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

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

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

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

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

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%)

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

Search for shared TE-junctions

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

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

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

Linkage Disequilibrium 64 markers at the same genetic position

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

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

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

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) Gbrowse @ URGI

Pseudomolecule vs GenomeZipper 3B-CSS 3B-MTP

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