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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...
Autores principales: | , , , , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8478411/ https://www.ncbi.nlm.nih.gov/pubmed/34581669 http://dx.doi.org/10.7554/eLife.68479 |
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author | Jaworski, Elizabeth Langsjoen, Rose M Mitchell, Brooke Judy, Barbara Newman, Patrick Plante, Jessica A Plante, Kenneth S Miller, Aaron L Zhou, Yiyang Swetnam, Daniele Sotcheff, Stephanea Morris, Victoria Saada, Nehad Machado, Rafael RG McConnell, Allan Widen, Steven G Thompson, Jill Dong, Jianli Ren, Ping Pyles, Rick B Ksiazek, Thomas G Menachery, Vineet D Weaver, Scott C Routh, Andrew L |
author_facet | Jaworski, Elizabeth Langsjoen, Rose M Mitchell, Brooke Judy, Barbara Newman, Patrick Plante, Jessica A Plante, Kenneth S Miller, Aaron L Zhou, Yiyang Swetnam, Daniele Sotcheff, Stephanea Morris, Victoria Saada, Nehad Machado, Rafael RG McConnell, Allan Widen, Steven G Thompson, Jill Dong, Jianli Ren, Ping Pyles, Rick B Ksiazek, Thomas G Menachery, Vineet D Weaver, Scott C Routh, Andrew L |
author_sort | Jaworski, Elizabeth |
collection | PubMed |
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 | Online Article Text |
id | pubmed-8478411 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-84784112021-09-30 Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants Jaworski, Elizabeth Langsjoen, Rose M Mitchell, Brooke Judy, Barbara Newman, Patrick Plante, Jessica A Plante, Kenneth S Miller, Aaron L Zhou, Yiyang Swetnam, Daniele Sotcheff, Stephanea Morris, Victoria Saada, Nehad Machado, Rafael RG McConnell, Allan Widen, Steven G Thompson, Jill Dong, Jianli Ren, Ping Pyles, Rick B Ksiazek, Thomas G Menachery, Vineet D Weaver, Scott C Routh, Andrew L eLife Genetics and Genomics 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. eLife Sciences Publications, Ltd 2021-09-28 /pmc/articles/PMC8478411/ /pubmed/34581669 http://dx.doi.org/10.7554/eLife.68479 Text en © 2021, Jaworski et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited. |
spellingShingle | Genetics and Genomics Jaworski, Elizabeth Langsjoen, Rose M Mitchell, Brooke Judy, Barbara Newman, Patrick Plante, Jessica A Plante, Kenneth S Miller, Aaron L Zhou, Yiyang Swetnam, Daniele Sotcheff, Stephanea Morris, Victoria Saada, Nehad Machado, Rafael RG McConnell, Allan Widen, Steven G Thompson, Jill Dong, Jianli Ren, Ping Pyles, Rick B Ksiazek, Thomas G Menachery, Vineet D Weaver, Scott C Routh, Andrew L Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants |
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 | Genetics and Genomics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8478411/ https://www.ncbi.nlm.nih.gov/pubmed/34581669 http://dx.doi.org/10.7554/eLife.68479 |
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