Novel rhesus macaque immunoglobulin germline genes identified by three sequencing approaches

Abstract

Introduction: Rhesus macaques have long been a focus of research for understanding immune responses to human pathogens due to their close phylogenetic relationship with humans. As rhesus macaque antibody germlines show high degrees of polymorphism, the spectrum of database-covered genes expressed in individual macaques remains to be determined.

Methods: Here, four rhesus macaques infected with SHIV_SF162P3N became a study of interest because they developed broadly neutralizing antibodies against HIV-1. To identify the immunoglobulin heavy chain V-gene (IGHV) germlines in these macaques, we applied three sequencing approaches - genomic DNA (gDNA) TOPO sequencing, gDNA MiSeq, and messenger RNA (mRNA) MiSeq inference with IgDiscover, and illustrated the detection power of each method.

Results: Of the 197 new rhesus IGHV germline sequences identified, 116 (59%) were validated by at least two methods, and 143 (73%) were found in at least two macaques or two sample sources. About 20% of germlines in each macaque are missing from the current database, including a subset frequently expressed. Overall, gDNA MiSeq determined the greatest number of germline sequences, followed by gDNA TOPO sequencing and mRNA MiSeq inference by IgDiscover, with IgDiscover providing direct evidence of allele expression and usage.

Discussion: Our interdisciplinary study sheds light on germline sequencing, enhances the rhesus IGHV germline database, and highlights the importance of germline sequencing in rhesus immune repertoire studies.

Keywords: IGHV genes; NGS; antibody; database; rhesus macaque.

Figure 1

Workflow of three IGHV sequencing approaches. As illustrated, three sequencing approaches – namely, gDNA TOPO sequencing, gDNA MiSeq, and mRNA MiSeq with IgDiscover, are applied to samples from four rhesus macaques to obtain their IGHV germline sequences.

Figure 2

Phylogenetic trees of germline alleles identified from gDNA MiSeq in each macaque. The gene families are highlighted in red, orange, green, cyan, yellow, purple, and blue, corresponding to IGHV1 through IGHV7, respectively. The number in parentheses indicates the total number of alleles in each animal and gene family. .

Figure 3

Comparison of germline genes derived from gDNA by TOPO Sanger sequencing and MiSeq. (A–D) Pairwise comparison of germline genes obtained from gDNA MiSeq (x-axis) and TOPO Sanger sequencing (y-axis) from each macaque. Identical allele pairs are depicted as red squares, with different gene families denoted by distinct colors. The numbers of common germline sequences between the two methods are listed in each panel.

Figure 4

Comparative analysis of IGHV germline alleles identified through three methods and those in KIMDB. (A) Venn diagram illustrating the number of overlapping genes identified by TOPO sequencing (red), gDNA MiSeq (blue), IgDiscover (green), and KIMDB (yellow). (B) Heatmap showing the unsupervised clustering and similarity of germline alleles derived from all four macaques, as identified through gDNA TOPO sequencing, gDNA MiSeq, and IgDiscover.

Figure 5

Cross-donor prevalence and gene family distribution of rhesus IGHV genes. (A) Venn diagram of germline genes shared between this study and KIMDB. For each macaque, the germline genes identified by all three methods are combined. (B) Number of new rhesus IGHV alleles identified for all gene families. (C) Phylogenetic tree of the extended rhesus germline database, with the alleles in KIMDB colored in blue and the new alleles in red. (D) The updated rhesus germline database reduces the SHM levels of IgM repertoire when compared to previous databases.

Figure 6

Divergence and usage frequency of rhesus germline genes. (A) Venn diagram of all germline alleles between this study, IMGT, and KIMDB. (B) 142 IGHV alleles from the VRC study are found in IMGT and KIMDB. 45 alleles are unique to the VRC study. (C) Sequence identity of IGHV alleles from the VRC study in comparison to those identified in this study and those in KIMDB. (D) The majority of frequently used germline genes in each macaque are included in KIMDB. However, multiple individualized alleles are also frequently used in the expressed naïve BCR repertoires. The x-axis displays the top 100 most frequently used germline alleles in each macaque; the y-axis represents the frequency of germline usage in the naïve BCR repertoire of each macaque.