title: An internet-based bioinformatics toolkit for plant biosecurity diagnosis and surveillance of viruses and viroids.
abstract: BACKGROUND
  Detection and preventing entry of exotic viruses and viroids at the border is critical for protecting plant industries trade worldwide. Existing post entry quarantine screening protocols rely on time-consuming biological indicators and/or molecular assays that require knowledge of infecting viral pathogens. Plants have developed the ability to recognise and respond to viral infections through Dicer-like enzymes that cleave viral sequences into specific small RNA products. Many studies reported the use of a broad range of small RNAs encompassing the product sizes of several Dicer enzymes involved in distinct biological pathways. Here we optimise the assembly of viral sequences by using specific small RNA subsets.
  
  
  RESULTS
  We sequenced the small RNA fractions of 21 plants held at quarantine glasshouse facilities in Australia and New Zealand. Benchmarking of several de novo assembler tools yielded SPAdes using a kmer of 19 to produce the best assembly outcomes. We also found that de novo assembly using 21-25 nt small RNAs can result in chimeric assemblies of viral sequences and plant host sequences. Such non-specific assemblies can be resolved by using 21-22 nt or 24 nt small RNAs subsets. Among the 21 selected samples, we identified contigs with sequence similarity to 18 viruses and 3 viroids in 13 samples. Most of the viruses were assembled using only 21-22 nt long virus-derived siRNAs (viRNAs), except for one Citrus endogenous pararetrovirus that was more efficiently assembled using 24 nt long viRNAs. All three viroids found in this study were fully assembled using either 21-22 nt or 24 nt viRNAs. Optimised analysis workflows were customised within the Yabi web-based analytical environment. We present a fully automated viral surveillance and diagnosis web-based bioinformatics toolkit that provides a flexible, user-friendly, robust and scalable interface for the discovery and diagnosis of viral pathogens.
  
  
  CONCLUSIONS
  We have implemented an automated viral surveillance and diagnosis (VSD) bioinformatics toolkit that produces improved viruses and viroid sequence assemblies. The VSD toolkit provides several optimised and reusable workflows applicable to distinct viral pathogens. We envisage that this resource will facilitate the surveillance and diagnosis viral pathogens in plants, insects and invertebrates.
authors.affiliation: Centre for Comparative Genomics, Murdoch University, Murdoch, WA, 6150, Australia. rbarrero@ccg.murdoch.edu.au.
authors.forename: Roberto A
authors.initials: RA
authors.lastname: Barrero
authors.affiliation: Centre for Comparative Genomics, Murdoch University, Murdoch, WA, 6150, Australia.|Plant Biosecurity Cooperative Research Centre, Canberra, ACT, 2617, Australia.
authors.forename: Kathryn R
authors.initials: KR
authors.lastname: Napier
authors.affiliation: Department of Agriculture and Water Resources, Mickleham, VIC, 3064, Australia.
authors.forename: James
authors.initials: J
authors.lastname: Cunnington
authors.affiliation: Ministry for Primary Industries, Wellington, New Zealand.
authors.forename: Lia
authors.initials: L
authors.lastname: Liefting
authors.affiliation: The New Zealand Institute for Plant Food and Research Limited, Better Border Biosecurity, Lincoln, 7608, New Zealand.
authors.forename: Sandi
authors.initials: S
authors.lastname: Keenan
authors.affiliation: The New Zealand Institute for Plant Food and Research Limited, Better Border Biosecurity, Lincoln, 7608, New Zealand.
authors.forename: Rebekah A
authors.initials: RA
authors.lastname: Frampton
authors.affiliation: Centre for Comparative Genomics, Murdoch University, Murdoch, WA, 6150, Australia.
authors.forename: Tamas
authors.initials: T
authors.lastname: Szabo
authors.affiliation: The New Zealand Institute for Plant Food and Research Limited, Better Border Biosecurity, Lincoln, 7608, New Zealand.
authors.forename: Simon
authors.initials: S
authors.lastname: Bulman
authors.affiliation: Centre for Comparative Genomics, Murdoch University, Murdoch, WA, 6150, Australia.
authors.forename: Adam
authors.initials: A
authors.lastname: Hunter
authors.affiliation: Ministry for Primary Industries, Wellington, New Zealand.
authors.forename: Lisa
authors.initials: L
authors.lastname: Ward
authors.affiliation: Department of Agriculture and Water Resources, Mickleham, VIC, 3064, Australia.
authors.forename: Mark
authors.initials: M
authors.lastname: Whattam
authors.affiliation: Centre for Comparative Genomics, Murdoch University, Murdoch, WA, 6150, Australia. mbellgard@ccg.murdoch.edu.au.
authors.forename: Matthew I
authors.initials: MI
authors.lastname: Bellgard
mesh_terms: D001315:Australia; D019295:Computational Biology; D020407:Internet; D009520:New Zealand; D010935:Plant Diseases / Q000235:genetics / Q000821:virology*; D010942:Plant Viruses / Q000235:genetics*; D018749:RNA, Plant / Q000032:analysis*; D034741:RNA, Small Interfering / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis*; D014772:Viroids / Q000235:genetics*
chemical_list: D018749:RNA, Plant; D034741:RNA, Small Interfering; D012367:RNA, Viral
keywords: Bioinformatics; Next generation sequencing; Plant biosecurity; Plant viruses and viroids; Quarantine; Small RNA-Seq; Virus diagnosis; Workflows; Yabi; viRNAs