on ILs
T562
SSR26
SSR32
T1494
cLEC7H4
SSR125
• design primers and sequence PCR product from
two parents, S. pennellii and M82
Seed BAC selection
Criteria for selecting a seed BAC :
1) large insert size (>60kb, if possible, or with unknown
insert size)
2) BAC-marker association is reconfirm by sequencing,
overgo hybridization or PCR amplification
3) BAC physical location are tested using FISH or mapping
in IL lines
3) in a valid FPC BAC contig (optional)
Future Data Analysis
• Computational and manual data analysis of
ambiguous results
• More overgo results for COSII markers
• Updating FPC results
• Integration of mapping results from Keygene
• Feedback of BACs and markers from SOL
community