CAT #: 72270007
LymphoTrack® TRG Assay - S5/PGM™
This Research Use Only assay identifies clonal TRG Vγ–Jγ rearrangements, the associated Vγ–Jγ region DNA sequences and provides the distribution frequency of Vγ region and Jγ region segment utilization using the Ion S5 and Ion PGM platforms.
Summary and Explanation of the Test
This LymphoTrack TRG Assay – S5/PGM represents a significant improvement over existing clonality assays using fragment analysis as it efficiently detects the vast majority of TRG gene rearrangements using a single multiplex master mix and, at the same time, the assay identifies the DNA sequence specific for each clonal gene rearrangement. Therefore, this product has two important and complementary uses: it both aids in the detection of initial clonal populations, and it identifies sequence information required to track those clones in subsequent samples.
Each single multiplex master mix for TRG targets the conserved regions within the Vγ and the Jγ regions described in lymphoid malignancies. Primers included in the master mixes are designed with Thermo Fisher Scientific adapters and 12 different indices. This assay allows for a one-step PCR and pooling of amplicons from several different samples and targets (generated with other LymphoTrack Assays for the Ion S5 or Ion PGM, sold separately) onto one Ion S5 or PGM chip, allowing up to 12 samples per target to be analyzed in parallel, in a single sequencing run.
The associated LymphoTrack Software – S5/PGM provides a simple and streamlined method of analysis and visualization of data.
Positive and negative DNA controls are included in the kit.
Note: For a more thorough explanation of the locus and the targeted deep sequencing strategy, please refer to Principle of Immunoglobulin and T-Cell Receptor Gene Rearrangement.1
Principles of the Procedure
The human T-cell receptor gamma (TRG, previously known as TCRG) gene locus on chromosome 7 (7q14) includes 14 V (variable region) genes (Group I, II, III, and IV), 5 J (joining region) gene segments, and 2 C (constant region) genes spread over 200 kilobases.
Lymphoid cells are different from the other somatic cells in the body. During development, the antigen receptor genes in lymphoid cells undergo somatic gene rearrangement.2 For example, during T-cell development, genes encoding the TRG locus are assembled from multiple polymorphic gene segments that undergo rearrangements and selection, generating V-J combinations. Since leukemias and lymphomas originate from the malignant transformation of individual lymphoid cells, all leukemias and lymphomas generally share one or more cell-specific or “clonal” antigen receptor gene rearrangements. Therefore, tests that detect TRG clonal rearrangements can be useful in the study of B- and T-cell malignancies.
Initially, clonal rearrangements were identified using Restriction Fragment, Southern Blot Hybridization (RF-SBH) techniques. However, these tests proved cumbersome, labor-intensive, required large amounts of DNA, and were not suitable for analysis of many of the less diverse antigen receptor loci. During the last several decades, use of RF-SBH assays has been supplanted by PCR-based clonality tests, first developed by Alexander Morley,3 and are considered the gold standard method. PCR-based assays identify clonality on the basis of over representation of amplified V–D–J (or incomplete D–J) products, following their separation using gel or capillary electrophoresis. Though sensitive and suitable for testing small amounts of DNA, these assays cannot readily differentiate between clonal populations and multiple rearrangements that might lie beneath a single-sized peak and are not designed to identify the specific V–J DNA sequence that is required to track clonal populations in subsequent analyses. This second limitation can be of particular importance, as once the unique clone-specific DNA sequence is identified, the sequence can be used in subsequent tests to track and follow the clonal cell populations.
Polymerase Chain Reaction (PCR)
PCR assays are routinely used for the identification of clonal B- and T-cell populations. These assays amplify the DNA between primers that target the conserved VH regions and the conserved JH regions of antigen receptor genes. These conserved regions, where primers target, lie on either side of an area where programmed genetic rearrangements occur during the maturation of all B and T lymphocytes. Different populations of the B and T lymphocytes arise as a result of these genetic rearrangements.
The antigen receptor genes that undergo rearrangements are the immunoglobulin heavy chain (IGH) and light chains (IGK and IGL) in B-cells, and the T-cell receptor genes (TRA, TRB, TRG, and TRD) in T-cells. Each B- and T- cell has a single productive V – J rearrangement that is unique in both length and sequence. Therefore, when DNA from a normal or polyclonal population is amplified using DNA primers that flank the V-J region, amplicons that are unique in both sequence and length are generated, reflecting the heterogeneous population. In some cases, where lymphocyte DNA is not present, no amplicons will be generated. Samples containing TRG clonal populations yield one or two prominent amplified products of the same length and sequence that are detected with significant frequency of occurrence, within a diminished polyclonal background amplified at a lower frequency.
PCR amplicons are purified to remove excess primers, nucleotides, salts, and enzymes using the Agencourt® AMPure® XP system. This method utilizes solid-phase reversible immobilization (SPRI) paramagnetic bead technology for high-throughput purification of PCR amplicons. Using an optimized buffer, PCR amplicons that are 100 bp or larger are selectively bound to paramagnetic beads while contaminants such as excess primers, primer dimers, salts, and unincorporated dNTPs are washed away. Amplicons can then be eluted and separated from the paramagnetic beads resulting in a more purified PCR product for downstream analysis and amplicon quantification.
Purified amplicons are quantified utilizing capillary electrophoresis, which applies the principles of traditional gel electrophoresis to separate and quantify DNA on a chip based platform. Quantification is achieved by running a marker of known concentration alongside PCR amplicons and then extrapolating the concentration of the amplicons. Calculating the concentration of PCR amplicons allows equal amplicon representation in the final pooled library that is loaded onto the Ion S5 or Ion PGM for sequencing.
Next-Generation Sequencing (NGS)
Sanger sequencing methods represent the most popular in a range of ‘first-generation’ nucleic acid sequencing technologies. Newer methods, which leverage massively parallel sequencing approaches, are often referred to as Next-Generation Sequencing (NGS). NGS technologies can use various combination strategies of template preparation, sequencing, imaging, and bioinformatics for genome alignment and assembly.
NGS technologies used in this product rely on the amplification of genetic sequences using a series of consensus forward and reverse primers that include adapter and index tags. Amplicons generated with LymphoTrack master mixes are quantified, pooled, and loaded onto a chip for sequencing with a Thermo Fisher Scientific Ion S5 or Ion PGM platform. These platforms require the pooled library of DNA fragments to be bound to individual beads prior to sequencing, one unique sequence per bead. Once bound to the beads, the DNA fragments are amplified via emulsion PCR until they cover the surface of the bead. The beads are then loaded onto a semi-conductor chip where each bead occupies an individual well and sequencing occurs.
Sequencing is conducted by flooding the chip with individual, unincorporated nucleotides one base at a time. The sequencing instruments detect the addition of nucleotides when hydrogen ions are released during DNA polymerization causing a change in the pH of the wells, measured as a voltage proportional to the nucleotides added. After a nucleotide is incorporated, unincorporated nucleotides are washed away and the process begins again with a new dNTP.
These products were designed to allow for two different levels of multiplexing in order to reduce costs and time for laboratories. The first level of multiplexing originates from the multiple indices that are provided with the assays, up to 12. Each of these 12 indices can be considered to act as a unique barcode that allows amplicons from individual samples to be pooled together after PCR amplification to generate the sequencing library. Later, the resulting sequences can be sorted by the bioinformatics software to identify those that originated from an individual sample.
The second level of multiplexing originates from the ability of the accompanying software to sort sequencing data by both index and target. This allows amplicons generated with targeted primers (even those tagged with the same index) to be pooled together to generate the library to be sequenced on a single sequencing chip. An example would be to sequence products from several Invivoscribe LymphoTrack kits for the Ion S5 or PGM such as IGH FR1, IGH FR2, IGH FR3, IGK, and TRG together. When multiplexing amplicons of different gene targets it is important to use the appropriate sequencing chemistry. The number of sequencing cycles must be sufficient to sequence the largest amplicon in the multiplex.
The number of samples that can be multiplexed onto a single sequencing chip is also dependent on the chip that is utilized.
To determine the number of reads per sample, the total number of reads for the sequencing chip should be divided by the number of samples that will be multiplexed.
Minimal Residual Disease Evaluation
NGS-based minimal residual disease (MRD) testing is a proven tool that aids in the development of treatment strategies for hematologic malignancies. The LymphoTrack Clonality Assays can be used with the LymphoTrack MRD Software (Catalog # 75000008), LymphoQuant® Internal Controls and LymphoTrack Low Positive Controls to objectively track up to five clonal rearrangements in longitudinal studies with up to 10-6 sensitivity. For more information on our bundled MRD solution email email@example.com or visit www.invivoscribe.com/mrd-clonality.
This RUO assay tests genomic DNA. The input quantity is 50 ng of high quality DNA.
1. Miller JE, et al. (2013) Molecular Genetic Pathology (2nd Edition., sections 188.8.131.52 and 184.108.40.206).
2. Tonegawa, S, et al. (1983) Nature. 302, 575-581.
3. Trainor, KJ, et al. (1990) Blood. 75, 2220-2222.
This product is for Research Use Only; not for use in diagnostic procedures.
This product is covered by one or more patents and patent applications owned by or exclusively licensed to Invivoscribe, Inc., including United States Patent Number 7785783, United States Patent Number 8859748, United States Patent Number 10280462, European Patent Number EP 1549764B1 (validated in 16 countries, and augmented by related European Patents Numbered EP2418287A3 and EP 2460889A3), Japanese Patent Number JP04708029B2, Japanese Patent Application Number 2006-529437, Brazil Patent Application Number PI0410283.5, Canadian Patent Number CA2525122, Indian Patent Number IN243620, Mexican Patent Number MX286493, Chinese Patent Number CN1806051, and Korean Patent Number 101215194.
Use of this product may require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). Any necessary license to practice amplification methods or to use reagents, amplification enzymes or equipment covered by third party patents is the responsibility of the user and no such license is granted by Invivoscribe, Inc., expressly or by implication.
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