Saha UC Davis Plant Pathology seminar Infrastructure for battling the Citrus Greening (HLB) disease: High quality genomes and an open access integrated systems biology portal
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Rapidly spreading invasive diseases in systems with little or no prior experimental data or resources pose a unique set of challenges for growers, scientists as well as regulators. As a part of a USDA NIFA CAPS project focused on the psyllid, Diaphorina citri, we have released improved genomics resources including high quality genome assemblies and annotation. We have also created an open access web portal for analyses around the Citrus Greening/Huanglongbing disease complex. Citrusgreening.org includes pathosystem-wide resources and bioinformatics tools for multiple Citrus spp. hosts, the Asian citrus psyllid vector (ACP, Diaphorina citri), and multiple pathogens including Candidatus Liberibacter asiaticus (CLas). To the best of our knowledge, this is the first example of a database to use the pathosystem as a holistic framework to understand an insect transmitted plant disease. Users can submit relevant data sets to enable sharing and allow the community to leverage their data within an integrated system. The system includes the metabolic pathway databases CitrusCyc and DiaphorinaCyc with organism specific pathways that can be used to mine metabolomics, transcriptomics and proteomics results to identify pathways and regulatory mechanisms involved in disease response. The Psyllid Expression Network (PEN) contains expression profiles of ACP genes from multiple life stages, tissues, conditions and hosts. The Citrus Expression Network (CEN) contains public expression data from multiple tissues and conditions for various citrus hosts. All tools connect to a central database. The portal also includes electrical penetration graph (EPG) recordings, information about citrus rootstock trials and metabolomics data in addition to traditional omics data types with a goal of combining and mining all information related to the Huanglongbing pathosystem. User-friendly manual curation tools will allow the continuous improvement of knowledge base as more experimental research is published. The portal can be accessed at https://citrusgreening.org/.
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Saha UC Davis Plant Pathology seminar Infrastructure for battling the Citrus Greening (HLB) disease: High quality genomes and an open access integrated systems biology portal
- 1. www.citrusgreening.org Infrastructure for battling the Citrusgreening disease: High quality genomes and an integrated systems biology portal Surya Saha Boyce Thompson Institute, Ithaca, New York, USA ss2489@cornell.edu | @SahaSurya Feb 3rd, 2020
- 2. www.citrusgreening.org Mueller Lab (Boyce Thompson Institute) Mirella Flores Prashant Hosmani Stephanie Hoyt Main Lab (Washington State University) Jodi Humann Acknowledgements
- 3. www.citrusgreening.org Roadmap • Citrusgreening (HLB) disease • Psyllid genome assembly and annotation • Undergraduate driven manual gene curation • Metabolic pathway databases • Expression atlases • AgriVectors digital ecosystem
- 4. www.citrusgreening.org Citrus Greening: Huanglongbing • Most significant disease of citrus worldwide. 100% infection in Florida now • More than $5 billion in lost citrus production and more than 10,000 lost jobs • Associated with gram negative bacterium Candidatus Liberibacter asiaticus (CLas) • Spread by insect vector, Diaphorina citri (Asian citrus psyllid, ACP) Heck Lab September 2017, UC Riverside Extension
- 6. www.citrusgreening.org Mueller Lab @ Boyce Thompson Institute Open source toolkit
- 7. www.citrusgreening.org Vector Host Pathogen Metabolic pathway databases Expression Atlas with RNAseq, proteomics and metabolomics interactome networks Systems biology data portal for host, vector and pathogen interactions Genome assembly and annotation of protein coding and non-coding genes Dissecting the symbiosis with beneficial and pathogenic partners in invasive disease systems Identification of genetic and epigenetic factors influencing gene regulation in interaction networks Understanding evolutionary factors controlling transposon expansion and contraction in disease systems Identification of resistance and susceptibility in the population using genome wide association methods Analyze the role of plant, environmental and arthropod microbiomes in disease transmission and resistance
- 9. www.citrusgreening.org Psyllid genomics timeline2014 • Psyllid v1.1 genome 2015 2016 • MCOT de novo transcriptome • Psyllid annotation OGSv1.0 • Psyllid PacBio genome v1.9 2017 2019 • Psyllid annotation OGSv3.0 • IsoSeq de novo transcriptome 2018 • Psyllid PacBio genome 2.0 • Psyllid annotation OGSv2.0 • Carsonella and Profftella genomes from FL • Psyllid PacBio genome v3.0 • Wolbachia strains from FL Manual annotation
- 10. www.citrusgreening.org Genome Diaci1.1 Contigs 161,988 Total Length 485 Mb Longest 1 Mb Shortest 201bp Ns 19.3 Mb Diaci v1.1 Illumina assembly Scaffold N50: 109,898 bp Contig N50: 34,407bp Highly fragmented Many examples of misassemblies!! http://biobeans.blogspot.com/2012/11/bioinformatics-genome-assembly.html
- 11. 500ng input DNA from single male psyllid Duplicated contigs added to alternate assembly Error correction • DNA sequencing data • RNA sequencing data • Duplication removal • Scaffolding Chicago scaffolding www.citrusgreening.org v1.1 v2.0 REFERENCE v3.0 REFERENCE Number of contigs 161,988 1,906 13 + unplaced Total bases 485 Mb 498 Mb 474 Mb Longest 1 Mb 4.2 Mb 50.3 Mb Contig N50 34.4 Kb 749 Kb 40.5Mb Ns 19.3 Mb 4.5 Mb 13.4Mb Complete BUSCO (%) 65.9 75.9 88.3 Repeat (%) 26.37 31.9 30.2
- 13. www.citrusgreening.org Kmer analysis Pacbio error corrected 40x data Combined (ACP v3 beta) 677 Mb 13 chromosomes + 207 scaffolds 445 Mb
- 14. www.citrusgreening.org First endosymbiont genomes from Psyllid in FL Wolbachia Profftella Carsonella 10 scaffolds 1 chromosome and 1 plasmid 1 chromosome Largest 923 Kb 471 Kb - Smallest 19 Kb 4.7 Kb - Total Size 2 Mb 475.7 Kb 150 Kb Stephanie Hoyt Mueller lab Wolbachia Profftella Carsonella Number of reference genomes 8 2 9 Total number of conserved orthogroups 559 307 116 Number of conserved orthogroups in our assembly 557 307 106 Number of shared orthogroups (<50% genomes) 167 - 12 Orthology Analysis
- 15. www.citrusgreening.org Wolbachia Strains Scaffolds were removed from the Wolbachia assembly resulting in a large decrease in duplication, but a small decrease in conserved orthogroup coverage Based on these results we hypothesize that there are two strains of Wolbachia present in this sample: • Strain 1: Scaffolds 1 and 2 cover 534/559 conserved orthogroups • Strain 2: Scaffolds 1 and 3 cover 503/559 conserved orthogroups Comparing genomic sequences of our Wolbachia strain 2 and reference genomes to our Wolbachia strain 1
- 16. www.citrusgreening.org https://www.biorxiv.org/content/10.1101/869685v1 17 students among 30 authors
- 17. www.citrusgreening.org Psyllid genome annotation and manual curation using Apollo Prashant Hosmani Mueller Lab
- 18. Gene prediction overview for OGS v3.0 • RepeatModeler • Protein masking • RepeatMasker Repeat Masking • RNA-seq HISAT & StringTie • Iso-Seq - GMAP & Cupcake ToFU Transcriptome • Portcullis junctions • StringTie • Iso-Seq Mikado • Mikado Gene Loci • Portcullis junctions Maker • AHRD • Interproscan Functional annotation Augustus GeneMark www.citrusgreening.org
- 19. www.citrusgreening.org Build a collaboratory ecosystem • Build an ecosystem of resources and integrated toolkit • Identify curation targets according to project goals • Collaboration between scientists and students Train undergraduate annotators and formalize curation practices • Recruiting annotators – Early career researchers • Build teams according to expertise and annotation targets • Establish the protocols for curation Manual Curation Workflow
- 20. Diaphorina citri Apollo annotation editor Request access by contacting https://citrusgreening.org/contact/form www.citrusgreening.org
- 21. Pathway based manual curation • Development • Segmentation • Wnt and other signaling pathways • Hox genes • Immune response • Metabolic and cellular functions • Carbohydrate metabolism • Chitin metabolism • vATPase • Chromatin remodeling • Environmental/Sensory • Circadian rhythm • Phototransduction • Reproduction • ~1000 curated genes in OGSv3 • ~200 updated models from OGSv1 (Diaci v1.1) www.citrusgreening.org
- 22. www.citrusgreening.org Cumulative Annotation Outcomes Group Annotation 2016-2017 2017-2018 2018-2019 14 total student annotators 17 total student annotators 17 total student annotators 18 total students 20 total students 30 total students involved >250 gene models >250 gene models >300 gene models in pathways >400 gene models in v3 >30 gene families >10 pathways >15 pathways 13 gene reports for publication 10 pathway reports 7 pathway reports
- 23. High-quality manually curated genes Annotation set OGS1.0 OGS2.0 OGS3.0 Curated No. of genes 19,311 20,793 19,049 811 No. of transcripts 20,966 25,292 21,345 916 No. of Exons Per transcript 5.42 7.06 7.29 7.87 Avg. transcript length (bp) 1,317 1,944 2,034 2,503 Avg. exon length (bp) 243 275 279 318 non-canonical splice sites 6.05% 3.13% 2.47% 1.91% OGS: Official Gene Set www.citrusgreening.org
- 24. www.citrusgreening.org Pacbio Isoseq transcriptome Mirella Flores Mueller Lab
- 25. De novo transcriptome input datasets RNA-Seq • Gut Clas+ and Clas- (Heck lab) • Male, female (Slupsky lab) • Salivary glands (Heck Lab) • Antenna and terminal abdomen (SRA) Iso-Seq • Adult Clas + and CLas- • Nymph Clas + and CLas- www.citrusgreening.org
- 26. Workflow Total transcripts: 60,261 Iso-Seq transcripts and RNA-seq transcripts clustering Remove contamination (endosymbionts, archaea , virus, bacteria) RNA-Seq De novo transcriptome assembly Genome based transcripts filtering Pfam domains coding transcripts filtering PacBio Iso-Seq pipeline Illumina data correction Remove contamination Filtering by insecta trembl set 2,197,769 196,000 DcDTr (RNA-Seq transcripts): 41,457 DcDTi (Iso-Seq transcripts): 18,804 www.citrusgreening.org
- 27. De novo transcriptome statistics Genes 40,637 Transcripts 60,261 Average length 1,736.1 Smallest 108 Largest 35,954 N50 3,657bp Complete 79.9% Single-copy 53.2% Duplicated 26.7 Fragmented 0.1% Missing 20% BUSCO Hemiptera dataset Number of BUSCOs: 3350 www.citrusgreening.org
- 28. www.citrusgreening.org Got genome and annotation. Now what? Citrusgreening.org portal
- 29. www.citrusgreening.org Citrusgreening.org digital ecosystem Portal home page Metabolic pathway database Virus-induced gene silencing (VIGS) tool BLAST database Jbrowse and Apollo Expression Network with RNA-seq and proteomics data
- 30. www.citrusgreening.org Citrusgreening.org Timeline 2015 • DiaporinaCyc pathway database v1.1 • CitrusgreeningCyc pathogen pathway databases • Jbrowse and BLAST • Psyllid Expression Network v1.12016 • Gene pages • CitrusCyc at CitrusGenomeDB • Apollo for curation of psyllid genes 2017 2019 • Psyllid annotation OGSv3.0 • Psyllid Expression Network v3 2018 • Citrus Expression Network • Psyllid Expression Network v2 • Citrus Virus Induced Gene Silencing (VIGS) tool
- 31. www.citrusgreening.org Pathway/Genome Database Construction Annotated Genome e.g. C sinensis PathoLogic Software Reference Pathway Database (MetaCyc) Reactions Pathways Compounds Gene products Genes Pathway/Genome Database (CitrusCyc) Source: Peter Karp (SRI) • Predicts metabolic pathways • Predicts which genes code for missing enzymes in metabolic pathways • Infers transport reactions from transporter names Mirella Flores Mueller Lab
- 32. www.citrusgreening.org C sinensis Cyc v3.0 http://pathways.citrusgenomedb.org/organism-summary?object=CSINENSIS_ML Photosynthesis pathway
- 33. Citrus Expression Network (CEN) Citrus sinensis RNA seq data from NCBI Sequence Read Archive (SRA) corresponding to 6 experiments Tissues Fruit peel Fruit flesh Calyx Leaves Treatments Citric acid Pomegranate Peel extract Stages Immature fruit Mature fruit Young leaf Mature leaf Infection states CLas+ CLas- CTV-B2 CTV-B6 Accessions Bingtang Newhall Succari Xinhui Blood orange Navel orange www.citrusgreening.org Citrus clementina Expression Network is also available
- 34. Colorscale www.citrusgreening.org Citrus Expression Network (CEN) Cube Your data here!! Genescorrelatedwith querygene Mirella Flores Mueller Lab
- 35. Citrus Expression Network (CEN) Tissues: • Fruit flesh • Fruit peel • Calyx • Leaf www.citrusgreening.org
- 36. Citrus virus-induced gene silencing (VIGS) tool www.citrusgreening.org Citrus clementina coming soon!!
- 37. Tissues Gut Abdomen Antennae Whole body Terminal abdomen Leg Thorax Head Midgut Sexes Male Female Stages Egg Nymph Adult Infection states CLas- CLas+ CLas+ Low infection CLas+ High Infection Host C. sinensis C. medica C. reticulata C. macrophylla Psyllid Expression Network (PEN) Various tissue and conditions for ACP have been added. www.citrusgreening.org
- 38. www.citrusgreening.org DiaphorinaCyc: Overlay of RNAseq Results Cellular Overview of Diaphorina citri Based on NCBI v100 Membrane proteins Secretory proteins Membrane proteins Kruse et al. 2017
- 39. www.citrusgreening.org Pathways by class Genes by GO term DiaphorinaCyc: Pathway comparison
- 40. www.citrusgreening.org DiaphorinaCyc: Smart Tables Load and save gene lists and data sets
- 42. www.citrusgreening.org Got assembly and database. Now what? Comparative genomics within an open access Digital Ecosystem AgriVectors.org
- 43. www.citrusgreening.org Power of comparative genomics Species Common name Genome size Lead Cacopsylla pyricola Pear psylla 480-485Mb Rodney Cooper Leuronota fagarae Lime psyllid 465-483Mb Jawwad Qureshi, Liliana Cano Bactericera cockerelli Potato psyllid 421-426Mb Daisy Fu Pachypyslla venusta Hackberry petiole gall psyllid TBD Nancy Moran Bactericera maculipennis Bindweed psyllid 442-451Mb Rodney Cooper Circulifer tenellus Beet leafhopper ~1Gb Bob Gilbertson, Bill Winter Lygus lineolaris Tarnished plant bug TBD OP Perera Geocoris pallens Western big-eyed bug ~1Gb Rosenheim lab Macrosteles quadrilineatus Aster leafhopper TBD Astri Wayadande Graminella nigrifrons Black-faced leafhopper TBD Astri Wayadande Dalbulus maidis Maize leafhopper TBD Astri Wayadande AgriVectors.org Ag100Pest
- 44. www.citrusgreening.org Portal for all Agricultural Diseases and Vector Systems Citrusgreening Zebra chip Pierce’s disease Pathogens: Bacteria, virus and fungi AgriVectors Home Page AgriVectors.org
- 45. www.citrusgreening.org AgriVectors Knowledge Base Data Producers Data Consumers Public Repository (remote) Pathosystem Repository (local and remote) Topic Repository (local and remote) Researchers Extension agents Industry INRA USDA ARS / APHIS CRISPR / RNAi genes Bacterial effectors Microbiome Geospatial disease data Your Pathosystem Zebrachip Citrusgreening Pierce’s disease Researchers INRA / USDA ARS / APHIS IPM product development Outreach and extension Educators Secure portal Patents Commercial NCBI / EMBL / DDBJ Ag Data Commons i5k AgriVectors Data Schema AgriVectors.org
- 46. www.citrusgreening.org AgriVectors Data types Integrated pest management pathosystem-wide data • Inclusive of Vector, pathogen, host, environment and beyond • Gene family based data sets (P450, RNAi pathway) • E.g. Virus, Bacteria, or fungal infection assays • Electrical Penetration Graph (EPG) feeding data • Phenotyping data from disease trials • Ecological and climactic data • Behavioral assays • Toxicology, Insecticide resistance, etc. Publications, notes, posters, videos and extension abstracts………… AgriVectors.org
- 50. www.citrusgreening.org AgriVectors Knowledge Base Data Producers Data Consumers Public Repository (remote) Pathosystem Repository (local and remote) Topic Repository (local and remote) Researchers Extension agents Industry INRA USDA ARS / APHIS CRISPR / RNAi genes Bacterial effectors Microbiome Geospatial disease data Your Pathosystem Zebrachip Citrusgreening Pierce’s disease Researchers INRA / USDA ARS / APHIS IPM product development Outreach and extension Educators Secure portal Patents Commercial NCBI / EMBL / DDBJ Ag Data Commons i5k AgriVectors Data Schema Questions?? @Citrusgreening @SahaSurya