Preparing unbiased T-cell receptor and antibody cDNA libraries for the deep next generation sequencing profiling

Abstract

High-throughput sequencing has the power to reveal the nature of adaptive immunity as represented by the full complexity of T-cell receptor (TCR) and antibody (IG) repertoires, but is at present severely compromised by the quantitative bias, bottlenecks, and accumulated errors that inevitably occur in the course of library preparation and sequencing. Here we report an optimized protocol for the unbiased preparation of TCR and IG cDNA libraries for high-throughput sequencing, starting from thousands or millions of live cells in an investigated sample. Critical points to control are revealed, along with tips that allow researchers to minimize quantitative bias, accumulated errors, and cross-sample contamination at each stage, and to enhance the subsequent bioinformatic analysis. The protocol is simple, reliable, and can be performed in 1-2 days.

Keywords: BCR repertoires; IG repertoires; MiTCR; NGS applications; T-cell receptor; T-cell receptor repertoire; TCR repertoires; cDNA libraries.

Figure 1

Flow-chart of the library preparation protocol from RNA and to NGS-ready PCR product. XXXXX: optional sample barcodes (see Sample Barcoding in Appendix for details and Supplementary Material for barcodes). *For TCR alpha/beta profiling with 100 nt sequencing length, multiplexed J-segment-specific primers should be used as a reverse primer in the second PCR amplification step as described in section “Next Generation Sequencing Options.”

Figure 2

MiTCR-viewer outputs for the analyzed TCR beta dataset. (A) Table with clonotypes. (B) In silico spectratyping.