1. A pseudomolecule of 774 Mb: the 3B experience
Frédéric CHOULET
INRA GDEC – Clermont-Ferrand, France

2. Sequenced physical map
3B MTP-BAC sequencing
Sequenced physical map
 #MTP BACs
8452
 #BAC pools
922
 #Roche 8 kb MP lib.
922
 bp coverage (Roche/454)
36x 3B
 BAC-ends (Sanger)
42,551
 Whole Genome Prof. tags
327,282
 Whole 3B shotgun (Illumina)
82x

3. 3B physical map Physical map  #BACs 132,000 (19x)  #BAC-contigs 1282
900 Mb
Physical map
 #BACs
132,000 (19x)
 #BAC-contigs
1282
 #MTP-BACs
8452

4. Assembly and scaffolding
3B-v1
16,136 scaff
1,040 Mb 3B
ACGTAGACTACA

5. Assembly and scaffolding
3B-v1
16,136 scaff
1,040 Mb
Curation of the scaffolding
V. Barbe, S. Mangenot (Genoscope)
18% Ns
3B-v3
4,999 scaff
992 Mb
13% Ns
Integration of BAC-end match positions
Parsing of MP read positions
scaff00001
scaff00013
scaff00024
scaff00008
scaff00011
scaff00007
scaff00005

6. Assembly and scaffolding
3B-v1
16,136 scaff
1,040 Mb
Curation of the scaffolding
V. Barbe, S. Mangenot (Genoscope)
18% Ns
Gap filling
Seq. error corrections
JM. Aury, A. Couloux (Genoscope)
3B-v3
4,999 scaff
992 Mb
13% Ns
3B-v4 -
8% Ns
Illumina reads
Whole 3B Shotgun
109,914 gaps filled
126,290 bases corrected (error rate: 0.1%)

7. Assembly and scaffolding
3B-v1
16,136 scaff
Curation of the scaffolding
V. Barbe, S. Mangenot (Genoscope)
1,040 Mb
Gap filling
Seq. error corrections
JM. Aury, A. Couloux (Genoscope)
3B-v4
4,999 scaff
992 Mb
Redundancy removal and scaffold merging
S. Theil (INRA GDEC)
Pool_A
Pool_B
ctg1
ctg2
2,808 scaff
833 Mb
3B-v443
redundancy:
160 Mb
scaffAssembler.pl

8. Search for shared TE-junctions

9. Ordering scaffolds SNP discovery
3B-v443
2,808 scaff
833 Mb
SNP discovery
SureSelect® seq. capture (E. Paux, N. Cubizolles, E. Rey)
Bait TE
DNA captured from 10 genotypes
gene
52,265 baits
isbpProbeDesign.pl
39,077 SNPs

10. Ordering scaffolds SNP discovery Genotyping mapping pop 3,075 SNPs
Genetic mapping (P. Sourdille)
Anchor map: 384 indiv Cs x Renan
+ Neighbor map: 3865 markers
LD mapping (F. Balfourier)
367 lines from a core-collection

11. Ordering scaffolds 3B genetic map 366 bins LD map 554 bins 0 cM
19 LD blocks
554 bins

12. Linkage Disequilibrium
64 markers at the same genetic position

13. Ordering scaffolds SNP discovery Genotyping mapping pop
Integration of phys. map info
pseudomolBuilder.pl
1358 scaff
774 Mb
pseudomolecule
unlocalized
1450 scaff
59 Mb
93% 7% N

14. orientation unknown: 48% of the seq.3 cM 1 2 4 5 6 ? ? ? A B C D E
orientation unknown: 48% of the seq.
micro-order unknown: 554 bins / 1358 scaff
Future Improvements
RH map
Optical map
Long reads

15. Annotation CLARI-TE TRIANNOT 774 Mb 7264 protein coding genes
234,606 TEs

16. Bioinformatics Assembly Scaffolding/pseudomolecule construction
Newbler
gapCloser
ssrFinishing
Scaffolding/pseudomolecule construction
isbpProbeDesign.pl
scaffAssembler.pl
pseudomolBuilder.pl
Annotation
triAnnot (new modules: filtering, pseudogenes, transfer annotation)
clari-TE & clari-TE-lib
Data management
gowDB (Bio::DB::seqFeatureStore)
URGI

17. Pseudomolecule vs GenomeZipper
3B-CSS
3B-MTP

18. Acknowledgments M. Alaux L. Couderc V. Jamilloux H. Quenesville
Genoscope
M. Alaux
L. Couderc
V. Jamilloux
H. Quenesville
URGI
A. Alberti
V. Barbe
J. Poulain
C. Durand
S. Mangenot
JM. Aury
A. Couloux
P. Wincker
Catherine Feuillet
Sébastien Theil
Natasha Glover
Josquin Daron
CNRGV
IEB
Lise Pingault
H. Berges
A. Bellec
J. Dolezel
J. Safar
Hélène Rimbert
Nelly Cubizolles
Etienne Paux
TGAC
BIA
J. Rogers, M. Caccamo et al.
Pierre Sourdille
C. Gaspin
François Balfourier
VIB
Jacques Le Gouis
K. Vandepoele
Nicolas Guilhot
SAB
MIPS
P. Schnable
S. Rounsley
D. Ware
J. Rogers
K. Eversole
Philippe Leroy
K. Mayer et al.
Aurélien Bernard