A practical and accessible workflow for bulk TCR sequencing from buffy coat samples without T cell enrichment

Area of Science:

• Immunology
• Molecular Biology
• Bioinformatics

Background:

• T cell receptor (TCR) sequencing is crucial for adaptive immune repertoire analysis in clinical research.
• Current methods often require fresh cells, T cell purification, or specialized equipment, limiting accessibility.
• There is a need for practical workflows applicable to routine labs and biobank samples.

Purpose of the Study:

• To develop and validate a robust, accessible workflow for bulk TCR sequencing using buffy coat.
• To optimize pre-analytical conditions for RNA yield and integrity from buffy coat.
• To identify the most effective library preparation kit for TCR sequencing from buffy coat.

Main Methods:

• Developed a TCR sequencing workflow starting with buffy coat, eliminating the need for T cell enrichment.
• Assessed pre-analytical factors including red blood cell contamination, cryopreservation (DMSO), and blood collection tube type.
• Implemented a TRAC-targeting RT-qPCR assay for quality control and compared three commercial library preparation kits.

Main Results:

• Identified optimal conditions to minimize RNA degradation from buffy coat samples.
• The TRAC RT-qPCR assay proved effective for quantifying T cell-specific RNA.
• The NEBNext® Immune Sequencing Kit demonstrated superior clonotype recovery and lower noise without enrichment.

Conclusions:

• The presented buffy coat-based TCR sequencing workflow is practical and robust for clinical and research laboratories.
• This accessible protocol enables reliable adaptive immune repertoire profiling from variable sample qualities.
• It provides a viable alternative to more complex T cell purification methods.