Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: 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, McConnell, Allan, Widen, Steve, Thompson, Jill, Dong, Jianli, Ren, Ping, Pyles, Rick B., Ksiazek, Thomas, Menachery, Vineet D., Weaver, Scott C., Routh, Andrew
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987005/
https://www.ncbi.nlm.nih.gov/pubmed/33758846
http://dx.doi.org/10.1101/2021.03.10.434828
_version_ 1783668546961670144
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
McConnell, Allan
Widen, Steve
Thompson, Jill
Dong, Jianli
Ren, Ping
Pyles, Rick B.
Ksiazek, Thomas
Menachery, Vineet D.
Weaver, Scott C.
Routh, Andrew
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
McConnell, Allan
Widen, Steve
Thompson, Jill
Dong, Jianli
Ren, Ping
Pyles, Rick B.
Ksiazek, Thomas
Menachery, Vineet D.
Weaver, Scott C.
Routh, Andrew
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-7987005
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Cold Spring Harbor Laboratory
record_format MEDLINE/PubMed
spelling pubmed-79870052021-03-24 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 McConnell, Allan Widen, Steve Thompson, Jill Dong, Jianli Ren, Ping Pyles, Rick B. Ksiazek, Thomas Menachery, Vineet D. Weaver, Scott C. Routh, Andrew bioRxiv Article 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. Cold Spring Harbor Laboratory 2021-09-01 /pmc/articles/PMC7987005/ /pubmed/33758846 http://dx.doi.org/10.1101/2021.03.10.434828 Text en https://creativecommons.org/licenses/by-nd/4.0/This work is licensed under a Creative Commons Attribution-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, and only so long as attribution is given to the creator. The license allows for commercial use.
spellingShingle Article
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
McConnell, Allan
Widen, Steve
Thompson, Jill
Dong, Jianli
Ren, Ping
Pyles, Rick B.
Ksiazek, Thomas
Menachery, Vineet D.
Weaver, Scott C.
Routh, Andrew
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 Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987005/
https://www.ncbi.nlm.nih.gov/pubmed/33758846
http://dx.doi.org/10.1101/2021.03.10.434828
work_keys_str_mv AT jaworskielizabeth tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT langsjoenrosem tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT mitchellbrooke tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT judybarbara tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT newmanpatrick tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT plantejessicaa tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT plantekenneths tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT milleraaronl tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT zhouyiyang tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT swetnamdaniele tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT sotcheffstephanea tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT morrisvictoria tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT saadanehad tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT machadorafael tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT mcconnellallan tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT widensteve tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT thompsonjill tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT dongjianli tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT renping tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT pylesrickb tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT ksiazekthomas tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT menacheryvineetd tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT weaverscottc tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants
AT routhandrew tiledclickseqfortargetedsequencingofcompletecoronavirusgenomeswithsimultaneouscaptureofrnarecombinationandminorityvariants