The QIAseq Immune Repertoire RNA Library Kits use Unique Molecular Indices (UMI) with gene-specific primers to target specific RNAs for NGS sequencing. Each unique panel is carefully designed and laboratory-verified for sequencing performance with a UMI-aware alignment software for maximum sequencing performance and accurate results. The Human and Mouse T-cell Receptors Panel is used for sequencing the V(D)J region of the alpha, beta, delta and gamma genes, including the CDR3 regions. Online analysis through the GeneGlobe Data Analysis Center provides key sequencing QC metrics, as well as the frequency and identity of each clonotype sequenced.
|Chain||% Jurkat cells||Rank||Reads||UMIs|
|TCR-alpha|| 10 || 1 || 751,749 ||107,150 |
|TCR-beta||10||1|| 383,594 || |
|1||1||5,920 || |
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"). TCR reverse transcriptase and enrichment panel primers are provided, together with library reagents.
RNA samples are first reverse transcribed into cDNA with TCR-specific RT primers. Subsequently, second-strand synthesis occurs, which generates double-stranded cDNA (ds-cDNA). This ds-cDNA is then end-repaired and A-tailed in a single-tube protocol.
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. Statistically, this process provides 4^12 possible indices per adapter, and each DNA molecule in the sample receives a unique UMI sequence. In addition, this ligated adapter also contains the first sample index.
Following UMI assignment, target enrichment is performed to ensure that TCR cDNA molecules are sufficiently enriched in the sequenced library. 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. A universal PCR is ultimately carried out to amplify the library and introduce platform-specific adapter sequences, as well as additional sample indices.