Volume 117, Issue 5, September 2025, 111077Author links open overlay panel, , , , , , , , , , , , , , , , , Highlights•

Nanopore sequencing was compared with Illumina for SARS-CoV-2 genomic surveillance and outbreak analysis.

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No SNP differences were observed between obtained sequences for 94.3 % clinical samples.

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PANGO lineage assignments and genomic clustering were highly concordant.

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The Nanopore workflow showed reduced turnaround time and costs for 96-well sequencing runs.

Abstract

Whole-genome sequencing (WGS) has become a key element of SARS-CoV-2 genomic epidemiology. In Spain and many other countries, this is mostly driven by Illumina-based sequencing, while Oxford Nanopore Tecnologies' MinION Mk1C is a low-cost, faster, and user-friendly sequencer. We aimed to evaluate the applicability of the Mk1C device with a long-amplicon PCR approach for SARS-CoV-2 rapid genomic surveillance and outbreak characterization. We compared the technical and practical performance of this workflow with the short-amplicon strategy on Illumina's MiSeq platform. We processed 183 PCR-positive nasopharyngeal samples, one positive control derived from a clinical sample, and two synthetic SARS-CoV-2 controls for both sequencing workflows. Good quality sequences were obtained by both methods: their mean breadth coverage was 99 % for both techniques and while mean depth of coverage was 1.78-fold lower for MinION (990× vs 557×), no single nucleotide polymorphism (SNP) differences were observed between paired synthetic controls and neither for most of the clinical samples (149/158, 94.3 %). Pango lineage assignments were totally concordant, which were mainly from the Omicron variant. Only one sample showed differences in sublineage assignment. Nanopore workflow had 1.3-fold shorter turnaround time to results for large batches of surveillance samples (96-well plate), and 4.1-fold for the study of small batches of outbreak-related samples. Nanopore sequencing had also a 4.5-fold lower cost per sample, considering consumables and reagents. These results support the use of MinION-based workflow for a rapid response to nosocomial outbreaks as well for its implementation into SARS-CoV-2 genomic surveillance, as a reliable alternative to Illumina MiSeq-based workflow.

Keywords

Whole-genome sequencing

SARS-CoV-2

COVID19

Illumina

MiSeq

Oxford nanopore technologies

MinION

Data availabilityAs a result from our genomic surveillance and outbreak control activities, the consensus sequences generated were uploaded to the GISAID database (Khare et al., 2021) as soon as they were obtained —only the first sequence of each sample was uploaded (Supplementary Table 2). The 158 pairs of sequences included in the comparison of this article (generated by both Nanopore and Illumina) were submitted to GenBank at the end of this study (Supplementary Table 2).

© 2025 The Authors. Published by Elsevier Inc.