Download Chromosome 6 update - Sol Genomics Network

Mapping and sequencing chromosome 6
of Solanum lycopersicum cv. Heinz 1706
Sander Peters
April 2008
Acknowledgements
Willem Stiekema
Taco Jesse
René Klein Lankhorst
Applied Bioinformatics
Erwin Datema
Elio Schijlen
Mark Fiers
Marjo van Staveren
Sander Peters
Jan van Haarst
Laboratory of Genetics
Marleen Henkens
Dóra Szinay
Roeland van Ham
Hans de Jong
Bas te Lintel Hekkert
Ludmilla Khrustaleva
Joost de Groot
Song-Bin Chang
Thamara Hesselink
Xiaobo Zhong
Outline
► Strategy
► Sequencing and finishing
► Cytogenetic mapping
► Chr. 6 euchromatin/heterochromatin boundaries
► Sequencing & assembly status
► Chr. 6 map prototype
► Repeat and gene content
► Overall conclusions and prognosis
Seed BAC
Assembly BAC ends
Min. overlapping BACs
Alignment & scaffolding
BLASTN BAC ends
SNaPshot & Genemapper
Genoprofiler
FPC
Largest extending BAC
Fingerprinting & mapping
TOPAAS
Extended consensus
FISH
Seq. analysis
BAC seq. & assembly
FISH analysis
BAC selection and STC
Sequencing and finishing
► ABI3730 (Macrogen)
| 1922 reads (10 x 96 x 2 + 2 BAC ends seqs)
| 2 kb and 6 kb libraries
| Shotgun clones + BAC dna
► Planning 454 BAC sequencing in house
► Finishing by primer walking
| on average 3 sequence gaps per BAC
| Shotgun clones, direct BAC sequencing
► Sequence gap closure
| Overlapping BACs (13.3 kb per extension)
| TOPAAS assisted (read pairs, similarity hits)
► Editting consensus to error rate 1:10000 (Phred 40)
Cytogenetic mapping
► Multi-color BAC FISH
| 5 fluorophores, DAEC (blue), FITC (green), Cy3 (orange), Cy3.5 (red),
Cy5 (far red).
| Pachytene chromosomes show highly condensed DAPI stained
heterochromatin and less condensed euchromatin.
► Cytogenetic position of BACs
| comparison to the linear order of anchored BACs on the genetic
F2.2000 map.
| BAC contig scaffolding.
H016K14
H147H20
0-2cM
H144J05
H301C21
M82G10
M82G10
H002B19
H286N17
H023B17
H250I21
H167M06
H261A18
H0307J13
H024F02
H250I21
H288L09
H304P16
H308F14
H309K01
H040F08
H301C21
H307J13
H215M16
H242H19
H302A23
H020O17
H194N16
H068M22
H242H19
H059K09
10-12 cM
H046G10
H309D09
H091L20
H003K02
H042L06
H302A23
H120H21
H026E06
H309D09
H066A20
H309D09
H002C17
H177K13
H066A20
H042L06
40-47 cM
H020O17
H012O10
H052N09
H012O10
H052N09
H304I22
H036J15
H084A18
H066I09
H055E14
H109C03
H034C13
a
b
c
H055E14
H099H05
H116G14
H084A18
H304I22
H060A01
H060A01
H182D16
H098L02
H060A01
H301C21
H310B09
H117B06
M012J12
M012J12
H261A18
H066I09
H246E15
M012J12
H169D11
H177K13
H194N16
95-101cm
H315H13
H021K07
d
H023B17
H197N20
e
f
g
h
i
j
k
Cytogenetic mapping results
► FISHed 113 candidate BACs
| 84 BACs (74%) map on chr. 6 of which 54 are seeds
| 29 (26%) land on other chromosome
• 21 single focus on other chromosomes
• 7 multiple foci of which 3 BACs also on chr. 6
| 1 without signal
► Overall cytogenetic mapping order agrees with the
linear order of anchored BACs on Tomato EXPEN2000
► However, differences at distal ends, pericentromere and
in 40-50 cM
Sequencing and walking progress
► 66 seed BACs
| 61 Hind, 5 Mbo
| 22 bidirectional, 18 unidirectional, 19 not initiated, 7 have extension overlaps >= 50% insert
► 92 extension BACs identified
| 46 Hind, 17 Eco, 29 Mbo
► Sequencing & assembly
| 45 BACs phase 3, 110 BACs finishing pipeline, 3 sequening not initiated
► Average overlap 13.3 kb (42 extension overlaps)
| 100% match
► 16.7 Mb redundant sequence
| 2.6 Mb short arm (2.4 Mb non-redundant), 14.1 Mb long arm (10.2 Mb non-redundant)
► 67 chr. 6 markers identified (EXPEN2000 / EXPEN1992 / Arabidopsis)
confidence
LOD3
LOD2
Total
No LOD
LOD<=1
library
H
M
E
H
M
E
H
M
E
H
M
E
seed
31
1
0
4
1
0
19
3
0
7
*
0
0
66
extension
7
3
1
2
0
1
4
1
0
33
25
15
92
Chromorome 6 integrated map
► 161 BACs on the physical map
► 28 contigs and 10 singleton BACs
► 3 markers mapped on multiple chromosmes (9, 11 and 12)
► 5 markers mapped other chromosomes (3, 5, 7 and11)
► Map
Gene rich space of chromosome 6
► 2n, 12 chromosomes, 950 Mb
► 75% of DNA in heterochromatin
► 90% non transposon genes in euchromatin of 220Mb
► Chr. 6 20Mb euchromatin
| 2.7 Mb 6SE → 4.1 Mb
| 17.7 Mb 6LE → 26.9 Mb
► Delineate euchromatin domain
| BAC FISH
| sequence composition
Euchromatin and heterochromatin
bordering BACs on chromosome 6
BAC
single focus
H016K14
cM
Marker
S. Telo
S. TeloH016K14
0
H158P14
H54K13
H147H20
H304P16
S.Telo
Cen
H158P14
2
T0686
H147H20
0
CT216
H107A05
3
T1188
H112G05 5.5
Mi
H304P16 10
cLET-2-H1
S. Telo
Cen
H147H20
H176K10
S. Telo
H147H20
H091L20
H112G05
S. Telo
H020O17
H107A05
Cen
CT135*
H060A01
H020O17
H309D09
H310B09
H091L20
H060A01
H060A01 101 Ct_At1g20050
L.Telo
H315H13 98
TG193
H315H13
L. Telo
Pericentromeric and sub-telomeric BACs on chromosome 6
single focus
S. Telo
BAC
cM Marker BLASTN
H016K14 0
TTTAGGG
S.Telo
S.Telo
H242H19
Cen
Cen
H242H19 12 T1063 centr. linked
repeat chr. 1
A. thal.
H295L11 10
T0244 Rev. transcriptase
S. demissum
(AAT40500)
H309K01 10 cLET- CCS3 A.thal.
5-A4 (AF204876)
CCS5 A.thal.
(AF204878)
H003K02 10 TG178 gag/pol P.sativum
(AAQ82037)
RNaseH M. tunc.
(ABD28291)
H040F08 12 CD67 put. polyprotein
Ty3 like O. sativa
(AAL78097)
L. Telo
H003K02
H315H13 98 TG193 rib. Protein S4
S. lycopersicum
(YP_514854)
L.Telo
S.Telo
282A22
H295L11
Cen
091L20
040F08
H309K01
Cen
L.Telo
H032D19
Conclusions
► Efficient and reliable extension BAC selection using STC and HICF
► ‘Map-as-you go’ decreases the amount of fingerprints needed and
produces reliable physical mapping
► Annotated physical map for chr. 6
► Seed BAC bias
| aberrant mapped markers
| chromosome duplications
| Gene poor domains in 6LE
► Established the borders of euchromatic/ heterochromatic domains
► Constructed a framework for finishing chromosome 6
► 12.6 Mb non redundant sequence within 155 BACs
► focus on Ol-1, Ol-3, Ol-5, Ty-1, Ty-2 in CBSG2
► Finishing requires at least 100 BACs, funding for 60 BACs