A review of canine B cell clonality assays and primer set optimization using large-scale repertoire data

Mei Hua Hwang, Nikos Darzentas, Dorothee Bienzle, Peter F Moore, Franco Guscetti, Jodi Morrison, Stefan M. Keller

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Several molecular clonality assays have been developed to assess canine B cell proliferations. These assays were based on different sequence data, utilized different assay designs and employed different testing strategies. This has resulted in a complex body of literature and complicates evidence-based selection of primer sets. In addition, further refinement of primer sets is difficult because it is unknown how well current primer sets cover the expressed sequence repertoire. The objectives of this study were 1) to provide an overview of published IGH clonality assays that highlights key differences in assay design and testing strategy and 2) to propose a novel method for optimizing primer sets that leverages large-scale sequencing data. A review of previously published assays highlighted confounding factors that hamper a direct comparison of performance metrics between studies. These findings illustrate the need for a multi-institutional effort to harmonize veterinary clonality testing. A novel in silico analysis of primer sequences using a large dataset of expressed sequences identified shortfalls of existing primer sets and was used to guide primer optimization. Three optimized primer sets were tested and yielded qualitative sensitivity values between 80–90%. The qualitative sensitivity ranged from 1% to over 50% and was dependent on the size of the neoplastic clone and the sample DNA used. These findings illustrate that inclusion of high-throughput sequencing data for primer design can be a useful tool to guide primer design and optimization. This strategy could be applied to other antigen receptor loci or species to further improve veterinary clonality assays.

Original languageEnglish (US)
Pages (from-to)45-52
Number of pages8
JournalVeterinary Immunology and Immunopathology
Volume209
DOIs
StatePublished - Mar 1 2019

Fingerprint

Antigen Receptors
Computer Simulation
B-lymphocytes
Sequence Analysis
Canidae
B-Lymphocytes
Clone Cells
Cell Proliferation
dogs
DNA
assays
testing
Datasets
cell proliferation
clones
antigens
receptors
loci

Keywords

  • B cell lymphoma
  • Clonality testing
  • Dog
  • Lymphoma
  • PARR
  • Sensitivity
  • Specificity

ASJC Scopus subject areas

  • Immunology
  • veterinary(all)

Cite this

A review of canine B cell clonality assays and primer set optimization using large-scale repertoire data. / Hwang, Mei Hua; Darzentas, Nikos; Bienzle, Dorothee; Moore, Peter F; Guscetti, Franco; Morrison, Jodi; Keller, Stefan M.

In: Veterinary Immunology and Immunopathology, Vol. 209, 01.03.2019, p. 45-52.

Research output: Contribution to journalArticle

Hwang, Mei Hua ; Darzentas, Nikos ; Bienzle, Dorothee ; Moore, Peter F ; Guscetti, Franco ; Morrison, Jodi ; Keller, Stefan M. / A review of canine B cell clonality assays and primer set optimization using large-scale repertoire data. In: Veterinary Immunology and Immunopathology. 2019 ; Vol. 209. pp. 45-52.
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