Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants
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 nucle...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2021-09-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/68479 |
| _version_ | 1828167858274697216 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Elizabeth Jaworski |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T02:24:16Z |
| format | Article |
| id | doaj.art-4d7ebe7fd8ba48a3ba01181583b2b50c |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T02:24:16Z |
| publishDate | 2021-09-01 |
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| spelling | doaj.art-4d7ebe7fd8ba48a3ba01181583b2b50c2022-12-22T03:52:02ZengeLife Sciences Publications LtdeLife2050-084X2021-09-011010.7554/eLife.68479Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variantsElizabeth Jaworski0Rose M Langsjoen1Brooke Mitchell2Barbara Judy3Patrick Newman4Jessica A Plante5https://orcid.org/0000-0002-4768-7458Kenneth S Plante6Aaron L Miller7Yiyang Zhou8Daniele Swetnam9Stephanea Sotcheff10Victoria Morris11Nehad Saada12Rafael RG Machado13Allan McConnell14Steven G Widen15Jill Thompson16Jianli Dong17Ping Ren18https://orcid.org/0000-0002-4022-6667Rick B Pyles19Thomas G Ksiazek20Vineet D Menachery21Scott C Weaver22Andrew L Routh23https://orcid.org/0000-0002-2874-5990Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States; ClickSeq Technologies LLC, Galveston, United StatesDepartment of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United StatesWorld 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 StatesDepartment of Pediatrics, University of Texas Medical Branch, Galveston, United StatesDepartment of Pediatrics, University of Texas Medical Branch, Galveston, United StatesWorld 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 StatesWorld 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 StatesDepartment of Pediatrics, University of Texas Medical Branch, Galveston, United StatesDepartment of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United StatesDepartment of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United StatesDepartment of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United StatesDepartment of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United StatesWorld 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 StatesWorld 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 StatesWorld 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 StatesDepartment 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 StatesNext-Generation Sequencing Core, The University of Texas Medical Branch, Galveston, United StatesDepartment of Pediatrics, University of Texas Medical Branch, Galveston, United States; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, United StatesDepartment of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, United StatesDepartment of Pediatrics, University of Texas Medical Branch, Galveston, United StatesWorld 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 StatesWorld 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 StatesWorld 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 StatesDepartment 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 StatesHigh-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.https://elifesciences.org/articles/68479SARS-CoV-2ClickSeqGenomicsNanopore SequencingDefective RNAsNext-Generation Sequencing |
| spellingShingle | 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 Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants eLife SARS-CoV-2 ClickSeq Genomics Nanopore Sequencing Defective RNAs Next-Generation Sequencing |
| title | Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants |
| title_full | Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants |
| title_fullStr | Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants |
| title_full_unstemmed | Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants |
| title_short | Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants |
| title_sort | tiled clickseq for targeted sequencing of complete coronavirus genomes with simultaneous capture of rna recombination and minority variants |
| topic | SARS-CoV-2 ClickSeq Genomics Nanopore Sequencing Defective RNAs Next-Generation Sequencing |
| url | https://elifesciences.org/articles/68479 |
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