LymphoTrack^® IGH FR1 Assay - S5/PGM™

Product Details

• Summary and Explanation of the Test

  This LymphoTrack IGH FR1 Assay – S5/PGM represents a significant improvement over existing clonality assays using fragment analysis as it efficiently detects the majority of IGH 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 assay has two important and complementary uses: it provides critical information on the existence of clonality and identifies sequence information required to track those clones in subsequent samples.  The LymphoTrack IGH FR1 Assay additionally provides detailed sequence information on the degree of SHM.

  Our single multiplex master mixes target the conserved framework region 1 (FR1) and the joining (J) region. Primers included in the master-mixes are designed with Thermo Fisher Scientific adapters and twelve different indices.  This allows for a one-step PCR reaction and pooling of amplicons from several different samples for loading on the Ion S5 or PGM chips.

  Positive and negative 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 B-Cell Receptor Gene Rearrangement.¹

• Principles of the Procedure

  Background

  The immunoglobulin heavy chain (IGH) gene locus on chromosome 14 (14q32.3) includes 46-52 functional and 30 non-functional variable (V_H) gene segments, 27 functional diversity (D_H) gene segments, and 6 functional joining (J_H) gene segments spread over 1250 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 B-cell development, genes encoding the IGH molecules are assembled from multiple polymorphic gene segments that undergo rearrangements and selection.  These gene rearrangements of the V_H, D_H, and J_H generate V_H-D_H-J_H combinations of unique length and sequence for each cell.  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 IGH clonal rearrangements can be useful in the study of B-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, the use of RF-SBH assays has been supplanted by PCR-based clonality tests developed by Alexander Morley,³ 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) gene rearrangements following their separation using gel 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 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 framework of the 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 chain loci (IGK and IGL) in B-cells, and the T-cell receptor gene loci (TRA, TRB, TRG, and TRD) in T-cells.  Each B- and T-cell has one or two productive V – J rearrangements that are 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 IGH 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.

  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.  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 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 PGM platform.  The Ion S5 and Ion PGM requires that the pooled library of DNA fragments is 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

  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 together in the same run.  It is recommended to multiplex no more than three different gene targets together, such as IGH FR1, IGH FR2, and IGH FR3, due to the capacity of the Thermo Fisher Scientific Ion 316™ Chip v2. The Ion 316™ Chip v2 can generate 2-3 million reads. 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).

  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.  Two or more sequencing libraries generated from the same LymphoTrack gene target master mixes (e.g., two IGH FR1 sequencing libraries, either from the same or different kit lots) can also be multiplexed together into a single sequencing library as long as each index for that master mix is only included once per sequencing run.

  IGHV Somatic Hypermutation (SHM) Evaluation

  For evaluation of the somatic hypermutation rate of the IGHV region, the LymphoTrack IGH FR1 Master Mixes can be used; however, this only targets a portion of the IGHV region as the sequence upstream of the primer binding site will not be assessed. When analyzing the somatic hypermutation status of samples, the bioinformatics software will provide the mutation rate based upon the percent mismatch of the clonal amplicons as compared to germline reference genes, a prediction of whether the protein would be in or out of frame, a prediction of whether mutations or gene rearrangements result in a pre-mature stop codon, and the percentage of V_H gene coverage for the region targeted by the assay.

  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 marketing@invivoscribe.com or visit www.invivoscribe.com/mrd-clonality.

• Specimen Requirements

  This RUO assay tests genomic DNA. The input quantity is 50 ng of high quality DNA.