NET-Seq

TCR-LA-MC PCR

TCR-LA-MC PCR identifies TCR-alpha and -beta chains from T cells and uses sequencing to analyze the catalog of clonal TCR in vivo or in vitro from blood or tissue samples. This technique can be performed with as little as 10 ng of cDNA, while providing great sensitivity and accuracy for analyzing the diversity and mechanisms that affect TCR clonality. First-strand cDNA is generated using biotinylated primers that anneal to the constant gene of the TCR chains. RNA strands are removed by RNA digestion, and the single-stranded cDNA is captured magnetically using streptavidin beads. Single-stranded linker cassettes (ssLCs) containing primer sequences are ligated to the cDNA, and the samples are PCR-amplified. The double-stranded cDNAs are flanked with sequencing adapters and are ready for sequencing.

Similar methods: TCR chain pairing.

Pros:

• Identifies TCR diversity without sequence-associated or quantitative restrictions
• Can be used to study the diversity and mechanism of TCRs that influences clonality
• As little as 10 ng of cDNA can be used as input
• Identifies T-cells with 1:10,000 resolution capacity, or even single cells

Cons:

• None reported yet

• TCR-LA-MC PCR: Ruggiero E., Nicolay J. P., Fronza R., et al. High-resolution analysis of the human T-cell receptor repertoire. Nat Commun. 2015;6:8081

1. Hou D., Chen C., Seely E. J., Chen S. and Song Y. High-Throughput Sequencing-Based Immune Repertoire Study during Infectious Disease. Front Immunol. 2016;7:336

1. Oliveira G., Ruggiero E., Stanghellini M. T., et al. Tracking genetically engineered lymphocytes long-term reveals the dynamics of T cell immunological memory. Sci Transl Med. 2015;7:317ra198