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Open Access

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

Hayden N. Brochu, Elizabeth Tseng, Elise Smith, Matthew J. Thomas, Aiden M. Jones, Kayleigh R. Diveley, Lynn Law, Scott G. Hansen, Louis J. Picker, Michael Gale Jr. and Xinxia Peng
J Immunol May 6, 2020, ji1901256; DOI: https://doi.org/10.4049/jimmunol.1901256
Hayden N. Brochu
*Department of Molecular Biomedical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC 27607;
†Bioinformatics Graduate Program, North Carolina State University, Raleigh, NC 27695;
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Elizabeth Tseng
‡Pacific Biosciences, Menlo Park, CA 94025;
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Elise Smith
§Department of Immunology, University of Washington, Seattle, WA 98109;
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Matthew J. Thomas
§Department of Immunology, University of Washington, Seattle, WA 98109;
¶Center for Innate Immunity and Immune Diseases, University of Washington, Seattle, WA 98109;
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Aiden M. Jones
*Department of Molecular Biomedical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC 27607;
‖Genetics Graduate Program, North Carolina State University, Raleigh, NC 27695;
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  • ORCID record for Aiden M. Jones
Kayleigh R. Diveley
*Department of Molecular Biomedical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC 27607;
‖Genetics Graduate Program, North Carolina State University, Raleigh, NC 27695;
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Lynn Law
§Department of Immunology, University of Washington, Seattle, WA 98109;
¶Center for Innate Immunity and Immune Diseases, University of Washington, Seattle, WA 98109;
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Scott G. Hansen
#Vaccine and Gene Therapy Institute, Oregon Health & Science University, Beaverton, OR 97006;
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Louis J. Picker
#Vaccine and Gene Therapy Institute, Oregon Health & Science University, Beaverton, OR 97006;
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Michael Gale Jr.
§Department of Immunology, University of Washington, Seattle, WA 98109;
¶Center for Innate Immunity and Immune Diseases, University of Washington, Seattle, WA 98109;
**Washington National Primate Research Center, University of Washington, Seattle, WA 98121; and
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Xinxia Peng
*Department of Molecular Biomedical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC 27607;
†Bioinformatics Graduate Program, North Carolina State University, Raleigh, NC 27695;
††Bioinformatics Research Center, North Carolina State University, Raleigh, NC 27695
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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.

This article is distributed under The American Association of Immunologists, Inc., Reuse Terms and Conditions for Author Choice articles.

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The Journal of Immunology: 206 (3)
The Journal of Immunology
Vol. 206, Issue 3
1 Feb 2021
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Systematic Profiling of Full-Length Ig and TCR Repertoire Diversity in Rhesus Macaque through Long Read Transcriptome Sequencing
Hayden N. Brochu, Elizabeth Tseng, Elise Smith, Matthew J. Thomas, Aiden M. Jones, Kayleigh R. Diveley, Lynn Law, Scott G. Hansen, Louis J. Picker, Michael Gale, Xinxia Peng
The Journal of Immunology May 6, 2020, ji1901256; DOI: 10.4049/jimmunol.1901256

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Systematic Profiling of Full-Length Ig and TCR Repertoire Diversity in Rhesus Macaque through Long Read Transcriptome Sequencing
Hayden N. Brochu, Elizabeth Tseng, Elise Smith, Matthew J. Thomas, Aiden M. Jones, Kayleigh R. Diveley, Lynn Law, Scott G. Hansen, Louis J. Picker, Michael Gale, Xinxia Peng
The Journal of Immunology May 6, 2020, ji1901256; DOI: 10.4049/jimmunol.1901256
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Print ISSN 0022-1767        Online ISSN 1550-6606