Key Points
PacBio Iso-Seq enables generation of immune repertoire C region references.
Full-length CCS reads benchmark targeted immune repertoire assay efficiencies.
Abstract
The diversity of Ig and TCR repertoires is a focal point of immunological studies. 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. 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. 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. 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. We show that sequence diversity exists across the entire variable regions of rhesus Ig and TCR transcripts. 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. 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. 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.
Footnotes
This work was supported by funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services R21AI120713 (to X.P.). This project has been funded in part with funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under Contracts HHSN272201300010C and HHSN272201800008C (to M.G.) and the National Institutes of Health Office of the Director P51OD010425 (to M.G.).
Movie files used to generate results presented in this article have been submitted to the National Center for Biotechnology Information BioProject (https://www.ncbi.nlm.nih.gov/bioproject/) under accession number PRJNA389440. The sequence reads presented in this article have been submitted to Zenodo (DOI: 10.5281/zenodo.3634898). The C region sequences presented in this article have been submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under accession numbers MT043311–MT043331.
The online version of this article contains supplemental material.
- Received October 22, 2019.
- Accepted April 13, 2020.
- Copyright © 2020 by The American Association of Immunologists, Inc.
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