Open AccessUltrasensitive DNA Immune Repertoire Sequencing Using Unique Molecular Identifiers
Author(s) -
Gustav Johansson,
Melita Kaltak,
Cristiana Rîmniceanu,
Avadhesh Kumar Singh,
Jan Lycke,
Clas Malmeström,
Michael Hühn,
Outi Vaarala,
Susanna Cardell,
Anders Ståhlberg
Publication year - 2020
Publication title -
clinical chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.705
H-Index - 218
eISSN - 1530-8561
pISSN - 0009-9147
DOI - 10.1093/clinchem/hvaa159
Subject(s) - biology , immune system , computational biology , repertoire , dna sequencing , deep sequencing , polymerase chain reaction , t cell receptor , immunology , t cell , microbiology and biotechnology , genetics , dna , genome , gene , physics , acoustics
Background Immune repertoire sequencing of the T-cell receptor can identify clonotypes that have expanded as a result of antigen recognition or hematological malignancies. However, current sequencing protocols display limitations with nonuniform amplification and polymerase-induced errors during sequencing. Here, we developed a sequencing method that overcame these issues and applied it to γδ T cells, a cell type that plays a unique role in immunity, autoimmunity, homeostasis of intestine, skin, adipose tissue, and cancer biology. Methods The ultrasensitive immune repertoire sequencing method used PCR-introduced unique molecular identifiers. We constructed a 32-panel assay that captured the full diversity of the recombined T-cell receptor delta loci in γδ T cells. The protocol was validated on synthetic reference molecules and blood samples of healthy individuals. Results The 32-panel assay displayed wide dynamic range, high reproducibility, and analytical sensitivity with single-nucleotide resolution. The method corrected for sequencing-depended quantification bias and polymerase-induced errors and could be applied to both enriched and nonenriched cells. Healthy donors displayed oligoclonal expansion of γδ T cells and similar frequencies of clonotypes were detected in both enrichment and nonenriched samples. Conclusions Ultrasensitive immune repertoire sequencing strategy enables quantification of individual and specific clonotypes in a background that can be applied to clinical as well as basic application areas. Our approach is simple, flexible, and can easily be implemented in any molecular laboratory.