Comparing Oxford Nanopore to Illumina sequencing for citrus virus surveillance

ONT sequencing has been previously evaluated for its ability to detect plant viruses and viroids. Its advantages of longer read lengths and real-time analysis contend with extensively validated Illumina platforms for possible incorporation into routine pathogen detection. The continuous development and improvement of ONT sequencing along with the discontinuation of older equipment and reagents, necessitate the renewed comparison of these sequencing platforms specifically in citrus where only limited research is available. This study compared the ability of Oxford Nanopore Technologies (ONT) sequencing, using the MinION flow cell, with Illumina sequencing, on the NovaSeqX platform, to accurately detect three viruses and three viroids in citrus. Both technologies were able to identify all pathogens using both reference-dependent and independent methods. While Illumina sequencing re-established the high sensitivity, coverage and accuracy seen previously, ONT compensated for fewer pathogen reads and lower depth with longer reads that enabled reasonable genome coverage and sequencing identities comparable to that of Illumina. Moreover, the pooling of data from different ONT Barcodes from a single sample, improved comparability to Illumina results as small variations in library preparation, sample loading and flow cells can lead to a significant decrease in sequencing data. Reference gene expression profiles were also investigated to evaluate internal controls and check outlier samples. The ONT platform also had a shorter turnaround compared to Illumina sequencing. The use of ONT sequencing may offer advantages for small-scale routine pathogen detection. It has the potential to accurately detect pathogens and discover novel viral agents. This comparison between Illumina and ONT platforms highlights the strengths associated with each approach and offers new insights into the possible application of high-throughput sequencing (HTS) within plant health surveillance and biosecurity programs.