Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants

Abstract

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.

Nucleocapsid mutations in SARS-CoV-2 augment replication and pathogenesis

“…Hamsters were then separated and SARS-CoV-2 present in the nasal cavities of the donors sampled by nasal wash. On day 2, each recipient hamster underwent nasal washing to sample transmitted SARS-CoV-2. Donor and recipient hamsters then underwent nasal washing and harvesting of lung tissue on days 4 and 5, respectively, and the ratio of WT to KR mt in all samples was determined by NGS(14). Neither WT nor the KR mt consistently dominated in the donor washes on day 1; similarly, the day 2 nasal washes from the recipients showed no distinct advantage between KR mt and WT for transmission ( Fig 2G and H ).…”

“…The raw Illumina data of the Tiled-ClickSeq libraries were processed with previously established bioinformatics pipelines (14). One modification is the introduction of ten wild cards (“N”) covering the KR and AA mutations in the reference genome to allow bowtie2 (46) to align reads to wild type or variant genomes without bias.…”

ViReMa: A Virus Recombination Mapper of Next-Generation Sequencing data characterizes diverse recombinant viral nucleic acids

“…ViReMa has been independently validated by Alnaji et al (33) and Boussier et al (34) who provided a series of condition and parameter optimizations using simulated and experimental data to map RNA recombination events in DVGs of influenza A and B. ViReMa has been used and validated to study viral recombination in range a of plant viruses (Polygonum ringspot virus (PolRSV) (35), Tomato yellow leaf curl virus (TYLCV) (36), Potato X Virus (37)), insect viruses (Flock House virus (FHV) (38), Cricket Paralysis virus (CrPV) (39)), arboviruses virus (Zika virus (ZIKV) (40), Chikungunya virus (CHIKV) (41, 42)), human pathogens (Influenza virus (43), Reovirus (44), MERS-CoV and SARS-CoV-2 (45, 46)), and retroviruses (Human Immunodeficiency Virus (HIV) (7)).…”

ViReMaShiny: An Interactive Application for Analysis of Viral Recombination Data

“…ViReMa (Viral-Recombination-Mapper) (Routh and Johnson, 2014) is a python package enabling viral read mapping from Next-Generation Sequencing (NGS) data that was developed for this purpose. ViReMa has been used to characterize the full gamut of different recombination events in multiple studies, including sub-genomic mRNA production in coronaviruses (Gribble, et al, 2021), drug-resistance development in HIV samples (Wang, et al, 2022), the evolution of D-RNAs during serial virus passaging of Flock House virus in culture (Jaworski and Routh, 2017), demonstrated differences in D-RNA abundance between intra- and extra-cellular compartments of alphaviruses (Langsjoen, et al, 2020), and compared recombination events from experimental and clinical isolates of SARS-CoV-2 (Jaworski, et al, 2021).…”