Next-generation sequencing (NGS) techniques have become an indispensable tool for the study and control of the SARS-CoV-2 virus pandemic1. The sequencing of the complete viral genome has been vital not only to the development of vaccines and specific drugs, but also to the discovery and monitoring of the variants of interest which, since the appearance of what is now known as the alpha variant2, have occurred and been related to the specific resurgences of the pandemic. However, and despite the efforts made by microbiology laboratories all over the world, there is insufficient sequencing capacity to characterise all SARS-CoV-2 positive samples with this technology. For this reason, different strategies have been developed for the characterisation of the virus by means of RT-PCR of known combinations of mutations3–5. However, the limitations involved in this type of study, which can lead to an erroneous variant assignment, must not be forgotten6,7.
Currently, in our department, all the first samples of an episode with Cts under 32 are characterised using the Allplex™ Variants I&II (Seegene, Korea) panels, which study the most relevant spike mutations in the latest variants of interest (H69/V70-, W152C, K417N, K417T, L452R, E484K and N501Y). As of February 2022, in a scenario totally dominated by the Omicron variant, the samples that presented the H69/V70-, K417N and N501Y mutations were classified as Omicron by RT-PCR. If the deletion was not present, they were considered to belong to the BA.2 subvariant by RT-PCR.
Given the appearance of a sample with H69/V70- and K417N, but without N501Y, the decision to do sequencing was taken, since these results did not allow it to be assigned to any variant by RT-PCR. To do this, an adaptation of the ARTIC protocol for Oxford Nanopore was used by amplifying the entire genome with the VarSkip scheme8. The resulting library was processed in a MinION mk1c sequencer (Oxford Nanopore). The readings obtained were subsequently assembled using the ARTIC pipeline for data obtained from sequencers with Oxford Nanopore technology and analysed using the Nextstrain web app9 with consistency verified using the Integrative Genomics Viewer (IGV) program10.
The sequence obtained (deposited in GISAID as EPI_ISL_10968563) belongs to the 21K clade (Omicron) and to the BA.1.17 lineage and presented, among other characteristic spike mutations, the three mutations used by the Allplex Variants I&II scheme: H69/V70-, K417N and N501Y; although, the N501Y mutation, in addition to the usual A23063T change, also had T23065C. All mutations were found with sufficient coverage. In this case, T23065C accompanying A23063T produces a silent mutation since the codon they form continues to encode a tyrosine and maintains the N501Y mutation.
Therefore, this mutation is not expected to involve an evolutionary advantage for the virus or a fundamental change in its behaviour. However, it may pose a problem for characterisation schemes based on the study of specific mutations, such as the one we currently use in our laboratory; since if this change had occurred, for example, in a variant of the BA.2 lineage in the Allplex Variants I&II panels, only the K417N mutation would be positive.
In summary, we believe that this case is a further example of the enormous utility offered by NGS in Clinical Microbiology laboratories today, since it allows us not only to correctly assign circulating variants but also to monitor the evolution of the same variants.
