The SUNRISE on the sunflower genome sequence

Transcription

1 The SUNRISE on the sunflower genome sequence Stéphane Muños Baptiste Mayjonade: molecular biology Jérôme Gouzy: bioinformatics ANR-11-BTBR-0005

2 Toulouse: a unique place for sunflower genetics and genomics A lot of facilities available for phenotyping, physiology, genomics, bioinformatics, microscopy Major sunflower seed companies (Syngenta Seeds, Pionneer, Biogemma, SOLTIS, Maïsadour ) are located arround Toulouse INRA National Seeds Ressources Center Three main topics in our team : water stress tolerance, broomrape resistance, downy mildew resitance. 2 Barcelona, November 10th th EMEA User Group Meeting 2

3 Sunflower (Helianthus annuus) history XXth century: modern breeding XVIIIth century: begin of the sunflower breeding in Russia years before JC: domestication XVIth century: imported by spanish in Europe. Sunflower oil has been successfull because of the orthodox christianism. Sunflower oil was the only one allowed to be consumed during Lent. Source: National Sunflower Association ( Photo NASA Barcelona, November 10th th EMEA User Group Meeting 3

4 Diversity in cultivated sunflower lines Very few diversity in the elite lines due to breeding. Introgression from wild Elite lines Cadic et al., 2013 Barcelona, November 10th th EMEA User Group Meeting 4

5 Sunflower crops should be highly affected by climate changes Oilseed crop cultivated in dry and marginal land High impact of climate change Yield losses: Moriondo et al to 50% in Mediterranean region 0.4q /ha /day of stress France: ha 8 M / day World: ha >100M /day Definition of a new ideotype Combination of phenotypes, genetically realistic and adapted to crop managements Source: Intergovernmental Panel on Climate Change (IPCC) Fourth Assessment Report Moriondo et al., Climatic Change, 2010 FAO 2013 Barcelona, November 10th th EMEA User Group Meeting 5

6 The SUNRISE project million project 7 millions from ANR (french government) 10 partners 6 private partners 8 public labs Coordinated by Nicolas Langlade (INRA, LIPM) Nicolas.Langlade@toulouse.inra.fr Goal: improve the stability of oil yield under water stress Barcelona, November 10th th EMEA User Group Meeting 6

7 Female GWA-hybrid genetic design 36 males x 36 females = 1296 possible hybrids 471 hybrids produced Male Male/ female X X X X X X X X X X X X X X X 15 2 X X X X X X X X X X X X X X X 15 3 X X X X X X X X X X X X X X X 15 4 X X X X X X X X X X X X X X X 15 5 X X X X X X X X X X X X X X X 15 6 X X X X X X X X X X X X X X X 15 7 X X X X X X X X X X X X X X X 15 8 X X X X X X X X X X X X X X X 15 9 X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X 13 Female/ Male Barcelona, November 10th th EMEA User Group Meeting 7

8 Molecular characterization of the 72 parental lines Goal: identification of SNPs in the 72 parental lines to deduce the genotypes in the 471 hyrids for association mapping analysis SNPs have been identified by resequencing experiments and by mapping data on the available sunflower genome sequence The sunflower genome: Diploid with 2n = 17 chromosome pairs 3.6Gb Barcelona, November 10th th EMEA User Group Meeting 8

9 Assembly of the XRQ sunflower line genome obtained by INRA 127X depth of HiSeq sequences used PE and MP sequences (300, 2300, 6700, 18500) NUM MIN 250 MAX N50 BP N50 NUM MEAN 1888 MEDIAN 392 BP ========================= NUM-noN MIN-noN 250 MAX-noN N50 BP-noN 9402 N50 NUM-noN MEAN-noN 1545 MEDIAN-noN 392 BP-noN % of the genome Barcelona, November 10th th EMEA User Group Meeting 9

10 Use of the XRQ assembly to identify SNPs from resequencing experiments 1 lane of HiSeq (2x100nt)/sunflower line Total sequences produced (Q30) : Gb (727 X the sunflower genome size) Mean depth: 10,1X %GC: 38 Combination of bwa; mpileup, varscan: SNPs identified on genomic scaffolds But SNPs are not exhaustive AND no structural variations have been identified Sunflower genome sequence need to be improved! Barcelona, November 10th th EMEA User Group Meeting 10

11 The international consortium for the sunflower genome initiative (HA412 sunflower line) Coordinated by Loren Rieseberg (University of British Columbia, Canada) Obtained by combining 454 and HiSeq data, togethers with an hightroughput genetic map and a physical map (BAC clones finger printing) A good assembly. But difficult to improve (due to the repeated sequences) NUM 21 MIN 9814 MAX N50 BP N50 NUM 8 MEAN MEDIAN BP ========================= NUM-noN 21 MIN-noN 9814 MAX-noN N50 BP-noN N50 NUM-noN 7 MEAN-noN MEDIAN-noN BP-noN % of the genome Barcelona, November 10th th EMEA User Group Meeting 11

12 Number of hits Why is it so difficult to assemble the sunflower genome? Analysis of the composition ot the LTR retrotransposons with LTRharvest (D. Ellinghaus et al. 2008, default parameters) 30% of the sunflower genome sequence is composed of LTR retrotransposons. 8.8% of the human genome. And the repeats are highly conserved in sunflower. Length of LTR retrotransposons (nt) There is a lot of repeated sequences in the sunflower genome. They are large (9-12kb) and highly conserved. For de novo assembly: it is important to have very long reads that fully cross the length of the repeats. Barcelona, November 10th th EMEA User Group Meeting 12

13 Why PacBio sequencing could help to improve the sunflower genome assembly? 1.Jerôme gouzy et al. obtained very good results on other organisms (bacteria and fungi) 2. In map-based cloning projects on sunflower: BAC clones sequencing has been greatly improved thanks to PacBio sequencing (coll. LIPM-CNRGV). We obtained systematically only one contig without any N, even by mixing several overlapping BAC clones. We decided to improve the sunflower genome assembly of the XRQ line by sequencing it at 100X depth with PacBio sequences only. Barcelona, November 10th th EMEA User Group Meeting 13

14 First PacBio sequencing machine has been installed in France by the end of march 2015 It is located in the GeT-PlaGe platform (INRA-Toulouse) Barcelona, November 10th th EMEA User Group Meeting 14

15 PacBio Data obtained for the XRQ sunflower line In 3 months, 407 SMRT cells produced. 407 SMRT Cells with P6/C4 chemistry (GeT-PlaGe; IGM; Lauzanne Univ.) Subreads statisitcs: ) IGM 202 (San SMR Diegao, cells USA) 202 SMR cells NUM MAX N50 NUMBP N50 MAX NUM N90 N50 BP N90 N50 NUM N90 MEAN BP N90 MEDIAN NUM BP/SMRTcell MEAN MEDIAN BP/SMRTce moyenne moyenne ,906 M ,906 M max max ,36Gb ,36Gb Swiss) Lausanne 59 SMRT University cells (Swiss) 59 SMRT cells moyenne moyenne ,15Gb ,15Gb max max ,6GB ,6GB Get-PlaGe 146 SMRT (France) cells 146 SMRT cells moyenne 77086,6301 moyenne 52317, , , , , , , , , , , , , , Mb 9152, Mb max max ,3Gb ,3Gb Improvements of the molecular biology steps have increased the length of the Pacio Sequences (B. Mayjonade) # MAX N50 BP NUM >= N50 MEAN 37,5M 80,9kb 367 Gb (102x) BP Toulouse, 12 et 13 novembre th EMEA User Group Meeting 15

16 Preliminary and quick pre-filtering of the raw data: removal of repeats. Max depth of each repeats (max=3750) Mapping of 1x of data on 2x of long reads (>= 20Kb) Analysis of the coverage of the long reads (only hits > 3kb are analyzed) Repeats pattern identification (MHAP/MinHash) Maxcov = 750 Example of pattern ~9Kb Length of the repeat (nt, max 36kb) Construction of a database containing repeated sequences Toulouse, 12 et 13 novembre th EMEA User Group Meeting 16

17 Removal of repeats from the PacBio raw data (102X) Repeat in database Specfific sequences PacBio subreads: suppressed Kept: # MAX N50 BP NUM >= N50 MEAN BP 32,8M 80,9kb 13,7kb 9,1M 10,3kb 339 Gb (94x) 8% of the raw data sequences are removed for next steps in the assembly! Toulouse, 12 et 13 novembre th EMEA User Group Meeting 17

18 Assembling protocol with 100% PacBio data 2 pipelines are close Reads are first corrected by WGS(CABOG) Main differences are the default parameters used in the different versions of the softwares. HGAP 3 (PacBio=PB) PBcR (Koren et al.) Correction of the reads Alignement PB/BLASR MHAP (Berlin et al.) or PB/BLASR Correction PB/dagcon PBcR (PB/falconcns PB/dag con) Contiging Overlap CA/overlap CA/overlap For any questions, contact Jérôme Gouzy: Jerome.Gouzy@toulouse.inra.fr Layout CA/unitigger CA/unitigger (bogart) Consensus CA/utgcns CA/utgcns (pbutgcns) Correction of the assembly Polishing PB/Quiver PB/Quiver We used PBcR to assemble the sunflower genome Toulouse, 12 et 13 novembre th EMEA User Group Meeting 18

19 CR1 Correction of the data during the first assembly process 2 strategies for filtering of the hsp Reads >= 12kb + MIN_Overlap>=5000nt Max_Overhang<2000nt # MAX N50 BP MEAN BP 11,2M 59kb 13,6kb 11,2kb 125 Gb (34x) CR2 Reads >= 3kb, MIN_Overlap >= 3000nt, LEN_Overlap >= 50% of the short sequence # MAX N50 BP MEAN BP 19,7M 58kb 11,5kb 9kb 180 Gb (50x) CR2 strategy is less stringent but seems to be accurate (evaluated on previously characterized genomic regions from sequenced BAC clones) Toulouse, 12 et 13 novembre th EMEA User Group Meeting 19

20 Sunflower genome assembly evolution according to PacBio sequecing depth Effect of CR2 correction strategy with only 18X depth and 2 days of computation (PBcR 8.3rc1), we obtained an assembly with metrics similar to the previous assembly obtained with 127X of HiSeq data Coverage of the genome and N50 of the contigs increase with the depth of the raw data. Toulouse, 12 et 13 novembre th EMEA User Group Meeting 20

21 Assembly with full data set (102X) Once the raw data (102X) are corrected, the assembly metrics seem very good! #ctg MAX N50 BP # > N50 MEDIAN Gb M 498 kb kb % of the sunflower genome is covered Better assembly than ever! The coverage is twice the one obtained with 127X of HiSeq Data (4 sisings of PE and MP) 3Gb of sequences with non, only contig with almost 500kb for the N50. Toulouse, 12 et 13 novembre th EMEA User Group Meeting 21

22 Does the assembly contain the gene content? We have obtained a reference transcriptome from the same XRQ sunflower line that can be accessed using this web link: efp browser Jérôme Gouzy, Sébastien Carrère, Nicolas Langlade (LIPM, INRA-Toulouse) Barcelona, November 10th th EMEA User Group Meeting 22

23 Does the genome assembly contain the gene content? The gene space is almost complete: 98% of the cdnas from the reference transcriptome can be fully (gmap) Toulouse, 12 et 13 novembre th EMEA User Group Meeting 23

24 What s next to obtain a sequence of the 17 sunflower chromosomes sequences of the nuclear genome? Several hightroughput genetic maps and the physical map (finger printing of the BAC clones) will be used to produce the 17 pseudomolecules of the nuclear genome. We are confident that we should be able to anchor 90% of the PacBio contigs. Chris Grassa (INRA): Chris.Grassa@toulouse.inra.fr This sequence is expected for the end of 2015 But the task is not easy because of the repeats and we already know that a part of the contigs are chimeric. Toulouse, 12 et 13 novembre th EMEA User Group Meeting 24

25 Accuracy of the assembly is our priority We want the sunflower genome sequence to be accurate and reliable. Metrics of an assembly are one thing, accuracy is another! And the quality of the genome sequence is important for genetics and breeding! Toulouse, 12 et 13 novembre th EMEA User Group Meeting 25

26 Conclusions Using PacBio sequences only, we have improved the coverage of the sunflower genome: from 43% (127X HiSeq) to 84% (102X PacBio) and the size of the contigs have been highly increased. The softwares (smrtanalysis from PacBio or PBcR) are «easy» to use and efficient for small or simple genomes. But for complex genomes, it is difficult. It should be easier with more longer sequences (majority of sequences >30kb or 40kb are needed)! Toulouse, 12 et 13 novembre th EMEA User Group Meeting 26

27 Comparison of different softwares Sequencing of a new sunflower line: PSC8 (52x) From corrected reads by PBcR PBcR/WGS # MAX N50 BP MEAN BP 7,5M 59kb 13,6kb 9kb 70,1 Gb (19,6x) #ctg MAX N50 BP # > N50 MEDIAN Gb M 223 kb kb 3.1 FALCON-default parameters: #ctg MAX N50 BP # > N50 MEDIAN Gb M 101 kb kb 2.05 FALCON-control of the repeats at the end of the reads desactivated: #ctg MAX N50 BP # > N50 MEDIAN Gb M 202 kb kb 3.2 Toulouse, 12 et 13 novembre th EMEA User Group Meeting 27

28 Perspectives The genome of Orobanche cumana (19 chromosomes pairs, 1.42Gb) will be sequenced (100X PacBio). Sequencing has begun. Assembling of heterozygous genomes (sunflower wild types for example) Evaluation of other de novo assembling softwares Toulouse, 12 et 13 novembre 2014

29 Many thanks LIPM: Jérôme Gouzy Baptiste Mayjonade Nicolas Langlade Chris Grassa Sébastien Carrere Erika Sallet Ludovic Legrand Marie-Claude Boniface Nicolas Pouilly Get-PlaGe: Cécile Donadieu Gérald Salin Céline Vandecasteele Denis Milan CNRGV: Hélène Bergès William Marande Sonia Vautrin Toulouse, November 12 th & 13 th 2014 SUNRISE 29