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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.
Afiliación
  • Jaworski E; Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States.
  • Langsjoen RM; ClickSeq Technologies LLC, Galveston, United States.
  • Mitchell B; Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States.
  • Judy B; World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, United States.
  • Newman P; Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, United States.
  • Plante JA; Department of Pediatrics, University of Texas Medical Branch, Galveston, United States.
  • Plante KS; Department of Pediatrics, University of Texas Medical Branch, Galveston, United States.
  • Miller AL; World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, United States.
  • Zhou Y; Department of Pathology, University of Texas Medical Branch, Galveston, United States.
  • Swetnam D; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, United States.
  • Sotcheff S; World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, United States.
  • Morris V; Department of Pathology, University of Texas Medical Branch, Galveston, United States.
  • Saada N; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, United States.
  • Machado RR; Department of Pediatrics, University of Texas Medical Branch, Galveston, United States.
  • McConnell A; Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States.
  • Widen SG; Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States.
  • Thompson J; Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States.
  • Dong J; Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, United States.
  • Ren P; World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, United States.
  • Pyles RB; Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, United States.
  • Ksiazek TG; World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, United States.
  • Menachery VD; Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, United States.
  • Weaver SC; World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, United States.
  • Routh AL; Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, United States.
Elife ; 102021 09 28.
Article en En | MEDLINE | ID: mdl-34581669
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ARN / Secuencia de Bases / Genoma Viral / Coronavirus / SARS-CoV-2 Límite: Humans Idioma: En Revista: Elife Año: 2021 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ARN / Secuencia de Bases / Genoma Viral / Coronavirus / SARS-CoV-2 Límite: Humans Idioma: En Revista: Elife Año: 2021 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido