AUTHOR=Mamedov Ilgar Z., Britanova Olga V., Zvyagin Ivan V., Turchaninova Maria A., Bolotin Dmitriy A., Putintseva Ekaterina V., Lebedev Yuriy B., Chudakov Dmitriy M. TITLE=Preparing Unbiased T-Cell Receptor and Antibody cDNA Libraries for the Deep Next Generation Sequencing Profiling JOURNAL=Frontiers in Immunology VOLUME=4 YEAR=2013 URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2013.00456 DOI=10.3389/fimmu.2013.00456 ISSN=1664-3224 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.