Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants
Elizabeth Jaworski,
Rose M Langsjoen,
Brooke Mitchell,
Barbara Judy,
Patrick Newman,
Jessica A Plante,
Kenneth S Plante,
Aaron L Miller,
Yiyang Zhou,
Daniele Swetnam,
Stephanea Sotcheff,
Victoria Morris,
Nehad Saada,
Rafael RG Machado,
Allan McConnell,
Steven G Widen,
Jill Thompson,
Jianli Dong,
Ping Ren,
Rick B Pyles,
Thomas G Ksiazek,
Vineet D Menachery,
Scott C Weaver,
Andrew L Routh
Affiliations
Elizabeth Jaworski
Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States; ClickSeq Technologies LLC, Galveston, United States
Rose M Langsjoen
Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States
Brooke Mitchell
World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, United States; Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, United States
Barbara Judy
Department of Pediatrics, University of Texas Medical Branch, Galveston, United States
Patrick Newman
Department of Pediatrics, University of Texas Medical Branch, Galveston, United States
World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, United States; Department of Pathology, University of Texas Medical Branch, Galveston, United States; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, United States
Kenneth S Plante
World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, United States; Department of Pathology, University of Texas Medical Branch, Galveston, United States; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, United States
Aaron L Miller
Department of Pediatrics, University of Texas Medical Branch, Galveston, United States
Yiyang Zhou
Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States
Daniele Swetnam
Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States
Stephanea Sotcheff
Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States
Victoria Morris
Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States
Nehad Saada
World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, United States; Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, United States
Rafael RG Machado
World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, United States; Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, United States
Allan McConnell
World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, United States; Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, United States
Steven G Widen
Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States; Next-Generation Sequencing Core, The University of Texas Medical Branch, Galveston, United States
Jill Thompson
Next-Generation Sequencing Core, The University of Texas Medical Branch, Galveston, United States
Jianli Dong
Department of Pediatrics, University of Texas Medical Branch, Galveston, United States; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, United States
Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, United States
Rick B Pyles
Department of Pediatrics, University of Texas Medical Branch, Galveston, United States
Thomas G Ksiazek
World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, United States; Department of Pathology, University of Texas Medical Branch, Galveston, United States
Vineet D Menachery
World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, United States; Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, United States; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, United States
Scott C Weaver
World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, United States; Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, United States; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, United States
Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, United States; Sealy Centre for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, United States
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.
