tetano
Editor, Senior Moderator
Eur J Clin Microbiol Infect Dis
. 2023 Apr 5.
doi: 10.1007/s10096-023-04590-0. Online ahead of print.
A comparison of five Illumina, Ion Torrent, and nanopore sequencing technology-based approaches for whole genome sequencing of SARS-CoV-2
Ellen C Carbo[SUP] 1 [/SUP], Kees Mourik[SUP] 1 [/SUP], Stefan A Boers[SUP] 1 [/SUP], Bas Oude Munnink[SUP] 2 [/SUP], David Nieuwenhuijse[SUP] 2 [/SUP], Marcel Jonges[SUP] 3 [/SUP], Matthijs R A Welkers[SUP] 3 [/SUP], Sebastien Matamoros[SUP] 3 [/SUP], Joost van Harinxma Thoe Slooten[SUP] 1 [/SUP], Margriet E M Kraakman[SUP] 1 [/SUP], Evita Karelioti[SUP] 4 [/SUP], David van der Meer[SUP] 4 [/SUP], Karin Ellen Veldkamp[SUP] 1 [/SUP], Aloys C M Kroes[SUP] 1 [/SUP], Igor Sidorov[SUP] 1 [/SUP], Jutte J C de Vries[SUP] 5 [/SUP]
Affiliations
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[SUB]10[/SUB] 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.
Keywords: Benchmark; SARS-CoV-2; Whole genome sequencing.
. 2023 Apr 5.
doi: 10.1007/s10096-023-04590-0. Online ahead of print.
A comparison of five Illumina, Ion Torrent, and nanopore sequencing technology-based approaches for whole genome sequencing of SARS-CoV-2
Ellen C Carbo[SUP] 1 [/SUP], Kees Mourik[SUP] 1 [/SUP], Stefan A Boers[SUP] 1 [/SUP], Bas Oude Munnink[SUP] 2 [/SUP], David Nieuwenhuijse[SUP] 2 [/SUP], Marcel Jonges[SUP] 3 [/SUP], Matthijs R A Welkers[SUP] 3 [/SUP], Sebastien Matamoros[SUP] 3 [/SUP], Joost van Harinxma Thoe Slooten[SUP] 1 [/SUP], Margriet E M Kraakman[SUP] 1 [/SUP], Evita Karelioti[SUP] 4 [/SUP], David van der Meer[SUP] 4 [/SUP], Karin Ellen Veldkamp[SUP] 1 [/SUP], Aloys C M Kroes[SUP] 1 [/SUP], Igor Sidorov[SUP] 1 [/SUP], Jutte J C de Vries[SUP] 5 [/SUP]
Affiliations
- PMID: 37017810
- DOI: 10.1007/s10096-023-04590-0
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[SUB]10[/SUB] 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.
Keywords: Benchmark; SARS-CoV-2; Whole genome sequencing.