LymphoTrack^® Dx TRG Assay Panel - MiSeq^TM

Product Details

• Summary and Explanation of the Test

  This LymphoTrack Dx TRG Assay Panel – MiSeq 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. Therefore, this assay 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.

  Our single multiplex master mixes target all conserved regions within the variable (V) and the joining (J) region genes described in lymphoid malignancies. This is critical for comprehensive analysis of samples, as some T-cell lymphoproliferative disorders involve V and J segments that would not be identified with existing assays or Vγ and Jγ primer sets. Primers included in the Master Mixes are designed with Illumina^® adapters and 24 different indices. This allows for a one-step PCR reaction and pooling of amplicons from several different samples for loading on the MiSeq flow cell. The average size of the TRG gene rearrangement PCR amplicons generated using this assay was designed to be compatible with testing fragmented DNA isolated from more challenging samples (e.g., FFPE sections). The associated LymphoTrack Dx Software – MiSeq provides a simple and streamlined analysis and visualization of data generated from this assay.

  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

  Background

  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 rearrangement². 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.

  During the last several decades, the use of RF-SBH assays has been supplanted by PCR-based clonality tests developed by Alexander Morley³ and are considered the current gold standard method. PCR-based assays identify clonality on the basis of over-representation of amplified V-J or 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 that may differ in sequence but are the same size, nor can they detect multiple rearrangements that might lie beneath a single-sized peak. These assays are also 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, this sequence can be used in subsequent tests to track and follow these 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 V and 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, 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 using the KAPA Library Quantification Kits for Illumina^® platforms. Purified and diluted PCR amplicons and a set of six pre-diluted DNA standards are amplified by quantitative (qPCR) methods, using the KAPA SYBR^® FAST qPCR Master Mix and primers. The primers in the KAPA kit target Illumina P5 and P7 flow cell adapter oligo sequences.

  The average Ct score for the pre-diluted DNA Standards are plotted against log10 to generate a standard curve, which can then be used to calculate the concentration (pM) of the PCR amplicons derived from sample DNA. Calculating the concentration of PCR amplicons allows equal amplicon representation in the final pooled library that is loaded onto the MiSeq 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 the LymphoTrack Dx master mixes are quantified, pooled, and loaded onto a flow cell for sequencing with an Illumina MiSeq sequencing platform. Specifically, the amplified products in the library are hybridized to oligonucleotides on a flow cell and are amplified to form local clonal colonies (bridge amplification). Four types of reversible terminator bases (RT-bases) are added and the sequencing strand of DNA is extended one nucleotide at a time. To record the incorporation of nucleotides, a CCD camera takes an image of the light emitted when fluorescently labeled nucleotides are added to the sequencing strand. A terminal 3’ blocker is added after each cycle of the sequencing process and any unincorporated nucleotides are removed prior to the addition of four new RT-bases.

  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 24. Each of these 24 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 flow cell. An example would be to sequence products from several Invivoscribe LymphoTrack Dx MiSeq kits such as IGH FR1 and TRG. 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. For example, when multiplexing IGH FR1, IGK and TRG amplicons, the MiSeq v2 (500 cycle) sequencing kit should be used. For further detail, please refer to the Instructions for Use (IFU).

  The number of samples that can be multiplexed onto a single flow cell is also dependent on the flow cell that is utilized. Illumina’s standard flow cells can generate 12-15 million reads. To determine the number of reads per sample, the total number of reads for the flow cell should be divided by the number of samples that will be multiplexed. The number of reads per sample (depth) must be sufficient to achieve the thresholds provided in Instructions for Use (IFU). Illumina also manufacturers other flow cells that utilize the same sequencing chemistry, but generate fewer reads. When using these alternative flow cells one must consider that fewer total reads either means less depth per sample or fewer samples can be run on the flow cell to achieve the same depth per sample.

• 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.