Detection of SARS-CoV-2 Δ426 ORF8 Deletion Mutant Cluster in NGS Screening

Abstract

Next-generation sequencing (NGS) from SARS-CoV-2-positive swabs collected during the last months of 2022 revealed a large deletion spanning ORF7b and ORF8 (426 nt) in six patients infected with the BA.5.1 Omicron variant. This extensive genome loss removed a large part of these two genes, maintaining in frame the first 22 aminoacids of ORF7b and the last three aminoacids of ORF8. Interestingly, the deleted region was flanked by two small repeats, which were likely involved in the formation of a hairpin structure. Similar rearrangements, comparable in size and location to the deletion, were also identified in 15 sequences in the NCBI database. In this group, seven out of 15 cases from the USA and Switzerland presented both the BA.5.1 variant and the same 426 nucleotides deletion. It is noteworthy that three out of six cases were detected in patients with immunodeficiency, and it is conceivable that this clinical condition could promote the replication and selection of these mutations.

Keywords: NGS; SARS-CoV-2; deletion; genomic surveillance; variants.

Figure 1

RNA secondary structure with predicted lowest Gibbs free energy (ΔG = −137.30 kcal/mol) of Omicron BA.5.1 variant sequence on the region involved in the deletion. Here are highlighted in red the breakpoints of our deletion, in blue the TRSs, arrows indicate the start and end of ORF7b and ORF8.

Figure 2

Alignments of reads generated by NGS sequencing. (A) Alignment of patients’ reads with the reference sequence; (B) IGV visualization of the single reads for each sample. Red lines indicate hybrid reads.

Figure 3

Chromatograms alignment of the Sanger sequencing product with the reference genome. Chromatograms were obtained from PCR reactions from our six samples. Red lines show the breakpoint site and in the lower part of the figure it is shown the deleted nucleotides with the indication of the ORF.

Figure 4

Phylogenetic tree. (A) UShER phylogenetic analysis of the sequences with similar size deletions of ORF7b and ORF8. Arrows indicate the locations of sequences carrying a big deletion of ORF8. In the enlargement, branches with our 6 patient samples are shown; (B) Phylogenetic analysis specific for the 15 strains having ORF8 deletion found on NCBI and our sequences. Red boxes indicated our six sequences.