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.
【저자키워드】 viruses, SARS-CoV-2, Genomics, Next-generation sequencing, nanopore sequencing, ClickSeq, Defective RNAs, 【초록키워드】 Evolution, Variation, Sequencing, NGS, Genome, Transmission, virus, variants, RNA, nucleic acid, sensitivity, specificity, pathogenicity, Illumina, SNV, Critical, platform, Structural variant, RNA recombination, Amplicon, cDNA, sequence, Final, primer, PCR reaction, genome coverage, SNVs, while, driving, virus genome, sgmRNAs, Affect, Complete, limitations, approach, sub-genomic mRNA, event, junction, primer pairs, cDNA synthesis, robust, clinical sample, identify, approach, generate, provide, SARS-CoV-2 isolate, single-nucleotide, SVs, Tiled-ClickSeq, UMI, Unique Molecular Identifier, 【제목키워드】 Sequencing, variant, RNA recombination, Complete, coronavirus genome, Tiled-ClickSeq,