Toward a better understanding of plant genomes structure: combining NGS and optical mapping technology to improve the sunflower assembly
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1 Toward a better understanding of plant genomes structure: combining NGS and optical mapping technology to improve the sunflower assembly Céline CHANTRY-DARMON 1
2 CNRGV The French Plant Genomic Center Created in 2004 by INRA A dedicated structure to assist plant genomic programs Distribute the genomic resources at the international level Provide high quality research material and efficient tools and services Develop genomic projects in collaboration Host scientists Develop innovative solutions ISO 9001:2008 Octobre
3 Interactions with laboratories around the world More than 3 millions BAC clones distributed during the last 5 years 3
4 Plants project diversity More than 40 species 4
5 The goal for the Plant Genomic Center Large genome size Repeats elements Polyploidy Manage genome size and diversity Decrease genome complexity Target genomic region of interest 5
6 Focus on the optical mapping with BioNano The Bionano Irys system: a new tool to study complex genomes Advantages of BioNano optical mapping: Direct visualization of long DNA molecules (>100 kb) Provides real physical distance information 6
7 The Workflow 50Gb data generated per flowcell (=> 100Gb / chip) 7
8 HMW DNA molecules in nanochanels Free DNA Solution DNA in a Microchannel DNA in a Nanochannel Gaussian Coil Partially Elongated Linearized HMW DNA molecules from 100kb to >2Mb 8
9 The Sunflower : an important crop for Europe 39 Million tons of seed produced worldwide 30 Million hectares worldwide 80% in Europe 71% in Europe Societal challenge The global production of sunflower seeds has to increase to meet growing demand (human food, animal feed, green chemistry...) 9
10 The Sunflower genome Helianthus annuus 3.6 Gb 2n=34 chromosomes 10
11 Occurrence Sunflower genome contains long repeated sequences Length distribution of LTR retrotransposons Sunflower J. Gouzy Human Repeats = 33% of the sunflower genome Repeats = 8% of the Human genome Retrotransposon length (bp) LTRharvest (Ellinghaus et al. 2008, default parameters) Two major repeats in the sunflower genome: 8 kb and 11.5 kb The repeats make the assembling very difficult 11
12 Development of long-fragment libraries The longer the PacBio sequences are, the better it is to cross the LTR : - New DNA extraction protocol - Optimization of fragmentation, purification, loading - Increase running time from 4 to 6h «Filtered_subreads» distribution depending on librairy preparation protocole Enrichment for long fragments (>20kb) B. Mayjonade 12
13 PacBio Genome Assembly N. Langlade XRQ sunflower line Genome sequence >100X PacBio # contigs LEN Max N50 BP #>N50 MEDIAN Gb ,35 Mb 524 kb kb 2,93 80% of the genome inside contigs 13
14 BioNano analyses HMW DNA Extraction of fresh young dark treated leaves 2 nicking enzymes (BspQ1 & BssS1) BspQ1 Bsss1 Theoretical nb labels / 100kb 7,2 17,2 Real nb labels / 100kb 6,4 12,8 Raw data (Gb) 846 (235X) 845 (235X) Filtered data >100kb (Gb) 635 (176X) 600 (167X) Molecules N50 (kb)
15 Example of a BioNano map 176X coverage, molecules from 150kb to 2,3Mb 15
16 Visualization of the Sunflower repeats Very long tandem repeats 2 majors repeats ~10 kb The two major repeats in the sunflower genome: 8 kb and 11.5 kb LTR retrotransposons 16
17 PacBio assembly and BioNano Maps PacBio Assembly Count Median length (Mb) N50 length (Mb) Max length (Mb) 3.35 Total length (Mb) 2930 BioNano BspQ1 Assembly BioNano BssS1 Assembly % genome coverage 81% 88% 86% 17
18 Hybrid Assembly PacBio Assembly A G G T G C T C T T C T A C A G C C A A T C C A C G A G A A G A T G T C G G T T Nickase sites on NGS contigs Hybrid scaffold BioNano Mapping Nickase event Fingerprint 18
19 Sunflower Hybrid Assembly PacBio Assembly BioNano BspQ1 Assembly Hybrid scaffold Count Median length (Mb) N50 length (Mb) Max length (Mb) Total length (Mb) % genome 81% 88% 81% 19
20 Sunflower Hybrid Assembly PacBio Assembly BioNano BspQ1 Assembly Hybrid scaffold Count Median length (Mb) N50 length (Mb) Max length (Mb) More than 5 fold increase Total length (Mb) % genome 81% 88% 81% 20
21 Sunflower Hybrid Assembly PacBio Assembly BioNano BspQ1 Assembly BspQ1 Hybrid Hybrid scaffold scaffold Count Median length (Mb) N50 length (Mb) Max length (Mb) Total length (Mb) % genome 81% 88% 81% Hybrid scaffold + not scaffolded PacBio contigs : 3611Mb 21
22 2 enzymes Hybrid scaffolding 22
23 2 Step Hybrid Assembly PacBio Assembly BioNano BspQ1 Assembly Hybrid scaffold BspQ1 BioNano BssS1 Assembly Hybrid scaffold 2 Step Count Median length (Mb) N50 length (Mb) Max length (Mb) Total length (Mb) % genome 81% 88% 81% 86% 82% 23
24 2 Step Hybrid Assembly PacBio Assembly BioNano BspQ1 Assembly Hybrid scaffold BspQ1 BioNano BssS1 Assembly Hybrid scaffold 2 Step Count Median length (Mb) N50 length (Mb) Max length (Mb) Improvement Total length (Mb) % genome 81% 88% 81% 86% 82% 24
25 2 Step Hybrid Assembly PacBio Assembly BioNano BspQ1 Assembly Hybrid scaffold BspQ1 BioNano BssS1 Assembly Hybrid scaffold 2 Step Count Median length (Mb) N50 length (Mb) Max length (Mb) More than 7 fold increase Total length (Mb) % genome 81% 88% 81% 86% 82% 25
26 Improvement of the sunflower assembly More than 7 fold improvement of the N50 length The 2 step hybrid scaffolding strategy improves significantly the resulting N50 Characterized a region of interest 26
27 Fine mapping of the QRM1 QTL in Sunflower S. Munos S. Vautrin QRM1 controls quantitative resistance to downy mildew Spring 2013: 7455 F2 segregating for QRM1 only. 901 recombinant plants identified. Phenotypic analysis (232 recombinant plants) Susceptible Resistant Strong effect on LG10 Explain 65% of the phenotypic variability 2 Near Isogenic Lines: Susceptible (PSC8) Resistant (XRQ) In silico physical mapping reduction of the genetic mapping to a 0.4 cm window 27
28 Map-based cloning of QRM1 HA412 BAC sequence Sequencing of the 6 HA412 BAC clones by Pacbio AX AX AX cM AX AX AX AX HA008507_93 AX AX AX AX AX AX AX AX AX AX QRM1 BAC 1 BAC 2 BAC 3 BAC 4 Contig (500 kb) A contig of 740kb assembled in one unique sequence AX AX AX AX AX AX BAC 5 Highly accurate sequence AX AX AX AX AX AX LG10 28
29 Map-based cloning of the QRM1 QTL XRQ WHOLE GENOME PACBIO SEQUENCE Resistant genotype XRQ Retrieval of a 2Mb sequence on chromosome 10 (based on 20 markers alignment) This 2 Mb sequences is composed of 14 scaffolded Pacbio contigs separating by N gaps (10k missing nucleotides) Comparison of the HA412 sequence (BAC clones) and the XRQ resistant line (full genome sequence) 29
30 HA412 contig (740 kb) Comparison of the XRQ genome vs HA412 BAC clones 600kb insertion 200kb insertion ctg9 ctg10 ctg11 ctg13 ctg14 XRQ chromosome 10 (2Mb) Low collinearity Fragmented alignment / orientation inconstancies : Tél: 05 Scaffolding / Fax: errors OR true variability? 30
31 XRQ Optical map data allowed to correct XRQ scaffolding Alignment of the contig cmaps against the BioNano assembly of XRQ genome S. Cauet C-map obtained on a unique DNA map ctg10 inv ctg13 ctg14 ctg11 ctg9 inv XRQ contigs On this targeted region, Optical Bionano map allowed: to orientate some contigs to correct scaffolding of the PacBio contigs 31
32 HA412 contig (740 kb) Manually curated genome XRQ sequence vs BAC clones contig HA412 Genetically anchored QTL limit 600kb insertion? Still need to be validated 200kb insertion validated Contig order validated by optical map data Genetically anchored QTL limit XRQ chromosome 10 (2Mb) The scaffoling of PacBio contigs is more accurate (improved collinearity between the two sequences on QRM1 QTL) But still high variability the two sunflower lines : 2 major insertions of several hundreds of kb in XRQ 32
33 Summary for the QRM1 QTL The mapping of the QRM1 QTL controlling downy mildew resistance in sunflower has been restricted to a 2Mb sequence The optical map allowed to validate major rearrangements between the 2 sunflower genotypes Annotation of the 2 sequences and comparative analysis are under progress but 9 candidates genes have been identified 33
34 At the full genome scale The optical map (2 enzymes, >150 X) will improve the sunflower genome sequence (orientation of PacBio contigs and scaffolding) More than 7 fold improvement of the N50 length The 2 step hybrid scaffolding strategy improves significantly the resulting N50 We hope to obtain more improvement with the use of the new 1 step 2 enzymes hybrid scaffolding tool from BioNano Now we must look to the conflicts between the NGS assembly and the BioNano assembly more in details 34
35 Acknowledgements Marie-Claude BONIFACE Félicity VEAR Nicolas POUILLY Patrcik VINCOURT Jérôme GOUZY Nicolas LANGLADE John BAETEN Kees-Jan FRANCOIJS Hélène BERGES Nadège ARNAL Arnaud BELLEC Genséric BEYDON Caroline CALLOT Stéphane CAUET Céline CHANTRY-DARMON Joëlle FOURMENT Nadine GAUTIER Laetitia HOARAU Céline JEZIORSKI William MARANDE Elisa PRAT David PUJOL Nathalie RODDE Roseana RODRIGUES Sonia VAUTRIN 35
de novo sequencing of the sunflower genome
de novo sequencing of the sunflower genome Stéphane Muños LIPM INRA Toulouse stephane.munos@toulouse.inra.fr @stephane_munos @SUNRISE_France 39 Sunflower, an important cropfor Europe Million tons of seed
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