Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols

Surveillance of the SARS-CoV-2 variants including the quickly spreading mutants by rapid and near real-time sequencing of the viral genome provides an important tool for effective health policy decision making in the ongoing COVID-19 pandemic. Here we evaluated PCR-tiling of short (~400-bp) and long...

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Autores principales:	Broňa Brejová, Kristína Boršová, Viktória Hodorová, Viktória Čabanová, Askar Gafurov, Dominika Fričová, Martina Neboháčová, Tomáš Vinař, Boris Klempa, Jozef Nosek
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/9d28bdf74f8f4d45a6155697dcadd81a
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spelling	oai:doaj.org-article:9d28bdf74f8f4d45a6155697dcadd81a2021-11-04T07:42:08ZNanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols1932-6203https://doaj.org/article/9d28bdf74f8f4d45a6155697dcadd81a2021-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8555800/?tool=EBIhttps://doaj.org/toc/1932-6203Surveillance of the SARS-CoV-2 variants including the quickly spreading mutants by rapid and near real-time sequencing of the viral genome provides an important tool for effective health policy decision making in the ongoing COVID-19 pandemic. Here we evaluated PCR-tiling of short (~400-bp) and long (~2 and ~2.5-kb) amplicons combined with nanopore sequencing on a MinION device for analysis of the SARS-CoV-2 genome sequences. Analysis of several sequencing runs demonstrated that using the long amplicon schemes outperforms the original protocol based on the 400-bp amplicons. It also illustrated common artefacts and problems associated with PCR-tiling approach, such as uneven genome coverage, variable fraction of discarded sequencing reads, including human and bacterial contamination, as well as the presence of reads derived from the viral sub-genomic RNAs.Broňa BrejováKristína BoršováViktória HodorováViktória ČabanováAskar GafurovDominika FričováMartina NeboháčováTomáš VinařBoris KlempaJozef NosekPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Broňa Brejová Kristína Boršová Viktória Hodorová Viktória Čabanová Askar Gafurov Dominika Fričová Martina Neboháčová Tomáš Vinař Boris Klempa Jozef Nosek Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols
description	Surveillance of the SARS-CoV-2 variants including the quickly spreading mutants by rapid and near real-time sequencing of the viral genome provides an important tool for effective health policy decision making in the ongoing COVID-19 pandemic. Here we evaluated PCR-tiling of short (~400-bp) and long (~2 and ~2.5-kb) amplicons combined with nanopore sequencing on a MinION device for analysis of the SARS-CoV-2 genome sequences. Analysis of several sequencing runs demonstrated that using the long amplicon schemes outperforms the original protocol based on the 400-bp amplicons. It also illustrated common artefacts and problems associated with PCR-tiling approach, such as uneven genome coverage, variable fraction of discarded sequencing reads, including human and bacterial contamination, as well as the presence of reads derived from the viral sub-genomic RNAs.
format	article
author	Broňa Brejová Kristína Boršová Viktória Hodorová Viktória Čabanová Askar Gafurov Dominika Fričová Martina Neboháčová Tomáš Vinař Boris Klempa Jozef Nosek
author_facet	Broňa Brejová Kristína Boršová Viktória Hodorová Viktória Čabanová Askar Gafurov Dominika Fričová Martina Neboháčová Tomáš Vinař Boris Klempa Jozef Nosek
author_sort	Broňa Brejová
title	Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols
title_short	Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols
title_full	Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols
title_fullStr	Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols
title_full_unstemmed	Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols
title_sort	nanopore sequencing of sars-cov-2: comparison of short and long pcr-tiling amplicon protocols
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/9d28bdf74f8f4d45a6155697dcadd81a
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Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols

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