All three viroids found in this study were fully assembled using either 21вЂ“22В nt or 24В nt viRNAs.

Most of the viruses were assembled using only 21вЂ“22В nt long virus-derived siRNAs (viRNAs), except for one Citrus endogenous pararetrovirus that was more efficiently assembled using 24В nt long viRNAs. Among the 21 selected samples, we identified contigs with sequence similarity to 18 viruses and 3 viroids in 13 samples. Such non-specific assemblies can be resolved by using 21вЂ“22В nt or 24В nt small RNAs subsets. We also found that de novo assembly using 21вЂ“25В nt small RNAs can result in chimeric assemblies of viral sequences and plant host sequences. Benchmarking of several de novo assembler tools yielded SPAdes using a kmer of 19 to produce the best assembly outcomes. We sequenced the small RNA fractions of 21 plants held at quarantine glasshouse facilities in Australia and New Zealand. Here we optimise the assembly of viral sequences by using specific small RNA subsets. 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. 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.

Existing post entry quarantine screening protocols rely on time-consuming biological indicators and/or molecular assays that require knowledge of infecting viral pathogens. Detection and preventing entry of exotic viruses and viroids at the border is critical for protecting plant industries trade worldwide.

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