LymphoTrack^® IGHV Leader Somatic Hypermutation Assay Kit A - MiSeq^®

Product Details

• Summary and Explanation of the Test

  This LymphoTrack IGHV Leader Somatic Hypermutation Assay – MiSeq 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, identifies the DNA sequence specific for each clonal gene rearrangement.   Therefore, this assay has three important and complementary uses: it provides critical information in the existence of clonality, identifies sequence information required to track those clones in subsequent samples and provides detailed sequence information on the degree of SHM.

  Our single multiplex master mix targets the Leader (VHL) and the joining (J) gene regions of IGH.  Primers included in the Master Mixes are designed with Illumina adapter and 24 different indices.  This method allows for a one-step PCR reaction and pooling of amplicons from several different samples for loading on the MiSeq flow cell.  The associated LymphoTrack Software – MiSeq provides a simple and streamlined analysis and visualization of data generated from this assay as well as the LymphoTrack Assays.

  A somatic hypermutation positive control, a clonal positive control, and polyclonal negative control 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 1,250 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 proteins are assembled from multiple polymorphic gene segments that undergo rearrangements and selection, generating V_H-D_H-J_H combinations that are unique in both length and sequence for each cell.  An additional level of diversity is generated by point mutations in the variable regions, somatic hypermutations (SHM).  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.  In addition, immunoglobulin variable heavy chain gene hypermutation status provides important prognostic information for patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL).  The presence of IGH somatic hypermutation (SHM) is defined as greater or equal to 2% difference from the germline V_H gene sequence, whereas less than 2% difference is considered evidence of no somatic hypermutation.  The status of somatic hypermutation for clone(s) has clinical relevance, as there is a clear distinction in the median survival of patients with and without somatic hypermutation.  Hypermutation of the IGH variable region is strongly predictive of a good prognosis, while lack of mutation predicts a poor prognosis.³

  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,⁴ 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 rearrangement 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_H – J_H 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 V and J regions of antigen receptor genes.  These primers target the conserved regions and 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 absent, no amplicons will be generated.  Samples containing IGH clonal populations yield one or two prominent amplified products of the same length and sequence which are detected with significant frequency within a diminished polyclonal background.

  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 products 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 tremendously parallel sequencing approaches, are often referred to as NGS.  These technologies can use various combination strategies of template preparation, sequencing, imaging, and bioinformatics for genome alignment and assembly.

  NGS technologies used in this assay 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 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 as each RT-base is added, and then cleaved to allow incorporation of the next base.

  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.  Each of these 24 indices acts as a unique barcode that allows amplicons from individual samples to be pooled together after PCR amplification to generate the sequencing library; the resulting sequences are sorted by the bioinformatics software, which identifies 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 flow cell.  An example would be to sequence a combination of products from several Invivoscribe LymphoTrack Assay kits for the MiSeq, such as IGHV Leader, IGH FR1, IGH FR2, IGH FR3, IGK, TRB 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 a combination of IGH FR1, IGH FR2, IGH FR3, IGK, TRB and TRG amplicons together, use the MiSeq v2 (500 cycle) sequencing kit for up to 4 targets or v3 (600 cycle) sequencing kit for up to 7 targets.  When multiplexing any of these amplicons together with IGHV Leader, use the MiSeq v3 (600 cycle) sequencing kit.  If multiplexing only IGH FR3 and TRG amplicons together, which both have shorter amplicon sizes, use the MiSeq v2 (300 or 500 cycle) sequencing kit and adjust the cycle settings in the sample sheet.

  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 (MiSeq v3) can generate 20-25 million reads.  To determine the number of reads per sample, divide the total number of reads for the flow cell by the number of samples that will be multiplexed.  Illumina also manufacturers other flow cells that utilize the same sequencing chemistry, but these 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

  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 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.