Identification of Variable and Joining Germline Genes and Alleles for Rhesus Macaque from B Cell Receptor Repertoires

Abstract

The rhesus macaque is a valuable preclinical animal model to estimate vaccine effectiveness and is also important for understanding Ab maturation and B cell repertoire evolution responding to vaccination. However, incomplete mapping of rhesus Ig germline genes hinders the research efforts. To address this deficiency, we sequenced the BCR repertoires of 75 Indian rhesus macaques. Using a bioinformatic method that has been validated with BCR repertoire analysis of three human donors, we were able to infer rhesus variable (V) and joint (J) germline alleles. We identified a total of 122 V and 20 J germline alleles, of which 91 V and 13 J alleles were novel, with 40 V novel genes, of which 8 were located at a novel genomic region not, to our knowledge, previously recorded. The novelty of these newly identified alleles was supported by two observations. First, the 50 V and 5 J novel alleles were observed in the whole genome sequencing data of 10 rhesus macaques. Second, using alignment reference including the novel alleles, the mutation rate of the rearranged repertoires significantly declined in nine other irrelevant samples, and all our identified novel V and J alleles were 100%-identity mapped by rearranged repertoire data. These identified novel alleles, along with the previously reported alleles, provide an important reference for future investigations of rhesus immune repertoire evolution in response to vaccination or infection. In addition, the method outlined in our study offers a powerful foundation for the identification of novel Ig alleles in the future.

Rhesus and cynomolgus macaque immunoglobulin heavy-chain genotyping yields comprehensive databases of germline VDJ alleles

“…Computational inference from high-throughput sequencing of expressed recombined VDJ (variability, diversity, and joining) repertoires (Rep-seq) has emerged as a powerful approach for the identification of functional germline Ig alleles in outbred populations. In macaques, two tools were used to identify germline Ig alleles, IgDiscover (Corcoran et al, 2016) and IMPre (Zhang et al, 2019). IgDiscover includes modules that support the tracing of antigen-specific Ab lineages in Rep-seq data (Kong et al, 2019;Martinez-Murillo et al, 2017;Phad et al, 2020).…”

Systematic Profiling of Full-Length Ig and TCR Repertoire Diversity in Rhesus Macaque through Long Read Transcriptome Sequencing

“…A more recent rhesus-specific MPCR design aimed to expand coverage of the Ig repertoire (32). There were also attempts to improve rhesus V and J germline gene annotation using 59 RACE sequencing (RACE-seq) (18,33). The recently developed IgDiscover tool (18) now makes it possible to leverage germline databases of related species to improve those of model organisms, for example using human germline databases to study Indian-and Chinese-origin rhesus macaques.…”

Systematic profiling of full-length immunoglobulin and T-cell receptor repertoire diversity in rhesus macaque through long read transcriptome sequencing

“…We then assessed the sequence quality of these Ig and TCR transcripts, by quantifying insertion and deletion events within the alignments of their constant region sequences and the corresponding reference cDNA sequences. We elected not to evaluate the rate of substitutions, as they are difficult to discern from allelic variation given our sequencing depth and are generally less common than insertions and deletions in CCS reads [32]. The rate of insertions in Ig constant region sequences was very low, with a mean of 0.11% and median of 0.06%.…”

“…A more recent rhesus-specific MPCR design aimed to expand coverage of the Ig repertoire [31]. There were also attempts to improve rhesus V and J germline gene annotation using 5’ RACE sequencing (RACE-seq) [17, 32]. The recently developed IgDiscover tool [17] now makes it possible to leverage germline databases of related species to improve those of model organisms, for example using human germline databases to study Indian- and Chinese-origin rhesus macaques.…”