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Multiple sequence alignment, generation of consensus sequences, and identification of the conserved DRACH sites

MB Mahmoud Bayoumi
MM Muhammad Munir
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Multiple sequence alignments (MSA) were generated to the long open reading frame of each tested HA subtype (i.e. H1-H18, separately) using the MUSCLE algorithm of the MSA tool implemented in IRD44. The computed and visualized MSA results together with the derived consensus sequences downloaded in FASTA format. Geneious software (v9.1.4) was used to further confirm the alignment and the generated consensus sequences utilizing the same MSA algorithm52. The latter software can provide consensus sequences based on the given threshold frequency (TF) percentage and display the non-consistent bases as IUPAC degenerate nucleotides. Each HA subtype consensus sequence was aligned with the reference H1N1 HA sequence using the Clustal W algorithm, Lasergene software package, version 3.18 (DNASTAR, Madison, WI) for identification of mutation hotspots, and prediction of nucleotide changes, then visualized using BioEdit program, v7.2.5 (Ibis Biosciences, Carlsbad, CA)53. A putative m6A site was considered specific and conserved if has the full 5′-DRACH-3′ motif sequence (D = A, G or T; R = A or G; H = A, C or T) when compared with the reference HA sequence and presented by the lowest conservation percentage of the total number sequences of the analysed subtype.

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