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In 2019, a new disease characterized by yellow mottling on leaves was observed on Metaplexis japonica (Thunb.) Makino plants in Liaoning, China. Using RNA sequencing and PCR with overlapping primers, the complete nucleotide sequence of the circular double-stranded DNA genome of a new virus similar to caulimoviruses in diseased samples was determined. The genome comprised 7733 base pairs and was predicted to encode six proteins. It had the closest relationship to a caulimovirus, strawberry vein banding virus, based on sequence comparisons and phylogenetic analysis. Species-level sequence divergences from caulimoviruses were also observed in the conserved genomic regions. A complete episomal viral genome sequence in symptomatic M. japonica plants was identified, and the name "metaplexis yellow mottle-associated virus" is proposed for this virus. [ABSTRACT FROM AUTHOR]

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called 'Tiled-ClickSeq', which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay. [ABSTRACT FROM AUTHOR]