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Ultrasensitive DNA Immune Repertoire Sequencing Using Unique Molecular Identifiers.

Clinical chemistry (2020-08-21)
Gustav Johansson, Melita Kaltak, Cristiana Rîmniceanu, Avadhesh K Singh, Jan Lycke, Clas Malmeström, Michael Hühn, Outi Vaarala, Susanna Cardell, Anders Ståhlberg
ABSTRACT

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. 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. 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. 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Protease from Streptomyces griseus, Type XIV, ≥3.5 units/mg solid, powder
Sigma-Aldrich
Deoxynucleotide Mix, 10 mM, Molecular Biology Reagent