Targeted next generation sequencing (NGS) allows rapid, cost-effective, in-depth analyses of specific areas of the genome. rhTCRseq is a next generation sequencing technique involving rhAmp PCR to characterize the immune system by targeting T cell receptor (TCR) segments.
For the immune system to adapt and respond to antigens, a wide variety of unique TCRs must be recruited. Therefore, the TCRs are a marker of immune system adaptability. The parts of TCRs that recognize antigens and determine TCR diversity are called complementarity determining regions 3 (CDR3), composed of two chains, alpha and beta. These chains consist of segments; alpha comprises V and J segments and beta comprises V, D, and J segments . The arrangement of these segments gives each TCR a unique identity and the ability to identify unique antigens. In addition to improving understanding of basic immunology, knowledge gained from sequencing CDR3 contributes to understanding autoimmune diseases, transplantation response, cancer, HIV, and infectious diseases [2–4].
rhAmp PCR is an RNase H-dependent, nucleic acid amplification method that increases target specificity compared to traditional PCR. In addition to the DNA polymerase, rhAmp PCR requires the enzyme, RNase H2, and uses blocked primers (rhPrimers or rhAmp Primers) in place of conventional PCR primers. The authors developed rhTCRseq using a variant of the IDT rhAmpSeq system, which is an amplicon sequencing method based on rhAmp PCR.
Li and colleagues demonstrate rhTCRSeq on both RNA derived from a single cell and “bulk” RNA. Single cells are first sorted in a 384-well plate before library construction. For bulk RNA, the researchers use 1–50 ng of total RNA per reaction and run 4–8 replicates. Next, they prepared a full-length cDNA library. They amplify the TCRs specifically using rhAmp Primers that contain a unique molecular identifier (UMI), which acts as a tag to identify specific molecules within a sample. rhAmp technology relies heavily on the unique design of the rhAmp primers and the inherent characteristics of RNase H2. As opposed to DNA-only primers used in traditional PCR and qPCR, rhAmp primers contain a single RNA base and a 3′ blocking group that must be removed by RNase H2 before extension by DNA polymerase can occur. The added specificity achieved by this mechanism consistently yields preferential amplification of the fragments you are targeting. After amplification, the TCR sequences are selected using several magnetic bead cleanups before being sequencing on the Illumina MiSeq® sequencing platform.
The authors report that cDNA libraries produced from single-cell RNA had a concentration of 0.5–6.1 ng/µL. The single-cell runs produced 5760–17,619 reads. The results achieved using bulk RNA were dependent on several conditions: the amount of RNA analyzed, the fraction of RNA that is T cells, and the type of sample. For example, fresh frozen tissue always yields more reliable results than formalin-fixed paraffin-embedded (FFPE) material.
The rhTCRseq technology enabled the researchers to increase data yield by producing a higher percentage of complete CDR3 sequences. The method provides highly specific and efficient characterization of the immune system using a simple workflow.