Fig. 2: Construction of a high ED synthetic variant library based on Omicron BA.1 RBD.

a, The RBD sequence was split into 11–12 fragments, each being on average 48 nucleotide in length. For each fragment, a ssODN library with either zero, one or two mutations was designed. b, To introduce mutations, NNK codons were tiled across the fragments (1). Each fragment was flanked by BsmBI sites (2). The ssODNs were flanked by primer binding sites for double-stranded synthesis through PCR (primers are represented by black arrows, and primer binding sites are peach coloured) (3). The type II-S restriction enzyme BsmBI gives rise to orthogonal four nucleotide overhangs, which are used by a ligase to assemble individual fragments into full-length RBD sequences (4). c, The use of GGA for library construction required the presence of constant regions for ligation between fragments (in black), thereby restricting the library diversity. To overcome this drawback, four staggered sub-libraries were constructed. Due to limitations in sequencing length, it was further necessary to split the RBD into two separate libraries. The extent of seq-library A is indicated in orange and seq-library B in cyan. The primer binding sites for deep sequencing are indicated using orange and cyan arrows. d, Targeted sequencing of seq-libraries A and B showed comprehensive mutational coverage for both libraries. The same colour scheme as in c was used to indicate the extent of both libraries. e, To adjust the mutational rate of the library, different ratios of fragments with zero, one or two mutations (60%/20%/20%, 70%/15%/15% and 80%/10%/10%) were pooled, yielding libraries with average number of mutations of 3.59, 2.07 and 1.41, respectively.