Template-Choice Bias in Deep Sequencing of Antibody Phage Display Libraries

Deep sequencing of antibody phage-display selections commonly uses intracellular double-stranded phagemid DNA (dsDNA), although selected libraries are propagated as virions containing single-stranded phagemid DNA (ssDNA). Whether these two templates yield equivalent quantitative profiles remains uncertain.

We sequenced matched intracellular dsDNA and virion ssDNA from a single-chain variable fragment library at the input and after each of four rounds of bio-panning.

A total of 1052 ssDNA-derived and 1093 dsDNA-derived unique heavy-chain complementarity-determining region 3 (HCDR3) clonotypes were analyzed, including 1003 shared clonotypes. The dsDNA and ssDNA profiles were closely concordant before strong enrichment, with Spearman correlations of 0.972-0.978 and Morisita-Horn overlap of 0.994-0.998 through round 2. Once a small number of clonotypes began to dominate the selection, however, the two profiles separated sharply: abundance-weighted overlap fell to 0.501 at round 3 and 0.379 at round 4, whereas rank order was preserved and the rank correlation remained high throughout (ρ ≥ 0.90). This divergence was concentrated among enriching trajectories, in which dominant clonotypes reached substantially higher relative abundance in virion ssDNA than in intracellular dsDNA. Antigen-reactive clonotypes were also preferentially shifted toward the ssDNA-biased fraction, with a median log₁₀(ssDNA/dsDNA) ratio of 0.44 compared with -0.17 for non-reactive clonotypes at round 3 (p = 6.3 × 10^-8) and round 4 (0.41 vs. -0.12, p = 1.1 × 10^-6).

dsDNA and ssDNA tell nearly the same story early in bio-panning but can provide markedly different accounts once selection becomes concentrated. In this selection campaign, template choice materially affected interpretation of antibody phage-display sequencing, suggesting that virion ssDNA or paired-template analysis may be preferable to dsDNA alone when late-round abundance is used for candidate prioritization. Because these results come from a single campaign against one antigen, they are descriptive of this dataset and require validation across antigens, scaffolds and display systems.