An open protocol for modeling T Cell Clonotype repertoires using TCRβ CDR3 sequences.

Area of Science:

• Immunology
• Genomics
• Bioinformatics

Background:

• T cell receptor (TCR) repertoire profiling using next-generation sequencing (NGS) is crucial for monitoring adaptive immune system dynamics.
• Genomic DNA-based bulk sequencing is cost-effective but suffers from variable amplification efficiencies due to multiple primer pairs.
• This variability introduces bias in measuring TCR repertoire changes.

Purpose of the Study:

• To develop a cost-effective and open-source method for accurate TCR repertoire profiling.
• To address and correct for amplification bias inherent in multiplex target amplification during bulk sequencing.
• To provide a viable alternative to expensive commercial solutions for TCR repertoire analysis.

Main Methods:

• Utilized an equimolar primer mixture for multiplex target amplification.
• Implemented a single statistical normalization step to correct for amplification bias post-sequencing.
• Compared results from the developed open protocol with a commercial sequencing solution.

Main Results:

• The proposed statistical normalization effectively corrected for amplification bias.
• High concordance was observed between bulk clonality metrics obtained from the open protocol and the commercial solution.
• The developed method demonstrated cost-effectiveness and efficiency.

Conclusions:

• The developed open-source protocol offers an inexpensive and accurate method for TCR repertoire profiling.
• This approach provides a reliable alternative to commercial solutions, enhancing accessibility for researchers.
• The normalization strategy effectively mitigates bias in NGS-based bulk sequencing of TCR repertoires.