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CAT #: 71210149

LymphoTrack® IGH FR1 Assay Panel B - MiSeq®

Product Use

This assay identifies clonal IGH VH-JH rearrangements, the DNA sequences of these rearrangements, and provides the distribution frequency of V and J segment utilization using the Illumina® MiSeq® platform. LymphoTrack IGH FR1 Assays contain primers that target the conserved framework 1 (FR1) region within the VH and the JH regions described in lymphoid malignancies. Invivoscribe LymphoTrack MiSeq Software is included with this product for bioinformatics data analysis, visualizations and PDF reports.

Product Details

  • Summary of Explanation of the Test

    The Invivoscribe portfolio of LymphoTrack® IGH – MiSeq® Assays represent a significant improvement over existing clonality assays using fragment analysis as they efficiently detect the majority of IGH gene rearrangements using a single multiplex master mix and, at the same time, identify the DNA sequence specific for each clonal gene rearrangement. Therefore, this product has two important and complementary uses: it aids both in the detection of initial clonal populations and identifies sequence information required to track those clones in subsequent samples. 

    LymphoTrack IGH FR1 Assays contain primers that target the conserved framework 1 (FR1) region within the VH and the JH regions described in lymphoid malignancies. Our single multiplex master mix for IGH targets the conserved framework region 1 (FR1) within the VH and the JH regions described in lymphoid malignancies. Primers included in the mastermix are designed with Illumina® adapters and 24 unique indices allowing amplicons generated from the different IGH master mixes to be pooled onto one flow cell. This allows for a one-step PCR reaction and pooling of amplicons from several different samples for loading onto the MiSeq® flow cell.

    Positive and negative controls are included in the kit.

    The associated LymphoTrack MiSeq® Software provides a simple and streamlined analysis and visualization of data generated from this assay. LymphoTrack MiSeq Software further enables the identification of population clonotypes. This sequence specific information may be seamlessly imported into LymphoTrack MRD Software for identification of residual clonotype sequences in subsequent samples. Please see the LymphoTrack MRD Software section of the catalog to learn more.

    Catalog #71210149 contains 24 unique indexes. However, Invivoscribe offers multiple kit configurations with their LymphoTrack IGHFR1 – MiSeq® Assays. For each kit, primers included in the master mixes are designed with Illumina® adapters and unique indices as follows: Kit A: PN 7-121-0009 (8 indices), Panel: PN 7-121-0039 (24 indices inclusive of the 8 indices subset found in Kit A), Panel B: PN 7-121-0149 (24 additional indices). The combined use of Panel and Panel B kit configurations provides end users a total of 48 unique sample indices. Together, these offerings allow laboratories to employ 48-sample indexing in their laboratory, or choose the optimal kit(s) for the sample throughput needs of their laboratory.

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

  • 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 (VH) gene segments, 27 functional diversity (DH) gene segments, and 6 functional joining (JH) gene segments spread over 1250 kilobases.

    During development of lymphoid cells, the antigen receptor genes in lymphoid cells undergo somatic gene rearrangement (Tonegawa S. et al., 1983). For example, during B-cell development, genes encoding the IGH molecules are assembled from multiple polymorphic gene segments that undergo rearrangements and selection, generating VH-DH-JH 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 IGH 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 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 (FR) of the 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. It is a result of these genetic rearrangements that different populations of the B and T lymphocytes arise.

    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 (TRATRBTRG 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 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 (nM) 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 this LymphoTrack 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

    The LympohTrack IGH FR1 – MiSeq® Assays 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 48. Each of these 48 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. Invivoscribe offers multiple kit configurations for their LymphoTrack IGH FR1 – MiSeq® Assays. Catalog # 7-121-0149 (LymphoTrack IGH FR1 Panel B – MiSeq) contains 24 unique indices. Please contact your sales representative or view the catalog for additional indices options.

    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 MiSeq® kits such as IGH FR1, 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. For example, when multiplexing IGH FR1, IGK, and TRG amplicons, the MiSeq® v2 (500-cycle) or v3 (600-cycle) sequencing kit should be used.

    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 15-25 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. 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.

    IGHV Somatic Hypermutation (SHM) Evaluation

    For evaluation of the somatic hypermutation rate of the IGHV region, the LymphoTrack IGH FR1 Assay – MiSeq® can be used; however, this assay only targets a portion of the IGHV region. 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 translation 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 VH gene coverage for the region targeted by the assay.

    Minimal Residual Disease Evaluation

    For additional information related to how to reach a desired level of sensitivity for MRD studies using the LymphoTrack Assays, please request a LymphoTrack MRD Technical Bulletin by emailing marketing@invivoscribe.com.

  • Specimen Requirements

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

Disclaimer

©2019 Invivoscribe, Inc All rights reserved. The trademarks mentioned herein are the property of Invivoscribe, Inc and/or its affiliates, or (as to the trademarks of others used herein) their respective owners.

ILLUMINA® and MISEQ® are registered trademarks of Illumina, Inc.

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