Fig. 1: Deep-screening workflow.

a, Antibody library preparation involves the addition of 5′ and 3′ untranslated regions (UTRs) that flank the library protein-coding region. The assembled library is then clustered and the N₂₈ UMI sequenced on a HiSeq 2500, which reports the UMI sequence and its physical x–y coordinates on the flow cell. b, In deep screening, DNA clusters are converted into RNA clusters using engineered polymerase TGK²¹ and the DNA template is removed. The RNA clusters are labelled with a complementary Atto 647N-labelled oligonucleotide before IVT into protein (antibody) clusters. Cluster binding is determined by equilibrium binding of an increasing concentration of biotinylated antigen and AF532-labelled Streptavidin (SA), followed by kinetic dissociation from the highest antigen concentration. c, If the binding assay reports hits within the library, a second sequencing experiment is performed to determine the UMI and CDRs with internal sequencing primers. CDRs are then paired with binding data using the common UMIs between the two experiments. d,e, Paired CDR–binding data is analysed for hits and/or a ML model is trained to predict hits, which can be used to generate libraries for subsequent rounds of deep screening (d) or short-list hit candidates for characterization via conversion into an appropriate antibody format, expression, purification (using methods like nickel-nitrilotriacetic acid (NiNTA) resin and size exclusion chromatography (SEC)) (e).