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...
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eLife Sciences Publications Ltd
2021
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oai:doaj.org-article:4d7ebe7fd8ba48a3ba01181583b2b50c2021-11-25T12:31:18ZTiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants10.7554/eLife.684792050-084Xe68479https://doaj.org/article/4d7ebe7fd8ba48a3ba01181583b2b50c2021-09-01T00:00:00Zhttps://elifesciences.org/articles/68479https://doaj.org/toc/2050-084XHigh-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.Elizabeth JaworskiRose M LangsjoenBrooke MitchellBarbara JudyPatrick NewmanJessica A PlanteKenneth S PlanteAaron L MillerYiyang ZhouDaniele SwetnamStephanea SotcheffVictoria MorrisNehad SaadaRafael RG MachadoAllan McConnellSteven G WidenJill ThompsonJianli DongPing RenRick B PylesThomas G KsiazekVineet D MenacheryScott C WeaverAndrew L RoutheLife Sciences Publications LtdarticleSARS-CoV-2ClickSeqGenomicsNanopore SequencingDefective RNAsNext-Generation SequencingMedicineRScienceQBiology (General)QH301-705.5ENeLife, Vol 10 (2021) |
| institution |
DOAJ |
| collection |
DOAJ |
| language |
EN |
| topic |
SARS-CoV-2 ClickSeq Genomics Nanopore Sequencing Defective RNAs Next-Generation Sequencing Medicine R Science Q Biology (General) QH301-705.5 |
| spellingShingle |
SARS-CoV-2 ClickSeq Genomics Nanopore Sequencing Defective RNAs Next-Generation Sequencing Medicine R Science Q Biology (General) QH301-705.5 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 |
| 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. |
| format |
article |
| 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 |
| title |
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_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_sort |
tiled-clickseq for targeted sequencing of complete coronavirus genomes with simultaneous capture of rna recombination and minority variants |
| publisher |
eLife Sciences Publications Ltd |
| publishDate |
2021 |
| url |
https://doaj.org/article/4d7ebe7fd8ba48a3ba01181583b2b50c |
| work_keys_str_mv |
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