Figure 7: Genomic validation of novel rhesus and human alleles.

(a) Windowed cluster plots (upper left histograms) and Linkage cluster plots (lower left panels) of rhesus macaque F124 VH sequences assigned to starting database sequences VH2.12 and VH2.62, respectively. The upper left histograms and lower left linkage cluster plots show that starting database gene VH2.12 is not expressed in the F124 library. In contrast, VH2.62 is expressed, as indicated by the presence of 426 exact matches that use 16 unique CDR3s. Three novel VH2 family alleles found by IgDiscover, VH2.12_S0085, VH2.12_S3618 and VH2.62_S8940, are shown in the upper right histograms and lower right linkage cluster plots. (b) Position of primers used to amplify novel VH2 family alleles. The primers were designed to encompass the genomic sequence of previously validated VH2 family genes, VH2.12 and VH2.62 (primer sequences in Supplementary Table 1) resulting in identification of VH2.12_S0085, VH2.12_S3618 and VH2.62_S8940 in the genomic DNA. The right lower panel shows a phylogenetic tree indicating the relationship between the previously published VH2 family sequences and the four VH2 family alleles expressed in rhesus macaque F124. The three novel alleles and one previously known allele identified are indicated with pink arrows and black arrow, respectively. (c) Left panels illustrate the initial assignment of sequences from the H1 human library to VH3-21*01. The centre four panels demonstrate IgDiscover identification of two distinct sequences within the initial assigned group that are used in multiple independent V, diversity and J rearrangements. The right panels show the position of genomic primers used for genomic validation and the resulting identification of an unrearranged genomic sequence corresponding to a novel IGHV3-21 allele that differs from IGHV3-21*01 by exactly three nucleotides.