Oral Presentation Science Protecting Plant Health 2017

An internet-based bioinformatics toolkit for Plant Biosecurity diagnosis and surveillance of viruses and viroids (4080)

Roberto A Barrero 1 , Kathryn R Napier 1 , James Cunnington 2 , Lia Liefting 3 , Sandi Keenan 4 , Rebekah A Frampton 4 , Tamas Szabo 1 , Simon Bulman 4 , Adam Hunter 1 , Lisa Ward 3 , Mark Whattam 2 , Matthew I Bellgard 1
  1. Centre for Comparative Genomics, Murdoch University, Murdoch, Western Australia, Australia
  2. Department of Agriculture and Water Resources, Mickleham, Victoria, Australia
  3. Ministry for Primary Industries, Auckland, New Zealand
  4. The New Zealand Institute for Plant Food & Research Limited, Lincoln, New Zealand

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. Here we optimise the assembly of viral sequences by using specific small RNA subsets. 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 set of 15,17 and19 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 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.