QIAseq Immune Repertoire RNA Library Kits
- Unique Molecular Indices (UMI) ensure accurate sequencing results
- Online data analysis through GeneGlobe
- Includes QIAseq Beads for reaction cleanup
- Automation-friendly protocol forhuman or mouse samples
- QIAseq Sample Indices ordered separately to multiplex up to 384 samples
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Cat No./ID:333705 QIAseq Immune Repertoire RNA Library Kit Go to GeneGlobe |
Product Details
Comprehensive view of the T-cell immune repertoire
The heatmaps allow for easy identification of enriched clonotypes across the sample.This figure shows the major clonotype of the Jurkat cell, as well as the diversity of the PBMC background.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.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. Table 1 shows the number of raw reads and the demultiplexed unique captures (UMIs) per Jurkat TCR-alpha and TCR-beta clonotype. Even when present at only 0.01%, the Jurkat RNA is readily quantifiably identified. For data analysis, UMIs and Raw Reads are used to ensure high precision around each clonotype sequence identified.| Chain | % Jurkat cells | Rank | Reads | UMIs |
|---|---|---|---|---|
| TCR-alpha | 10 | 1 | 751,749 | 107,150 |
| 1 | 1 | 146,959 | 20,692 | |
| 0.1 | 1 | 10,708 | 1,742 | |
| 0.01 | 10 | 1,306 | 217 | |
| TCR-beta | 10 | 1 | 383,594 | 40,943 |
| 1 | 1 | 5,920 | 7,541 | |
| 0.1 | 2 | 5,401 | 620 | |
| 0.01 | 61 | 457 | 60 |
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.
cDNA synthesis
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.
UMI assignment
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.
Target enrichment and final library construction
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.
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