Figure 1

A: Diagram of the IgH variable region. Dashed lines identify primer binding sites used for PCR amplification of IgG cDNA transcripts. FR = framework region; CDR = complementarity determining region; C1 = constant region 1; V, D, and J = variable, diversity, and joining segments. Color gradations flanking CDR3 indicate junction sequence diversity. B: Experimental design for single-molecule real-time (SMRT) sequencing, data analysis, and visualization of IgG antigen binding regions. 1: cDNA libraries are constructed from PCR amplicons of the variable region from expressed IgG transcripts. Differing lengths are primarily the result of variation in CDR3. 2: SMRTbells™ are prepared for sequencing by ligating adapters to individual double-stranded amplicons. Small arrows indicate multiple passes of bound DNA polymerase around the SMRTbell™ resulting in several reads of the template. These reads are concatenated into circular consensus (CCS) sequences. 3: CCS sequences are filtered based on quality and correct open reading frame is determined based on alignment with the conserved FR1. 4: Antigen binding regions (CDRs1–3) are identified from translated sequences and are extracted from each sequence. 5: Networks are constructed from all vs. all BLAST searches of thousands of antigen binding regions from each IgG repertoire.