LymphoTrack^® Dx TRG Assay - S5/PGM™

Product Details

• Summary and Explanation of the Test

  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.¹ For example, during T-cell development genes encoding the TRG molecules are assembled from multiple polymorphic gene segments that undergo rearrangements and selection, generating V-J combinations that are unique in both length and sequence. 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 and labor-intensive, they required large amounts of DNA, and they were not suitable for analysis of many of the less diverse antigen receptor loci.

  During the last several decades, the use of RF-SBH assays has been supplanted by PCR-based clonality tests developed by Alexander Morley (Trainor K.J. et al., 1990), and are considered the current 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 LymphoTrack Dx TRG Assay – S5/PGM™ represents a significant improvement over existing clonality assays using fragment analysis as it efficiently detects the majority of TRG gene rearrangements using a single multiplex master mix and, at the same time, identifies the DNA sequence specific for each clonal gene rearrangement. This product has two important and complementary uses. One, it aids in the detection of initial clonal populations. Two, it identifies sequence information required to track those clones in subsequent samples. Therefore the LymphoTrack Dx TRG Assay – S5/PGM provides critical evidence on the existence of clonality and the specific rearranged TRG genes involved.

  Our single multiplex master mix for TRG targets the conserved regions within the V and the J regions described in lymphoid malignancies. Primers included in the master mixes are designed with Life Technologies adapters and up to 12 different indices; thereby, allowing, amplicons generated from different TRG master mixes to be pooled together to generate a library of amplicons that can be loaded onto a single Ion S5™ or PGM™ chip for sequencing. The associated LymphoTrack Dx Software – S5/PGM provides direct interpretation of the data generated from this assay via a simple and streamlined method of analysis and visualization. The results of molecular clonality tests should always be interpreted in the context of clinical, histological and immunophenotypic data.

  Positive and negative controls for clonality are included in the kit.

  Note:  For a more thorough explanation of the locus and the targeted sequencing strategy, please refer to Miller J.E., 2013.

• Principles of the Procedure

  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 V regions and the conserved J 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 unique in both sequence and length, reflecting the heterogeneous population, are generated. In some cases, where lymphocyte DNA is not present, no amplicons will be generated. For samples containing clonal populations, the yield is 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.

  Amplicon Purification

  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.

  Amplicon Quantification

  Purified amplicons are quantified utilizing the Agilent Technologies 2100 Bioanalyzer or the Perkin Elmer LabChip^® GX. These are electrophoretic methods that utilize 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 samples and then extrapolating the concentration of samples. 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 Dx master mixes are quantified, pooled, and loaded onto a chip for sequencing with a Life Technologies Ion S5™ or Ion PGM platform. The Ion S5 and Ion PGM require that the pooled library of DNA fragments be bound to individual beads prior to sequencing, (one unique sequence per bead) through a process known as emulsion PCR. Once bound to the beads the DNA fragments are amplified until they cover the surface of the bead. Beads are then loaded onto a semi-conductor chip, where they find their own well to occupy and where sequencing occurs. Sequencing is conducted by flooding the chip with individual unincorporated nucleotides one base at a time (dATP, dCTP, dGTP, dTTP). The Ion PGM™ instrument detects the addition of nucleotides when hydrogen ions are released during DNA polymerization and causes a change in pH of the wells, which can be measured as a change in voltage. The voltage changes proportionally to the number of nucleotides added. After nucleotides are incorporated, unincorporated nucleotides are washed away and the process begins again with a new dNTP.

  Multiplexing Amplicons

  This product was 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 and sequenced on a single sequencing chip. An example would be to sequence products from several Invivoscribe LymphoTrack Dx – S5/PGM kits 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. Thermo Fisher Scientific Ion 316™ Chip v2 BC can generate 2-3 million reads so it is recommended to multiplex no more than three different gene targets together. Up to five different gene targets can be multiplexed together on the Ion PGM Ion 318™ Chip v2 BC (4-5.5 million reads), Ion S5 Ion 520™Chip (3-6 million reads) and Ion S5 Ion 530™ Chip (15-20 million reads). 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.

• Specimen Requirements
  • This assay tests extracted and purified genomic DNA. DNA must be quantified with a method specific for double stranded DNA (dsDNA) and free of inhibitors of PCR amplification.
  • Resuspend DNA in an appropriate solution such as 0.1X TE (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0, prepared with molecular biology grade water) or molecular biology grade water alone.
  • The minimum input quantity is 50 ng of high quality DNA.