A novel method for multiple water-borne plant virus detection combining Deep sequencing and Bioinformatics.

Transcription

1 A novel method for multiple water-borne plant virus detection combining Deep sequencing and Bioinformatics. WATER CONFERENCE OKLAHOMA CITY LIZBETH PEÑA-ZÚÑIGA 1, Jon Daniels 2, Andrés Espíndola 1, and Francisco M. Ochoa-Corona 1. 1 National Institute for Microbial Forensics & Food and Agricultural Biosecurity, Oklahoma State University, Stillwater, OK, USA. 2 USDA- APHIS, Houston, TX, USA. lizbeth.pena_zuniga@okstate.edu ochoaco@okstate.edu

2 INTRODUCTION Viral diversity & abundance Land Use Microbiota Environmental factors

3 (Mehle & Ravnikar, 2012)

4 VIRAL DETECTION METHODS Enzymatic Immunological Nucleic Acid based Next Generation Sequencing (NGS) - BIOINFORMATICS Electronic Diagnosis Nucleic acid Analysis EDNA Plant Waterborne virus

5 OBJECTIVE To apply & validate Next-Generation Sequencing and Bioinformatics (EDNA-Mi/Fi) as pathogen detection tool for plant waterborne viruses

6 Stable and widespread (remain infectious) Plant viruses GENERA Carmoviruses Cucumoviruses Diathoviruses Tobamoviruses Necroviruses Potexviruses Tombusviruses (Mehle & Ravnikar, 2012) TARGETED PLANT WATERBORNE VIROME TOMBUSVIRUS NC Pelargonium necrotic spot virus complete genome NC Plantago asiatica mosaic virus complete genome NC Tomato brown rugose fruit virus NC Streptocarpus flower break virus NC Cucumber fruit mottle mosaic virus complete genome NC Lily virus X complete genome NC Narcissus mosaic virus complete genome NC Cymbidium ringspot virus complete genome NC Potato aucuba mosaic virus complete genome NC Phaius virus X complete genome NC Hibiscus latent Singapore virus NC Yellow tailflower mild mottle virus isolate Cervantes complete genome NC Tobacco mild green mosaic virus AJ Neckar river virus cp gene for coat protein genomic RNA NC Cucumber necrosis virus complete genome NC Hibiscus latent Fort Pierce virus NC Alstroemeria virus x complete genome NC Cassava common mosaic virus complete genome NC Clover yellow mosaic virus complete genome NC Cucumber green mottle mosaic virus NC Alternanthera mosaic virus complete genome NC Cucumber mottle virus NC Brugmansia mild mottle virus NC Wasabi mottle virus complete genome NC Artichoke mottled crinkle virus complete genome NC Cucumber Bulgarian latent virus complete genome NC Moroccan pepper virus isolate MPV-PM75 complete genome NC Eggplant mottled crinkle virus complete genome NC Grapevine Algerian latent virus complete genome NC Plumeria mosaic virus TOBAMOVIRUS NC Rattail cactus necrosis associated virus NC Pelargonium leaf curl virus isolate T46 clone p complete genome NC Carnation Italian ringspot virus genomic sequence for ORF1-5 NC Clitoria yellow mottle virus AY Petunia asteroid mosaic virus isolate Lim 1 coat protein gene complete cds NC Papaya mosaic virus complete genome AY Havel river tombusvirus strain S coat protein and movement protein NC Tomato mottle mosaic virus AY Limonium flower distortion virus isolate Lim 2 coat protein gene complete cds NC Rehmannia mosaic virus NC Hosta virus X complete genome NC Tomato mosaic virus NC Tamus red mosaic virus isolate IT complete genome NC Bell pepper mottle tobamovirus NC Malva mosaic virus complete genome AY Sikte waterborne virus isolate Lim 6 coat protein gene complete cds NC Cactus mild mottle virus NC Passion fruit mosaic virus NC Youcai mosaic virus complete genome NC Tobacco mosaic virus NC Obuda pepper virus NC Paprika mild mottle virus NC Allium virus X complete genome NC White clover mosaic virus complete genome NC Frangipani mosaic virus NC Pepino mosaic virus complete genome NC Hydrangea ringspot virus complete genome NC Tulip virus X complete genome NC Bamboo mosaic virus complete genome NC Maracuja mosaic virus NC Ribgrass mosaic virus NC Turnip vein-clearing virus complete genome NC Odontoglossum ringspot virus NC Pepper mild mottle virus NC Tropical soda apple mosaic virus isolate Okeechobee complete genome NC Foxtail mosaic virus complete genome NC Strawberry mild yellow edge virus complete genome NC Nerine virus X complete genome NC Yam virus X isolate T551 complete genome NC Opuntia virus X complete genome NC Opuntia virus X NC Cymbidium mosaic virus complete genome NC Mint virus X complete genome NC Lettuce virus X complete genome NC Schlumbergera virus X complete genome NC Cactus virus X complete genome NC Zygocactus virus X complete genome NC Cassava virus X isolate Ven164 NC Potato virus X complete genome AB Lagenaria mild mosaic virus RdRP TGB1 TGB2 TGB3 CP genes partial and complete cds NC Tomato bushy stunt virus complete genome NC Kyuri green mottle mosaic virus NC Zucchini green mottle mosaic virus complete genome NC Asparagus virus 3 complete genome POTEXVIRUS Neighbor-Joining Phylogenomic tree of reported plant water-borne Virome (current research)

7 MATERIALS AND METHODS VIRUS SELECTION BUILDING A DATABASE ELECTRONIC PROBE SELECTION VIRUS DETECTION

8 Mi / Fi FRIENDLY WEB USER INTERFASE Andres Espindola, PhD. E-PROBE DATABASES AVAILABLE

9 RESULTS in silico OF MULTIPLE INFECTION POTEXVIRUS TOBAMOVIRUS TOMBUSVIRUS METAGENOME ID p-value PepMV # hits p-value PMMoV # hits p-value CuNV # hits Multiple E E E Multiple E E E Multiple E E E Multiple E E E Multiple E E E Multiple E E E Multiple E E E Multiple E E E Multiple E E E Multiple E E E Multiple E E E Multiple E E E Multiple E E E Multiple E E E Multiple E E E Multiple E-01 S E-02 S E-01 S 3 Multiple E-02 S E E-01-1 Multiple E-03 S E-01 S E-02 S 5 Negative ~less than 8 e-probes match 30 reads out of 10 million reads LIMIT OF DETECTION p<0.05

10 Water Research 137(2018) EDNA DETECTION WITH PUBLISHED DATA NCBI Sequence Read Archive (SRA) No. SRR SRX : Viral metagenomic sequencing of freshwater samples Illumina HiSeq 2500 run 11/32 Potexvirus detected in fresh water (Singapore) p<0.05

11 Water Research 137(2018) EDNA DETECTION WITH PUBLISHED DATA NCBI Sequence Read Archive (SRA) No. SRR SRX : Viral metagenomic sequencing of freshwater samples Illumina HiSeq 2500 run 10/27 Tobamovirus detected in fresh water (Singapore) p<0.05

12 Water Research 137(2018) EDNA DETECTION WITH PUBLISHED DATA NCBI Sequence Read Archive (SRA) No. SRR SRX : Viral metagenomic sequencing of freshwater samples Illumina HiSeq 2500 run 7/16 Tombusvirus detected in fresh water (Singapore) p<0.05

13 CONCLUSIONS Plant waterborne virus can be detected by EDNA-Water. Results of simulations of single and multiple viral infections take 10 minutes (10 million reads). EDNA-Mi/Fi accurately detect viral infections. The limit of detection of EDNA-Mi/Fi in-silico is 10-3 for single and multiple infections. Processing of metagenomic data by EDNA-Mi/Fi allows semi-quatitative detection of waterborne plant virus.

14 On going research Water sample NGS Sample sequence database in-vitro field Plant bio assay EDNA-Mi/Fi server Hit (Extremely sensitive 1e-3) NEGATIVE POSITIVE (quantification # of hits)

15 Special Thanks Ulrich Melcher, PhD. Beatriz Gallucci Mazziero, MS. VIRUS CHASERS ACKNOWLEDGMENTS Funding Thomas E. Berry Professorship Mi / Fi TM EDNA Plant Waterborne virus