The experimental protocol was performed exactly as described previously [56 (link)]. Briefly, an oligonucleotide pool was synthesized that contains sequences coding for peptides of 31 amino acids that tile along the length of the Wuhan-Hu-1 Spike protein sequence [88 (link)] in 1 amino acid increments. For each peptide with the wildtype sequence, 19 variations were included that have a single mutation at the middle amino acid, resulting in a total library size of 24,820 unique sequences. The oligonucleotide pool was cloned into T7 phage, followed by amplification of the phage library; this step was performed twice independently to generate biological duplicate phage libraries. The phage library was incubated with a serum or plasma sample, then bound antibody-phage complexes were immunoprecipitated using Protein A and Protein G Dynabeads (Invitrogen). Bound phage were lysed, and DNA was amplified by PCR and cleaned prior to sequencing on an Illumina MiSeq or HiSeq 2500 with single end reads. Demultiplexing and read alignment were also performed as described previously [57 (link)].
Free full text: Click here
