Evaluation of two SARS-CoV-2 whole genome amplification and sequencing workflows for genomic surveillance and outbreak analysis in the clinical microbiology laboratory.

Publication date: Jul 10, 2025

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 (990cD7 vs 557cD7), 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.

Semantics

Original Article