Cargando…
Whole-Genome Sequencing of SARS-CoV-2: Assessment of the Ion Torrent AmpliSeq Panel and Comparison with the Illumina MiSeq ARTIC Protocol
Fast and effective methods are needed for sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome to track genetic mutations and to identify new and emerging variants during the ongoing pandemic. The objectives were to assess the performance of the SARS-CoV-2 AmpliSeq r...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Microbiology
2021
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8601232/ https://www.ncbi.nlm.nih.gov/pubmed/34550806 http://dx.doi.org/10.1128/JCM.00649-21 |
_version_ | 1784601301249687552 |
---|---|
author | Plitnick, Jonathan Griesemer, Sara Lasek-Nesselquist, Erica Singh, Navjot Lamson, Daryl M. St. George, Kirsten |
author_facet | Plitnick, Jonathan Griesemer, Sara Lasek-Nesselquist, Erica Singh, Navjot Lamson, Daryl M. St. George, Kirsten |
author_sort | Plitnick, Jonathan |
collection | PubMed |
description | Fast and effective methods are needed for sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome to track genetic mutations and to identify new and emerging variants during the ongoing pandemic. The objectives were to assess the performance of the SARS-CoV-2 AmpliSeq research panel and S5 plug-in analysis tools for whole-genome sequencing analysis of SARS-CoV-2 and to compare the results with those obtained with the MiSeq-based ARTIC analysis pipeline, using metrics such as depth, coverage, and concordance of single-nucleotide variant (SNV) calls. A total of 191 clinical specimens and a single cultured isolate were extracted and sequenced with AmpliSeq technology and analysis tools. Of the 191 clinical specimens, 83 (with threshold cycle [C(T)] values of 15.58 to 32.54) were also sequenced using an Illumina MiSeq-based method with the ARTIC analysis pipeline, for direct comparison. A total of 176 of the 191 clinical specimens sequenced on the S5XL system and prepared using the SARS-CoV-2 research panel had nearly complete coverage (>98%) of the viral genome, with an average depth of 5,031×. Similar coverage levels (>98%) were observed for 81/83 primary specimens that were sequenced with both methods tested. The sample with the lowest viral load (C(T) value of 32.54) achieved 89% coverage using the MiSeq method and failed to sequence with the AmpliSeq method. Consensus sequences produced by each method were identical for 81/82 samples in areas of equal coverage, with a single difference present in one sample. The AmpliSeq approach is as effective as the Illumina-based method using ARTIC v3 amplification for sequencing SARS-CoV-2 directly from patient specimens across a range of viral loads (C(T) values of 15.56 to 32.54 [median, 22.18]). The AmpliSeq workflow is very easily automated with the Ion Chef and S5 instruments and requires less training and experience with next-generation sequencing sample preparation than the Illumina workflow. |
format | Online Article Text |
id | pubmed-8601232 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Society for Microbiology |
record_format | MEDLINE/PubMed |
spelling | pubmed-86012322021-12-07 Whole-Genome Sequencing of SARS-CoV-2: Assessment of the Ion Torrent AmpliSeq Panel and Comparison with the Illumina MiSeq ARTIC Protocol Plitnick, Jonathan Griesemer, Sara Lasek-Nesselquist, Erica Singh, Navjot Lamson, Daryl M. St. George, Kirsten J Clin Microbiol Virology Fast and effective methods are needed for sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome to track genetic mutations and to identify new and emerging variants during the ongoing pandemic. The objectives were to assess the performance of the SARS-CoV-2 AmpliSeq research panel and S5 plug-in analysis tools for whole-genome sequencing analysis of SARS-CoV-2 and to compare the results with those obtained with the MiSeq-based ARTIC analysis pipeline, using metrics such as depth, coverage, and concordance of single-nucleotide variant (SNV) calls. A total of 191 clinical specimens and a single cultured isolate were extracted and sequenced with AmpliSeq technology and analysis tools. Of the 191 clinical specimens, 83 (with threshold cycle [C(T)] values of 15.58 to 32.54) were also sequenced using an Illumina MiSeq-based method with the ARTIC analysis pipeline, for direct comparison. A total of 176 of the 191 clinical specimens sequenced on the S5XL system and prepared using the SARS-CoV-2 research panel had nearly complete coverage (>98%) of the viral genome, with an average depth of 5,031×. Similar coverage levels (>98%) were observed for 81/83 primary specimens that were sequenced with both methods tested. The sample with the lowest viral load (C(T) value of 32.54) achieved 89% coverage using the MiSeq method and failed to sequence with the AmpliSeq method. Consensus sequences produced by each method were identical for 81/82 samples in areas of equal coverage, with a single difference present in one sample. The AmpliSeq approach is as effective as the Illumina-based method using ARTIC v3 amplification for sequencing SARS-CoV-2 directly from patient specimens across a range of viral loads (C(T) values of 15.56 to 32.54 [median, 22.18]). The AmpliSeq workflow is very easily automated with the Ion Chef and S5 instruments and requires less training and experience with next-generation sequencing sample preparation than the Illumina workflow. American Society for Microbiology 2021-11-18 /pmc/articles/PMC8601232/ /pubmed/34550806 http://dx.doi.org/10.1128/JCM.00649-21 Text en Copyright © 2021 American Society for Microbiology. https://doi.org/10.1128/ASMCopyrightv2All Rights Reserved (https://doi.org/10.1128/ASMCopyrightv2) . https://doi.org/10.1128/ASMCopyrightv2This article is made available via the PMC Open Access Subset for unrestricted noncommercial re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. |
spellingShingle | Virology Plitnick, Jonathan Griesemer, Sara Lasek-Nesselquist, Erica Singh, Navjot Lamson, Daryl M. St. George, Kirsten Whole-Genome Sequencing of SARS-CoV-2: Assessment of the Ion Torrent AmpliSeq Panel and Comparison with the Illumina MiSeq ARTIC Protocol |
title | Whole-Genome Sequencing of SARS-CoV-2: Assessment of the Ion Torrent AmpliSeq Panel and Comparison with the Illumina MiSeq ARTIC Protocol |
title_full | Whole-Genome Sequencing of SARS-CoV-2: Assessment of the Ion Torrent AmpliSeq Panel and Comparison with the Illumina MiSeq ARTIC Protocol |
title_fullStr | Whole-Genome Sequencing of SARS-CoV-2: Assessment of the Ion Torrent AmpliSeq Panel and Comparison with the Illumina MiSeq ARTIC Protocol |
title_full_unstemmed | Whole-Genome Sequencing of SARS-CoV-2: Assessment of the Ion Torrent AmpliSeq Panel and Comparison with the Illumina MiSeq ARTIC Protocol |
title_short | Whole-Genome Sequencing of SARS-CoV-2: Assessment of the Ion Torrent AmpliSeq Panel and Comparison with the Illumina MiSeq ARTIC Protocol |
title_sort | whole-genome sequencing of sars-cov-2: assessment of the ion torrent ampliseq panel and comparison with the illumina miseq artic protocol |
topic | Virology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8601232/ https://www.ncbi.nlm.nih.gov/pubmed/34550806 http://dx.doi.org/10.1128/JCM.00649-21 |
work_keys_str_mv | AT plitnickjonathan wholegenomesequencingofsarscov2assessmentoftheiontorrentampliseqpanelandcomparisonwiththeilluminamiseqarticprotocol AT griesemersara wholegenomesequencingofsarscov2assessmentoftheiontorrentampliseqpanelandcomparisonwiththeilluminamiseqarticprotocol AT laseknesselquisterica wholegenomesequencingofsarscov2assessmentoftheiontorrentampliseqpanelandcomparisonwiththeilluminamiseqarticprotocol AT singhnavjot wholegenomesequencingofsarscov2assessmentoftheiontorrentampliseqpanelandcomparisonwiththeilluminamiseqarticprotocol AT lamsondarylm wholegenomesequencingofsarscov2assessmentoftheiontorrentampliseqpanelandcomparisonwiththeilluminamiseqarticprotocol AT stgeorgekirsten wholegenomesequencingofsarscov2assessmentoftheiontorrentampliseqpanelandcomparisonwiththeilluminamiseqarticprotocol |