Single-Cell BCR V(D)J Sequencing and Analysis

Full-length BCR V(D)J segments were enriched from amplified cDNA from 5′ libraries using a Chromium Single-Cell V(D)J Enrichment kit according to the manufacturer’s protocol. BCR sequences for each single B cell were assembled by Cell Ranger vdj pipeline (v.3.0.2). Only cells with both productive immunoglobulin heavy chains (IGH) and productive immunoglobulin light chains kappa (IGK) or lambda (IGL) were kept. If more than one heavy chain or light chain were detected in one cell, the chain with the highest amount of unique molecular identifiers was retained (Zheng et al., 2017).

A clonotype was defined as a unique combination of an IGH-IGK/IGL pair. A cell was considered to be clonally expanded if its clonotype was shared by at least two cells. The clonality of a clonotype could be indicated by the number of cells with the same clonotype. Using barcode information, B cells with prevalent BCR clonotypes were projected on a uniform manifold approximation and projection (UMAP) plot. The somatic hypermutation (SHM) rate of each B cell was defined as the percentage of mismatching nucleotides in the VH portion compared with the most similar germline gene (Kuri-Cervantes et al., 2020). Lineage analysis was performed on the protein sequences of the CDR3 region in both heavy and light chains. BCR sequences with the same V(D)J assignment were grouped together into a lineage if their CDR3 sequences differed by no more than one amino acid (Jiang et al., 2013). Plots of the lineage structures were generated with Cytoscape (v.3.5.1).