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![]() ![]() Second, abundant copies of mRNAs encoding TCR or IG chains comprise an essential portion of the total lymphocyte RNA. Multiplexing inevitably leads to dramatic bias in relative efficiency of amplification of different variable segments and thus to the loss of quantitative information, and complete loss of some of the rare clonotypes ( 10, 16, 22, 23). Moreover, a subsequent nested PCR amplification, which requires another set of multiplex primers, can be necessary to obtain pure TCR or IG library from genomic DNA. In contrast, the approaches starting with the genomic DNA require multiplex primer sets to be used both at the 5′ V gene segments’ end, and at the 3′ introns/J-segments end of the library ( 2). Starting with cDNA synthesis using 5′-template switching ( 16, 20, 21) has at least two decisive advantages in comparison with the genomic DNA-based approaches ( 2, 12).įirst, the whole diversity of variable chains (up to approximately 100 different V gene segment variants 1, can be amplified using just a pair (for TCRs) or a simple multiplex set (for IGs) of oligonucleotides, specific to the template switch adapter on the 5′-end and to the constant gene segments on the 3′-end of the library (Figure 1). Advantages of cDNA Libraries and 5′-Template Switch Here we report the upgraded and tested protocol in a ready-to-use format with the technical details required for the method to be easily and uniformly reproduced in any laboratory. Current protocol is optimal for the sequencing on Illumina MiSeq/HiSeq platforms and Roche 454 platforms. ![]() This approach allows efficient and unbiased amplification of millions of the TCR/IG mRNA molecules in only 27–30 (21–24 considering dilution factor, see below) PCR cycles, thus providing sufficient starting material for the deep NGS-analysis of complex lymphocyte samples. #THE HUMAN ABSTRACT FULL DISCOGRAPHY TORRENT PLUS#Subsequent two-step PCR amplification is performed with universal pairs of primers for the whole library using step-out plus PCR-suppression effect ( 18) on the 5′-end and nested PCR ( 19) on the 3′-end of the library ( 16). The protocol employs a specific oligonucleotide to prime cDNA synthesis, and template switching effect to form a universal 5′-adapter and to introduce sample barcode at the very first stage of library preparation. While studying autoimmunity and hematopoietic stem cell transplantation therapy ( 10, 14– 17), we have optimized cDNA-based protocol that allows unbiased pre-sequencing amplification of the human and murine, alpha- and beta-TCR, as well as IG heavy chain gene libraries. Multiple sampling bottlenecks, PCR biases, and cross-contamination at different stages lie in wait to trick a researcher on his way to get the deep, clear, and congruent data. Still, rational NGS-analysis of such immune repertoires is critically dependent on the library preparation protocols, starting from a lymphocytes/PBMC sample and ending with the amplification of individual TCR/IG segment encoding molecules on the solid phase of a sequencing machine. Next generation sequencing (NGS) technologies opened a breathtaking opportunity to perform deep analysis and comparative studies of the T-cell receptor (TCR) and antibody (IG) repertoires of the human donors and model animals, as well as of the various sorted, separated, or cultured lymphocyte subsets of interest ( 1– 13). The protocol is simple, reliable, and can be performed in 1–2 days. 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. 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. 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. ![]()
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