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<p>T cell receptor (TCR) sequencing enables high-resolution characterization of the adaptive immune repertoire and is increasingly applied in clinical research and disease monitoring. However, many existing workflows rely on fresh peripheral blood mononuclear cells (PBMCs), highly purified T cell populations, or specialized equipment such as cell sorters, limiting their accessibility in routine laboratory settings and biobank-derived samples. Here, we present a practical and robust workflow for bulk TCR sequencing using buffy coat as the starting material, without requiring T cell enrichment. We assessed key pre-analytical factors affecting RNA yield and integrity—red blood cell contamination, DMSO concentration during cryopreservation, and blood collection tube type—and identified conditions that minimize RNA degradation. We also introduce a TRAC-targeting RT-qPCR assay as a cost-effective quality control step to quantify T cell-specific RNA prior to library preparation. We compared three commercial library preparation kits and found that the NEBNext® Immune Sequencing Kit reproducibly yields the highest clonotype recovery and lowest noise, even without enrichment. Finally, we provide a streamlined, step-by-step workflow optimized for clinical and research laboratories processing whole blood-derived buffy coat samples. This accessible protocol supports reliable repertoire profiling across variable sample qualities and offers a practical alternative to more labor-intensive T cell purification workflows.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月14日

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  • Jolene Caifeng Ho,
  • Ah-Jung Jeon,
  • Ming Xue Wee,
  • Seng Chong Teo,
  • Suit-Fong Chan,
  • He Cheng

摘要

T cell receptor (TCR) sequencing enables high-resolution characterization of the adaptive immune repertoire and is increasingly applied in clinical research and disease monitoring. However, many existing workflows rely on fresh peripheral blood mononuclear cells (PBMCs), highly purified T cell populations, or specialized equipment such as cell sorters, limiting their accessibility in routine laboratory settings and biobank-derived samples. Here, we present a practical and robust workflow for bulk TCR sequencing using buffy coat as the starting material, without requiring T cell enrichment. We assessed key pre-analytical factors affecting RNA yield and integrity—red blood cell contamination, DMSO concentration during cryopreservation, and blood collection tube type—and identified conditions that minimize RNA degradation. We also introduce a TRAC-targeting RT-qPCR assay as a cost-effective quality control step to quantify T cell-specific RNA prior to library preparation. We compared three commercial library preparation kits and found that the NEBNext® Immune Sequencing Kit reproducibly yields the highest clonotype recovery and lowest noise, even without enrichment. Finally, we provide a streamlined, step-by-step workflow optimized for clinical and research laboratories processing whole blood-derived buffy coat samples. This accessible protocol supports reliable repertoire profiling across variable sample qualities and offers a practical alternative to more labor-intensive T cell purification workflows.