Ultrasensitive DNA Immune Repertoire Sequencing Using Unique Molecular Identifiers. (20th August 2020)

• Record Type: Journal Article
• Authors: Johansson, Gustav; Kaltak, Melita; Rîmniceanu, Cristiana; Singh, Avadhesh K; Lycke, Jan; Malmeström, Clas; Hühn, Michael; Vaarala, Outi; Cardell, Susanna; Ståhlberg, Anders
• Is Part Of: Clinical chemistry. Volume 66:Number 9(2020)
• Page Start: 1228
• Page End: 1237
• Publication Date: 2020-08-20
• Subjects: immune repertoire -- next-generation sequencing -- error-free sequencing -- unique molecular identifier -- UMI -- γδ T cell; Clinical chemistry -- Periodicals; Pharmaceutical chemistry -- Periodicals; Biochemistry -- Periodicals; Biochimie -- Périodiques; Diagnostics biologiques -- Périodiques; Biochemistry; Clinical chemistry; Pharmaceutical chemistry; Biochemistry; Laboratory Techniques and Procedures; Klinische chemie; Periodicals; 616.075605
• DOI: 10.1093/clinchem/hvaa159
• Languages: English
• ISSNs: 0009-9147

Abstract

Abstract: 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.