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Based on the results of virus survey with multiplex RT-PCR, hybrid wine grapes (Vidal and Baco), juice grapes (Niagara and Concord), and the table grape Coronation were selected for metagenomics analysis with HTS. Total RNAs from either a single sample or combined RNA preps from two or three vines were first subjected to removal of ribosomal RNAs (rRNAs) by using the Ribo-Zero rRNA Removal Kit from Novogene (Sacramento, CA, USA). The composition of samples used for HTS was as follows: Coronation, ON595; Vidal, ON936 and ON1030; Baco, ON562 and ON1193; Niagara, ON544, ON919, and ON1206; and Concord, ON602, ON721, and ON766. These samples were collected from six individual grape growers located in Niagara, Ontario.
The RNA samples after rRNA removal were used as templates to prepare a cDNA library using the Illumina TruSeq RNA Sample Prep Kit. Sequencing was carried out on an Illumina NovaSeq 6000 sequencer generating 150-bp pair-end reads at Novogene. The HTS data sets were first analyzed using CLC Genomics Workbench (Qiagen, Hilden, Germany). For de novo assembly, the raw sequencing reads were filtered to remove adaptor sequences and reads of low quality, and then mapped to the reference genome of V. vinifera (PRJEA18785) to eliminate host sequences. Non-grapevine sequence reads were then de novo assembled into contigs. De novo assembly was done by mapping reads back to contigs with the default parameter setting and a minimum contig length of 250nt. Resulting contigs were subsequently used as queries in a BLAST search against the complete reference sequences of viruses and viroids (http://www.ncbi.nlm.nih.gov/genome/viruses/ (accessed during 2018–2023 for several times)) to identify viruses and viroids that were present in the samples. The default threshold of 10 was used as the EXPECT value. To obtain the complete or partial genome sequences of the viruses and viral variants detected, de novo assembled viral contigs were compared manually with individual viral genome sequences available in GenBank.
The raw sequence data from HTS were deposited in the NCBI SRA database under BioProject accession PRJNA1003946 and SRA accessions SRR25590732-SRR25590735. The complete and near-complete genome sequences of viruses identified through HTS were deposited in GenBank under the following accession numbers: OR478438-OR47843841 for GRSPaV; OR478442-OR478446 for GLRAV-3; OR478447 for GLRaV-2; OR478448-OR478450 for GRBV; OR400565-OR400566 for GVA; and OR400567-OR400568 for GVE.
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