A Comparison of Five Illumina, Ion Torrent, and Nanopore Sequencing Technology-Based Approaches for Whole Genome Sequencing of SARS-CoV-2

Rapid identification of the rise and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern currently 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 are 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), 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 Oxford Nanopore protocol and Illumina Ampliseq protocol. Correlation of coverage with PCR Ct-values varied and was dependent on the protocol. Amplicon distribution signatures differed across the methods, with peak differences of up to 4 log10 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 highest for the EasySeq protocol. The hands-on time was 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 can assist laboratories when selecting protocols for their specific setting.

Note:

Funding Information: None.

Declaration of Interests: The authors have no conflicts of interest to declare.

Ethics Approval Statement: Approval was obtained from the ethical committee of the LUMC (B20.002, Biobank Infectious Diseases 2020-03), and the Institutional Review Board of the LUMC for observational Covid-19 studies (CoCo 2021-006).

Keywords: Whole genome sequencing, SARS-CoV-2, benchmark

Suggested Citation: Suggested Citation

Carbo, Ellen C. and Mourik, Kees and Boers, Stefan A. and Oude Munnink, Bas and Nieuwenhuijse, David and Jonges, Marcel and Welkers, Matthijs R.A. and Matamoros, Sébastien and van Harinxma thoe Slooten, Joost and Kraakman, Margriet E.M. and Karelioti, Evita and van der Meer, David and Veldkamp, Karin Ellen and Kroes, Aloys C.M. and Sidorov, Igor and de Vries, Jutte J.C., A Comparison of Five Illumina, Ion Torrent, and Nanopore Sequencing Technology-Based Approaches for Whole Genome Sequencing of SARS-CoV-2. Available at SSRN: https://ssrn.com/abstract=4264151 or http://dx.doi.org/10.2139/ssrn.4264151