A Robust Quantitative Multiplex PCR for T-cell Receptor Repertoire Sequencing from Formalin-Fixed Paraffin-Embedded...

Area of Science:

• Immunology
• Genomics
• Bioinformatics

Background:

• T-cell receptor (TCR) analysis is crucial for understanding the tumor microenvironment and immune responses to cancer therapies.
• Challenges exist in amplifying TCR libraries from degraded nucleic acids found in clinical tumor samples, particularly formalin-fixed paraffin-embedded (FFPE) tissues.
• Accurate TCR repertoire profiling is essential for insights into cancer progression, regression, and the efficacy of immune checkpoint inhibitors.

Purpose of the Study:

• To develop a robust, cost-effective protocol for generating high-quality TCR repertoire data from diverse and low-quality RNA sources.
• To enable TCR repertoire analysis from archived FFPE tumor samples, expanding the scope of cancer immunology research.
• To integrate this method with the Decombinator computational pipeline for accurate TCR annotation.

Main Methods:

• Developed FFPE-suitable Unique Molecular idEntifier-based TCRseq (FUME-TCRseq) protocol.
• Utilized multiplex PCR to amplify the TCRbeta CDR3 region with V and J gene sequences.
• Incorporated a 12-base pair unique molecular identifier (UMI) for error and bias correction during sequencing.

Main Results:

• The FUME-TCRseq method successfully generates TCR repertoires from various RNA sources, including low-concentration, poor-quality RNA from FFPE tissues.
• The protocol is robust, inexpensive, and suitable for integration with computational TCR annotation pipelines like Decombinator.
• The use of UMIs effectively corrects for PCR bias and sequencing errors, improving data accuracy.

Conclusions:

• FUME-TCRseq overcomes limitations of analyzing TCR repertoires from challenging clinical samples.
• This method unlocks the potential of archived tumor samples for detailed immunological studies.
• Enhanced TCR repertoire analysis can lead to a deeper understanding of tumor immunology and immunotherapeutic interventions.