Development and application of a next-generation sequencing protocol and bioinformatics pipeline for the comprehensive analysis of the canine immunoglobulin repertoire

Jonah N. Cullen, Jolyon Martin, Albert J. Vilella, Amy Treeful, David Sargan, Allan Bradley, Steven G. Friedenberg

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Profiling the adaptive immune repertoire using next generation sequencing (NGS) has become common in human medicine, showing promise in characterizing clonal expansion of B cell clones through analysis of B cell receptors (BCRs) in patients with lymphoid malignancies. In contrast, most work evaluating BCR repertoires in dogs has employed traditional PCR-based approaches analyzing the IGH locus only. The objectives of this study were to: (1) describe a novel NGS protocol to evaluate canine BCRs; (2) develop a bioinformatics pipeline for processing canine BCR sequencing data; and (3) apply these methods to derive insights into BCR repertoires of healthy dogs and dogs undergoing treatment for B-cell lymphoma. RNA from peripheral blood mononuclear cells of healthy dogs (n = 25) and dogs newly diagnosed with intermediate-to-large B-cell lymphoma (n = 18) with intent to pursue chemotherapy was isolated, converted into cDNA and sequenced by NGS. The BCR repertoires were identified and quantified using a novel analysis pipeline. The IGK repertoires of the healthy dogs were far less diverse compared to IGL which, as with IGH, was highly diverse. Strong biases at key positions within the CDR3 sequence were identified within the healthy dog BCR repertoire. For a subset of the dogs with B-cell lymphoma, clonal expansion of specific IGH sequences pre-treatment and reduction post-treatment was observed. The degree of expansion and reduction correlated with the clinical outcome in this subset. Future studies employing these techniques may improve disease monitoring, provide earlier recognition of disease progression, and ultimately lead to more targeted therapeutics.

Original languageEnglish (US)
Article numbere0270710
JournalPloS one
Issue number7 July
StatePublished - Jul 2022

Bibliographical note

Funding Information:
JC was funded by a United State Department of Agriculture National Institute of Food and Agriculture Predoctoral Fellowship as part of the Agriculture and Food Research Initiative ( (2020-67034-31739). JM was funded by a studentship from the BBSRC Cambridge Bioscience Doctoral Training Partnership ( and the Wellcome Trust Sanger Institute PhD Core Funding. Partial funding for this work was provided by a University of Minnesota Academic Health Center seed grant provided to SGF by the Office for the Vice President of Research. Partial support for SGF was provided by a National Institutes of Health ORIP K01 Mentored Research Scientist Development Award (K01-OD027058) ( The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Publisher Copyright:
Copyright: © 2022 Cullen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


  • Animals
  • Computational Biology
  • Dogs
  • High-Throughput Nucleotide Sequencing
  • Leukocytes, Mononuclear
  • Lymphoma, B-Cell
  • Receptors, Antigen, B-Cell/genetics

PubMed: MeSH publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Journal Article
  • Research Support, N.I.H., Extramural


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