![]() Standard databases (e.g., the international ImMunoGeneTics information system ) for these diverse sequences also remain fairly meager relative to their human counterparts, although there are new tools developed to address these gaps ( 18). As demonstrated by a recent vaccine-related study in rhesus macaque ( 17), correct assembly of these complex regions requires much longer sequencing reads. These gene segments are duplicated in several large loci in the genome, making their correct assembly a major technical challenge, one that has yet to be fully resolved in rhesus macaques despite significant improvements ( 16) and the recent release of a new genome assembly, rheMac10 (GCA_003339765.3). Ig and TCR have two domains: a C region and a V region, which is comprised of a variable (V), joining (J), and in some cases, a diversity (D) gene segment. Developing complete and accurate NHP genomic resources, especially for the immune system, is imperative for efficient translational interpretation ( 14, 15).Ĭritical for mounting adaptive immune responses, Ig and TCR repertoires house an enormous amount of diversity responsible for recognizing a near limitless array of Ags presented through environmental exposures. They are frequently used for vaccine development ( 5) and to model infection with human pathogens, such as Mycobacterium tuberculosis ( 6, 7), HIV ( 8, 9), influenza A virus ( 10), and Zika virus ( 11), among others ( 12, 13). NHPs are key to studying human biology and human diseases because of their close phylogenetic relationship and similar physiology to humans ( 1– 4). Rhesus macaque ( Macaca mulatta) is one of the most commonly used and best-studied nonhuman primate (NHP) animal models. Our improved approach will enable future studies to fully capture rhesus Ig and TCR repertoire diversity and is applicable for improving annotations in any model organism. To overcome these limitations, we designed new rhesus-specific assays that remove the need for primers conventionally targeting variable regions and allow single cell level Ig and TCR repertoire analysis. Consequently, existing strategies using targeted amplification of rearranged variable regions comprised of V(D)J gene segments miss a significant fraction (27–53% and 42–49%) of rhesus Ig/TCR diversity. We show that sequence diversity exists across the entire variable regions of rhesus Ig and TCR transcripts. We constructed, to our knowledge, the first complete reference set for the constant regions of all known isotypes and chain types of rhesus Ig and TCR repertoires. In this study, using long read sequencing, we sequenced four Indian-origin rhesus macaque tissues and obtained high-quality, full-length sequences for over 6000 unique Ig and TCR transcripts, without the need for sequence assembly. However, because of incomplete reference resources, the coverage and accuracy of the traditional targeted amplification strategies for profiling rhesus Ig and TCR repertoires are largely unknown. Rhesus macaques ( Macaca mulatta) are key for modeling human immune responses, placing critical importance on the accurate annotation and quantification of their Ig and TCR repertoires. The diversity of Ig and TCR repertoires is a focal point of immunological studies. ![]()
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