Recent years have also seen other computational methods introduced for immune repertoire construction from RNA-seq data, including V’DJer 10, MiXCR 11, CATT 12 and ImRep 13. Although less sensitive than TCR-seq and BCR-seq, TRUST is able to identify the abundantly expressed and potentially more clonally expanded TCRs/BCRs in the RNA-seq data that are more likely to be involved in antigen binding 9. When applied to The Cancer Genome Atlas (TCGA) tumor RNA-seq data, TRUST revealed profound biological insights into the repertoires of tumor-infiltrating T cells 6 and B cells 8, as well as their associated tumor immunity. Previously we developed the TRUST algorithm 6, 7, 8, utilized to de novo assemble immune receptor repertories directly from tissue or blood RNA-seq data. However, because repertoire sequences from V(D)J recombination and SHM are different from the germline, they are often eliminated in the read-mapping step. Alternatively, RNA-seq data contain expressed TCR and BCR sequences in tissues or peripheral blood mononuclear cells (PBMC).
Repertoire sequencing has been increasingly adopted in infectious disease 1, allergy 2, autoimmune 3, tumor immuology 4 and cancer immunotherapy 5 studies, but it is an expensive assay and consumes valuable tissue samples. Following antigen recognition, BCRs also undergo somatic hypermutations (SHMs) to further improve antigen-binding affinity.
A universal PCR is ultimately carried out to amplify the library and introduce platform-specific adapter sequences, as well as additional sample indices.Both T and B cells can generate diverse receptor (TCR and BCR, respectively) repertoires, through somatic V(D)J recombination, to recognize various external antigens or tumor neoantigens. For enrichment, ligated cDNA molecules are subjected to targeted PCR using one TCR constant-region-specific primer and one universal primer complementary to the adapter. Target enrichment and final library constructionįollowing UMI assignment, target enrichment is performed to ensure that TCR cDNA molecules are sufficiently enriched in the sequenced library. In addition, this ligated adapter also contains the first sample index. Statistically, this process provides 4^12 possible indices per adapter, and each DNA molecule in the sample receives a unique UMI sequence. Prior to target enrichment and library amplification, each original cDNA molecule is assigned a UMI by ligating an adapter containing a 12-base fully random sequence (i.e., the UMI) to the ds-cDNA. This ds-cDNA is then end-repaired and A-tailed in a single-tube protocol. Subsequently, second-strand synthesis occurs, which generates double-stranded cDNA (ds-cDNA). RNA samples are first reverse transcribed into cDNA with TCR-specific RT primers. TCR reverse transcriptase and enrichment panel primers are provided, together with library reagents. The QIAseq Immune Repertoire RNA Library Kit relies on a highly efficient, TCR-specific cDNA synthesis, TCR gene-specific primer enrichment and molecular indexing for accurate and sensitive TCR clonotype and diversity assessment (see figure " QIAseq Immune Repertoire RNA Library workflow"). For data analysis, UMIs and Raw Reads are used to ensure high precision around each clonotype sequence identified. Even when present at only 0.01%, the Jurkat RNA is readily quantifiably identified. Table 1 shows the number of raw reads and the demultiplexed unique captures (UMIs) per Jurkat TCR-alpha and TCR-beta clonotype. Sensitive to at least 0.01% RNA from Jurkat cells was spiked into RNA extracted from peripheral blood mononuclear cells (PBMCs Precision Medicine) at 10%, 1%, 0.1% and 0.01% and used to make an RNA-seq library. The data analysis included with the purchase of the QIAseq Immune Repertoire T-cell receptor panels includes an online portal that seamlessly integrates with Illumina BaseSpace and provides primary read mapping, UMI demultiplexing and reports on sequencing performance, TCR chain usage, CDR3 peptide sequence and length distributions, together with rarefaction and V/D/J usage heat maps. This figure shows the major clonotype of the Jurkat cell, as well as the diversity of the PBMC background. Comprehensive view of the T-cell immune repertoire The heatmaps allow for easy identification of enriched clonotypes across the sample.
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