Novel Rhesus Macaque Immunoglobulin Germline Genes Identified by Three Sequencing Approaches

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, it is unclear how the current germline database covers the antibody genes expressed in individual macaques. Here, four rhesus macaques infected with SHIVSF162P3N 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 mRNA MiSeq with IgDiscover, and illustrated the detection power of each method. Over 180 new rhesus IGHV germline sequences have been identified, and 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 with IgDiscover, although the last is the most convenient as the expressed transcript MiSeq data are usually readily available in most antibody repertoire studies. In summary, our study sheds light on germline sequencing, enhances the rhesus IGHV germline database, and highlights the importance of germline sequencing in rhesus immune repertoire studies.