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 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.