Fig. 3

Masking of the Delta variant’s signature mutations due to the bioinformatics tools used or due to systematic sequencing errors. (A), (B) and (C) shows reads aligned to the S protein of the SARS-CoV-2 Wuhan-Hu-1 isolate (NCBI accession: NC_045512.2) from three representative COVID-19 positive samples. The first and second rows above the reads show the reference and consensus sequences (from the read assembly), respectively. White and green backgrounds represent nucleotide and amino acid sequences, respectively. The number below the reference amino acid sequence represents the residue position in the S protein of the SARS-CoV-2 Wuhan-Hu-1 isolate (reference sequence). Reads aligned to the reference sequence are represented below the consensus sequence where bold black characters represent nucleotides differing from the reference sequence. Gaps are represented by dashes (-). (A) Shows residue positions 154 to 160. A non-codon-aligned deletion of six nucleotides is expected between 156 and 158, leading to the triple mutation E156/F157/R158G in the Delta sequences. All reads have the expected six nucleotide deletions (red rectangle), therefore the sequencing and read assembly were correct. (B) Shows residue positions 611 to 617. An A to G nucleotide substitution is expected at residue position 614, leading to the amino acid substitution D614G. Multiple reads exhibit a gap at this position, thereby lowering the frequency of reads exhibiting the nucleotide G at this position (red rectangle). (C) Shows residue positions 942 to 953. The G to A nucleotide substitution is expected at residue position 950, leading to the amino acid substitution D950N. Many reads exhibit the ARTIC Network SARS-CoV-2 V3 primer sequence at its 5’ end (black trapezoid, primer name in white) likely as a result of inefficient primer trimming when using Geneious. Consequently, primer sequences are introduced in the read alignment, thereby lowering the frequency of reads exhibiting nucleotide A (red rectangle). In this case, it masks the D950N amino acid substitution.