Figure 2. LIBRA-seq applied to a human B cell sample from HIV-infected donor NIAID45.
(A.) LIBRA-seq experiment setup consisted of three antigens in the screening library: BG505, CZA97, and H1 A/New Caledonia/20/99, and the cellular input was donor NIAID45 PBMCs.
(B.) After bioinformatic processing and filtering of cells recovered from single-cell sequencing, the LIBRA-seq score for each antigen was plotted (total, 866 cells). Each axis represents a range of LIBRA- seq scores for each antigen. Density of total cells is shown, with purple to yellow indicating lowest to highest number of cells, respectively.
(C.) 29 VRC01 lineage B cells were identified and examined for phylogenetic relatedness to known lineage members and for sequence features, with phylogenetic tree showing relatedness of previously identified VRC01 lineage members (black) and members newly identified using LIBRA-seq (red). Each row represents an antibody. Sequences were aligned using clustalW and a maximum likelihood tree was inferred using maximum likelihood inference. The resulting tree was visualized using an inferred VRC01 unmutated common ancestor (UCA) (accession MK032222) as the root. For each antibody isolated from LIBRA-seq, a heatmap of the LIBRA-seq scores for each HIV antigen (BG505 and CZA97) is shown; a scale of tan-white-purple represents LIBRA-seq scores from −2 to 0 to 2; in this heatmap, scores lower or higher than that range are shown as −2 and 2, respectively. Levels of somatic hypermutation (SHM) at the nucleotide level for the heavy and light chain variable genes as reported by IMGT are displayed as bars, with the numerical percentage value listed to the right of the bar; length of the bar corresponds to level of SHM. Amino acid sequences of the complementarity determining region 3 for the heavy chain (CDRH3) and the light chain (CDRL3) for each antibody are displayed. The tree was visualized and annotated using iTol (Letunic and Bork, 2019).