Figure 1

Experimental design for characterization of V1R diversity using PacBio circular consensus sequencing. 1) Phylogenetic analyses of existing data are performed to identify and select clades of closely related sequences (i.e. subfamilies). 2) Individual subfamilies are aligned and evolutionary conserved regions are identified for PCR primer design. 3) Double stranded PCR amplicons are used for library preparation and circular consensus sequencing is performed. 4) CCS sequences are filtered based on CCS pass and average Phred score. Sequences are demultiplexed based on length, phylogenetic clustering, or barcode. 5) Cluster analyses are performed on filtered CCS data, de novo chimera detection methods remove putative PCR chimeras, cluster alignments are checked for accuracy, and consensus sequences are generated. 6) Consensus sequences are validated based on comparisons across individuals or with existing sequence data.