A comparison of five Illumina, Ion Torrent, and nanopore sequencing technology-based approaches for whole genome sequencing of SARS-CoV-2
Abstract Rapid identification of the rise and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern remains critical for monitoring of the efficacy of diagnostics, therapeutics, vaccines, and control strategies. A wide range of SARS-CoV-2 next-generation sequencing (NGS) methods have been developed over the last years, but cross-sequence technology benchmarking studies have been scarce. In the current study, 26 clinical samples were sequenced using five protocols: AmpliSeq SARS-CoV-2 (Illumina), EasySeq RC-PCR SARS-CoV-2 (Illumina/NimaGen), Ion AmpliSeq SARS-CoV-2 (Thermo Fisher), custom primer sets (Oxford Nanopore Technologies (ONT)), and capture probe-based viral metagenomics (Roche/Illumina). Studied parameters included genome coverage, depth of coverage, amplicon distribution, and variant calling. The median SARS-CoV-2 genome coverage of samples with cycle threshold (Ct) values of 30 and lower ranged from 81.6 to 99.8% for, respectively, the ONT protocol and Illumina AmpliSeq protocol. Correlation of coverage with PCR Ct values varied per protocol. Amplicon distribution signatures differed across the methods, with peak differences of up to 4 $ log_{10} $ at disbalanced positions in samples with high viral loads (Ct values ≤ 23). Phylogenetic analyses of consensus sequences showed clustering independent of the workflow used. The proportion of SARS-CoV-2 reads in relation to background sequences, as a (cost-)efficiency metric, was the highest for the EasySeq protocol. The hands-on time was the lowest when using EasySeq and ONT protocols, with the latter additionally having the shortest sequence runtime. In conclusion, the studied protocols differed on a variety of the studied metrics. This study provides data that assist laboratories when selecting protocols for their specific setting..
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:42 |
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| Enthalten in: |
European journal of clinical microbiology & infectious diseases - 42(2023), 6 vom: 05. Apr., Seite 701-713 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Carbo, Ellen C. [VerfasserIn] |
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| Links: |
Volltext [kostenfrei] |
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| Themen: |
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| Anmerkungen: |
© The Author(s) 2023 |
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| doi: |
10.1007/s10096-023-04590-0 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a Abstract Rapid identification of the rise and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern remains critical for monitoring of the efficacy of diagnostics, therapeutics, vaccines, and control strategies. A wide range of SARS-CoV-2 next-generation sequencing (NGS) methods have been developed over the last years, but cross-sequence technology benchmarking studies have been scarce. In the current study, 26 clinical samples were sequenced using five protocols: AmpliSeq SARS-CoV-2 (Illumina), EasySeq RC-PCR SARS-CoV-2 (Illumina/NimaGen), Ion AmpliSeq SARS-CoV-2 (Thermo Fisher), custom primer sets (Oxford Nanopore Technologies (ONT)), and capture probe-based viral metagenomics (Roche/Illumina). Studied parameters included genome coverage, depth of coverage, amplicon distribution, and variant calling. The median SARS-CoV-2 genome coverage of samples with cycle threshold (Ct) values of 30 and lower ranged from 81.6 to 99.8% for, respectively, the ONT protocol and Illumina AmpliSeq protocol. Correlation of coverage with PCR Ct values varied per protocol. Amplicon distribution signatures differed across the methods, with peak differences of up to 4 $ log_{10} $ at disbalanced positions in samples with high viral loads (Ct values ≤ 23). Phylogenetic analyses of consensus sequences showed clustering independent of the workflow used. The proportion of SARS-CoV-2 reads in relation to background sequences, as a (cost-)efficiency metric, was the highest for the EasySeq protocol. The hands-on time was the lowest when using EasySeq and ONT protocols, with the latter additionally having the shortest sequence runtime. In conclusion, the studied protocols differed on a variety of the studied metrics. This study provides data that assist laboratories when selecting protocols for their specific setting. | ||
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| 700 | 1 | |a Mourik, Kees |4 aut | |
| 700 | 1 | |a Boers, Stefan A. |4 aut | |
| 700 | 1 | |a Munnink, Bas Oude |4 aut | |
| 700 | 1 | |a Nieuwenhuijse, David |4 aut | |
| 700 | 1 | |a Jonges, Marcel |4 aut | |
| 700 | 1 | |a Welkers, Matthijs R. A. |4 aut | |
| 700 | 1 | |a Matamoros, Sebastien |4 aut | |
| 700 | 1 | |a van Harinxma thoe Slooten, Joost |4 aut | |
| 700 | 1 | |a Kraakman, Margriet E. M. |4 aut | |
| 700 | 1 | |a Karelioti, Evita |4 aut | |
| 700 | 1 | |a van der Meer, David |4 aut | |
| 700 | 1 | |a Veldkamp, Karin Ellen |4 aut | |
| 700 | 1 | |a Kroes, Aloys C. M. |4 aut | |
| 700 | 1 | |a Sidorov, Igor |4 aut | |
| 700 | 1 | |a de Vries, Jutte J. C. |0 (orcid)0000-0003-2530-6260 |4 aut | |
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