Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants
| Autor: | Victoria Morris, Stephanea Sotcheff, Vineet D. Menachery, Yiyang Zhou, Rick B. Pyles, Jessica A. Plante, Thomas G. Ksiazek, Steven G. Widen, Andrew Routh, Scott C. Weaver, Brooke Mitchell, Allan McConnell, Daniele M. Swetnam, Rafael R. G. Machado, Jianli Dong, Kenneth S. Plante, Elizabeth Jaworski, Rose M. Langsjoen, Ping Ren, Barbara M. Judy, Nehad Saada, Patrick C. Newman, Aaron L. Miller, Jill K. Thompson |
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| Rok vydání: | 2021 |
| Předmět: |
Polymerase Chain Reaction
Genome Insert (molecular biology) Nanopores Genomic library Biology (General) Recombination Genetic Microbiology and Infectious Disease General Neuroscience Defective RNAs High-Throughput Nucleotide Sequencing General Medicine Genomics Amplicon Tools and Resources Viruses Medicine RNA Viral Next-Generation Sequencing DNA Complementary QH301-705.5 Science Genome Viral Computational biology Biology General Biochemistry Genetics and Molecular Biology DNA sequencing Article Complementary DNA Humans RNA Messenger Gene Library Whole genome sequencing General Immunology and Microbiology Base Sequence Whole Genome Sequencing SARS-CoV-2 COVID-19 RNA Genetics and Genomics Coronavirus Nanopore Sequencing ClickSeq Nucleic acid Nanopore sequencing Primer (molecular biology) |
| Zdroj: | bioRxiv article-version (status) pre article-version (number) 1 eLife, Vol 10 (2021) eLife |
| DOI: | 10.1101/2021.03.10.434828 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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