Updates on the ACP v3 genome and annotation from USDA NIFA project meeting
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ACP version 3 genome, official gene set version 3 and Isoseq transcriptome Prashant Hosmani, Mirella Flores-Gonzalez, Lukas Mueller, Surya Saha 5th Annual Meeting Indian River State College Fort Pierce, FL
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Updates on the ACP v3 genome and annotation from USDA NIFA project meeting
- 1. www.citrusgreening.org Objective 1 Data Integration and Analysis Genome, annotation and transcriptome Fifth Annual Meeting Ft. Pierce, FL Prashant Hosmani, Mirella Flores, Lukas Mueller and Surya Saha Boyce Thompson Institute
- 3. 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
- 4. www.citrusgreening.org https://www.biorxiv.org/content/10.1101/869685v1 17 students among 30 authors
- 6. 500ng input DNA for Dovetail Chicago from single male psyllid Duplicated contigs added to alternate assembly Asian citrus psyllid(ACP) reference genome 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 www.citrusgreening.org Chicago and Hi-C
- 7. Microbial contamination on Chr09 • Removing first 2.3Mb • 4-5000X depth of coverage • Coverage of >90% of endosymbiont genome Carsonella Chr09
- 8. www.citrusgreening.org Got assembly. Now what? Comparative genomics AgriVectors.org
- 9. 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 Bactericera cockerelli Potato psyllid 421-426Mb Daisy Fu Pachypyslla venusta Hackberry petiole gall psyllid TBD Nancy Moran Lygus lineolaris Tarnished plant bug TBD OP Perera Geocoris pallens Western big-eyed bug ~1Gb Nick Booster (Rosenheim lab) Circulifer tenellus Beet leafhopper ~1Gb Bob Gilbertson Macrosteles quadrilineatus Aster leafhopper TBD Astri Wayadande Graminella nigrifrons Black-faced leafhopper TBD Astri Wayadande Dalbulus maidis Maize leafhopper TBD Astri Wayadande
- 10. www.citrusgreening.org Psyllid genome annotation and manual curation using Apollo Prashant Hosmani Mueller Lab
- 11. 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
- 12. Diaphorina citri Apollo annotation editor Collaboratory system ● Indian River State College (IRSC) ● Kansas State University (KSU) ● University of Cincinnati (UC) ● BTI / Cornell University More than 40 registered annotators Login to Apollo at https://citrusgreening.org/
- 13. 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 • 811 curated genes in OGSv3 • 132 updated models from OGSv1 (genome v1.1)
- 14. 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
- 15. Completeness using BUSCO single copy markers BUSCO Hemiptera complete Duplicated Fragmented Missing OGS1 74.5 13.0 0.3 25.2 OGS2 81.6 37.3 0.2 18.2 OGS3 80.2 29.4 0.1 19.7
- 16. www.citrusgreening.org Pacbio Isoseq transcriptome Mirella Flores Mueller Lab
- 17. De novo transcriptome input datasets RNA-Seq • Gut Clas+ and Clas- (Heck lab) • Male, female, antenna and terminal abdomen (Slupsky lab) • Salivary glands (Heck Lab) Iso-Seq • Adult Clas +/CLas- • Nymph Clas +/-
- 18. Workflow Total transcripts: 60,261 Iso-Seq transcripts and RNA-seq transcripts clustering Remove contamination (endosymbionts, archaea , viral, 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 196k DcDTr (RNA-Seq transcripts): 41,457 DcDTi (Iso-Seq transcripts): 18,804
- 19. 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
- 21. Genome and annotation paper 2019-2020 • New v3.0 assembly • First hemipteran chromosomal length genome assembly • Curated genes from previous v1.1 and v2.0 assemblies • Meta-paper with 11-15 sub-papers led by students for each pathway • Isoseq transcriptome www.citrusgreening.org