Ultrasensitive DNA Immune Repertoire Sequencing Using Unique Molecular Identifiers

Area of Science:

• Immunology
• Molecular Biology
• Genomics

Background:

• T-cell receptor (TCR) sequencing identifies expanded clonotypes in immune responses and diseases.
• Current TCR sequencing methods suffer from amplification bias and sequencing errors.
• γδ T cells have critical roles in immunity, autoimmunity, and cancer, but their repertoire analysis is challenging.

Purpose of the Study:

• To develop an ultrasensitive immune repertoire sequencing method to overcome limitations of existing protocols.
• To apply this novel method for accurate analysis of γδ T-cell repertoires.
• To enable precise quantification of individual T-cell clonotypes.

Main Methods:

• Developed an ultrasensitive immune repertoire sequencing strategy using unique molecular identifiers (UMIs).
• Constructed a 32-panel assay to capture the full diversity of T-cell receptor delta loci in γδ T cells.
• Validated the protocol using synthetic molecules and blood samples from healthy donors.

Main Results:

• The 32-panel assay demonstrated a wide dynamic range, high reproducibility, and single-nucleotide resolution.
• The method effectively corrected for sequencing quantification bias and polymerase errors.
• Oligoclonal expansion of γδ T cells was observed in healthy donors, with consistent clonotype frequencies in enriched and non-enriched samples.

Conclusions:

• Ultrasensitive immune repertoire sequencing allows accurate quantification of specific clonotypes.
• This flexible and simple approach is applicable to both basic research and clinical settings.
• The method facilitates deeper understanding of immune responses and disease states involving T cells.