Deep mutational scanning library construction.

(A) Scheme for the generation of Omicron BQ.1.1 and XBB.1.5 mutant libraries. Site saturation mutagenesis oligonucleotide libraries were constructed by Twist Bioscience with constant flank sequences and cloned via three-fragment Gibson Assembly to create libraries of barcoded mutant variants. PacBio sequencing of the barcoded mutant library plasmid was used to create barcode:variant lookup tables, enabling subsequent Illumina sequencing of barcode fragments in experimental partitions to generate mutant phenotypes. (B-E) For pooled duplicate BQ.1.1 (top) and XBB.1.5 (bottom) libraries, we show (B) the per-variant rate of mutation types, (C) the distribution of number of amino acid mutants per barcoded variant, (D) the mutation rate of each type along each site in the RBD sequence, and (E) the distribution of total number of barcodes that were averaged for each amino acid mutant in the final ACE2-binding score. (PNG)