QIAseq Targeted RNA-seq Panel for T-cell Receptor

For RNA-seq library construction of T-cell receptor (TCR) alpha, beta, gamma and delta genes using 200 pg to 1000 ng of RNA from human or mouse samples.

S_1321_4_LS_GG_QIAseq_Targeted_RNA_Panel_Human_TCR_12

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

QIAseq Targeted RNA Panel Human TCR (12)

Cat. No. / ID:  334651

Includes enzymes, buffers and TCR-alpha, TCR-beta, TCR-gamma and TCR-delta panels
Copy order details
SGD 1,380.00
Log in To see your account pricing.
KitIndex
T-cell Receptor Library Kit
T-cell Receptor Index Kit
Sample type
Human
Mouse
Samples
12
96
Must use 334792 QIAseq 24-Index TCR UDI (24) or 334805 QIAseq 96-Index TCR UDI Set A (96) to complete library construction
Notice: The QIAseq Targeted RNAseq Panel for T-cell Receptor is an upgraded kit and replaces the QIAseq Immune Repertoire RNA Library Kit (cat. no. 333705).
The QIAseq Targeted RNA-seq Panel for T-cell Receptor is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • Unique molecular indexing (UMI) technology removes amplification, duplication and sequencing artifacts to increase accuracy
  • Profile single TCR receptors (TCR-alpha, TCR-beta, TCR-gamma, TCR-delta) or combine panels for a comprehensive immune repertoire
  • Single-tube library construction and versatile panels allow analysis of one, two, three or all four receptors at once
  • Unique dual indexing (UDI) minimizes sample index misassignment
  • Includes access to GeneGlobe Analyze for data analysis, including read alignment and clonotype report

 

Product Details

The QIAseq Targeted RNAseq Panel for T-cell Receptor enables targeted next-generation sequencing (NGS) of the human or mouse expressed T-cell receptor (TCR), which includes four distinct genes: TCR-alpha, TCR-beta, TCR-gamma and TCR-delta. This highly optimized solution incorporates unique molecular indices (UMIs) to facilitate ultrasensitive and accurate characterization of the immune repertoire in cells and tissues starting from 200 pg to 1000 ng of total RNA.

The adaptive immune system is composed of T and B lymphocytes that bind antigens via highly specific T-cell receptors (TCRs) and B cell receptors (BCRs) on their cell surfaces. To recognize a nearly infinite number of potential antigens, extensive sequence diversity of TCRs and BCRs is generated by somatic V(D)J recombination of the TCR and BCR loci, and by subsequent somatic hypermutation and class switch recombination of the BCR upon antigen stimulation. Accurate characterization of the TCR and BCR repertoires is key to understanding adaptive immune responses and has many applications across different fields, including vaccine development, autoimmunity, monitoring treatment response in lymphoid malignancies and immunotherapy.


Compared to traditional methods, NGS provides an unprecedented, high-resolution picture of the immune repertoire. However, many available immune repertoire sequencing methods involve multiplex PCR with primers targeting different V or J regions, which can introduce substantial amplification bias.


The QIAseq Targeted RNAseq Panel for T-cell Receptor utilizes unique molecular indices (UMIs) with QIAseq Enrichment Technology to robustly create targeted RNA-seq libraries for NGS instruments. This library construction approach greatly improves amplification uniformity compared to multiplexed PCR-based V and J primer pools. The incorporation of UMIs before the amplification step further reduces amplification bias and allows for accurate and sensitive TCR clonotype and repertoire diversity assessment.

The QIAseq Targeted RNAseq Panel for T-cell Receptor is a complete Sample to Insight solution for precise characterization of the TCR immune repertoire using NGS. The purchase of the TCR Panel includes access to GeneGlobe Analyze, which contains a highly optimized pipeline for read mapping and clonotype calling. 

 

 

Principle

The QIAseq Targeted RNAseq Panel for T-cell Receptor provides accurate and sensitive TCR clonotype and diversity assessment using QIAGEN Enrichment Technology. TCR-specific primers for reverse transcription and enrichment are provided along with all the necessary library construction reagents. The QIAseq Targeted RNAseq Panel for T-cell Receptor is designed to enrich TCR α, β, γ and σ subunits (individually or combined) in a single reaction using 200 pg to 1000 ng of RNA (see figure  "QIAseq Targeted RNAseq Panel for T-cell Receptor workflow").

 

See figures

Procedure

cDNA synthesis

Start with total RNA from peripheral blood leukocytes, whole blood, enriched T-cells or solid tumors. RNA samples are first reverse-transcribed into cDNA with gene-specific primers. Subsequently, second-strand synthesis occurs, which generates double-stranded cDNA (ds-cDNA). This ds-cDNA is then end-repaired with an A-addition in a single-tube protocol.

Unique Molecular Index (UMI) assignment

Prior to target enrichment and library amplification, each original cDNA molecule is assigned a unique molecular index (UMI) containing up to 18 bases. This is achieved by ligating a random sequence adapter containing the UMI to the ds-cDNA. Statistically, this process provides >268 million possible indices per adapter, with each DNA molecule in the sample receiving a unique UMI sequence.

Target enrichment and final library construction

Following UMI assignment, QIAseq Enrichment Technology is performed to capture TCR cDNA molecules with UMIs into the NGS library. For enrichment, ligated cDNA molecules are subjected to a TCR-specific enrichment using a TCR targeted panel. After enrichment, a universal PCR is performed to amplify the library and introduce unique dual indices. 

NGS adapter and Unique Dual Index (UDI) technologies

The QIAseq Targeted RNAseq Panel for T-cell Receptor requires the QIAseq 24-Index TCR UDI (24) (cat. no. 334792) or QIAseq 96-Index TCR UDI Set A (96) (cat. no. 334805), which contain the TUDI adapters and dual index primers. The UDI design significantly reduces the risk of index bleeding issues (“index hopping”) associated with next-generation sequencing instruments that utilize patterned flow cells. With unique dual indexing (UDI), each sample will be assigned two unique sample indices to overcome the error introduced by image analysis, sequencing error and demultiplexing, and to remove misassignment of sequencing data to the wrong samples.

Next-generation sequencing on Illumina NGS systems

The QIAseq Targeted RNAseq Panel for T-cell Receptor is compatible with Illumina NGS systems (MiSeq, NextSeq 1000/2000 and NovaSeq 6000). The best sequencing results are achieved using 500 or 600 cycle flow cells.

Data analysis

The QIAseq Immune Repertoire pipeline automatically performs all steps necessary to generate TCR diversity and clonotype reports from your raw NGS data. The QIAseq Immune Repertoire pipeline is available through cloud, desktop and server software packages. For cloud-based analysis, researchers can utilize the QIAseq section of GeneGlobe Analyze. For desktop and server support, QIAGEN Genomics Workbench can be used. 

Sample specifications
RNA types Total RNA, FFPE/fragmented RNA and enriched mRNA
Type of library Targeted RNA-seq with UMIs
Gene targets Human: TRA, TRB, TRD, TRG
Mouse: Tcra, Tcrb, Tcrd, Tcrg
Minimum concentration of RNA 40 pg/µL
Volume of RNA per reverse transcription reaction 5 µL
Total volume of cDNA reaction 6 µL
RIN requirements 1 to 10
Minimum DV 600% 30%

 

Sequencing specifications
Research goal
Sample type Survey with shallow sequencing Comprehensive with deep sequencing
Peripheral blood leukocytes: 200 pg to 1000 ng of total RNA 0.016–80 M reads 0.32–1600 M reads
Whole blood: 500 pg to 200 ng of total RNA 0.01–4 M reads 0.2–80 M reads
Tumor tissue: 100 ng to 1000 ng of total RNA 0.12–1.2 M reads 1.2–12 M reads
Purified T-cells: 10 to 10,000 cells of RNA 0.002–2 M reads 0.04–40 M reads

 

Product specifications
Supported index kits (required) 334792    QIAseq 24-Index TCR UDI (24)
334805    QIAseq 96-Index TCR UDI Set A (96)
Recommended control RNA for monitoring library performance (sold separately) QIAGEN XpressRef Universal Total RNA
Recommended library quantification kits (sold separately) QIAseq Library Quant Assay Kit
Cat. No. / ID: 333314
Online data analysis included with kit GeneGlobe Analyze
Standalone desktop data analysis (sold separately) CLC Genomics Workbench
Recommended NGS instruments Illumina MiSeq or NextSeq 1000/2000
Recommended sequencing cycles/length Paired-end, 600 cycles on Illumina NextSeq 1000/2000

Supporting data and figures

Resources

Safety Data Sheets (1)
Certificates of Analysis (1)

FAQ

Which panels are offered with this workflow?
We offer human and mouse TCR panels. You can target each individual alpha, beta, delta, and gamma chain, or any combination of them.
What are the major differences between QIAseq Targeted RNA Panel TCR kit and previous QIAseq Immune Repertoire RNA library kit?
The new QIAseq Targeted RNA Panel has several improvements. First, it was optimized to start with lower amounts of sample. The UMI used in this kit is longer, so that it can accommodate more T-cell clonotypes without risk of UMI collision. This is necessary when working with samples that have high amounts of T-cells. Lastly, an improvement in library design allows the kit to handle low diversity samples, but still obtain high sequencing Quality scores.
FAQ ID - 3999
Can libraries from different product line be mixed and run together in the same run?
Yes. We removed the custom read 1 primer required in the Illumina sequencing process, which means users are now free to mix and match QIAseq Targeted RNA Panel TCR libraries with other Illumina instrument compatible libraries in the same run, even non-QIAseq ones. The long reads kit is recommended such as 500 cycles and 600 cycles kit, so it can cover the needs for different libraries.
FAQ ID - 4005
Do you expect BCR panels with the QIAseq Targeted RNA Panel TCR kit technology to be available in the coming future?
Currently, QIAseq Targeted RNA Panel TCR kit technology is only applicable to TCR applications. A BCR kit may be released in the future.
FAQ ID - 4009
What is the lowest amount of RNA input I can use?
We recommend using 200 pg as it is usually a level that can be quantified, but it can work on lower input T cell RNAs or several T cells with direct cell lysis.
FAQ ID - 4002
What is the best sample QC method to use for the QIAseq Targeted RNA Panel TCR kit workflow?
RNA sample quality can be checked using the QIAxcel, Agilent Bioanalyzer, or Agilent TapeStation. The high-quality RNA provides full length TCR for full clonotype information identification and more sensitive clonotype detection. If FFPE RNA is used with QIAseq Targeted RNA Panel TCR kit, the DV600 is strongly recommended for determining the useful RNA signal from FFPE RNA sample. Quantification based on mass calculations (OD, NanoDrop) cannot reliably measure the amplifiable amounts of RNA that are important for a multiplex PCR-based targeted enrichment NGS workflow, such as with the QIAseq Targeted RNA Panel TCR kit.
FAQ ID - 4006
What software do you use to analyze data?
There are two options, the QIAGEN GeneGlobe QIAseq Targeted RNA Panel TCR Kit Pipeline or the CLC Genomics Workbench TCR plug-in.
FAQ ID - 4004
Is this workflow compatible with sequencers other than those from Illumina?
It is for Illumina sequencer only or any other sequencer that can support Illumina library structure or can convert Illumina sequencer library to their own sequencer.
FAQ ID - 4001
Why do we recommend long reads kit even you don’t report full length TCR information?
The 500 cycles or 600 cycles kit is recommended as it will reduce the ambiguous V gene call.
FAQ ID - 4003
Does QIAGEN have validated automation script for instrument like Hamilton NGS STAR?
QIAseq Targeted RNA TCR kit has no validated automation script for Hamilton NGS STAR for now. As we have other product line that works in similar workflow, it is possible to modify them for TCR kit. Please contact our tech support team to discuss the possibility.
FAQ ID - 4007
Have you tested the panels at very low input such as 10 pg input? What is the expected performance at lower input.
Yes, we can generate library with very low input PBMC or purified T cells RNA, though we don’t recommend that as the quality of very low concentration RNA sample is hard to be determined and it is difficult to troubleshoot if library generation failed. If you want to test lower than the recommended input, please verify the performance with your own test. It is better to use that very low input from limited cells. Otherwise, the very low input RNA from large population of cell will lose their representation.
FAQ ID - 4008