16 Next-Generation Sequencing (NGS) | CE-IVD Assays Invivoscribe 2019 | 17 NGS CE-IVD Assays LymphoTrackDx IGHV Leader Somatic Hypermutation Assay Assay Description The LymphoTrack Dx IGHV Leader Somatic Hypermutation Assay for the Illumina® MiSeq® is an in vitro diagnostic product intended for next-generation sequencing (NGS) based determination of the frequency distribution of IGH gene rearrangements, as well as the degree of somatic hypermutation (SHM) of rearranged genes in patients suspected of having lymphoproliferative disease. This assay helps identify lymphoproliferative disorders, and it offers an aid in the identification of disease prognosis. If you would like to test for IGHV somatic hypermutation using the Thermo Fisher® Ion PGM™ or Ion S5™ platform, please refer to the LymphoTrack Dx IGH FR1 Assay (9-121-0007). Summary and Explanation of the Test The NGS LymphoTrack Dx IGHV Leader Somatic Hypermutation Assay for the Illumina® MiSeq® represents a significant improvement over clonality assays using fragment analysis as it efficiently detects the majority of IGH gene rearrangements using a single multiplex master mix, identifies the DNA sequence specific for each clonal gene rearrangement, and assesses the somatic hypermutation rate of clonal samples in the same workflow. The single multiplex master mixes target the Leader (VHL) and the joining (JH) gene regions of the IGH locus and are designed with Illumina® adapters and indices (8 included in Kit A and 24 included in the Panel). This allows for a one-step PCR reaction and pooling of amplicons from several different samples and targets into a single Illumina® MiSeq® run. No post-PCR ligation step is required. The included LymphoTrack Dx Bioinformatics Software enables simplified analysis and visualization of individual sample data. Positive (clonal positive, SHM negative), negative (polyclonal) and SHM (clonal positive, SHM positive) DNA controls are included in the kits. Background The human immunoglobulin heavy chain (IGH) gene locus on chromosome 14 (14q32.3) includes 46-52 functional and 30 nonfunctional variable (VH) gene segments, 27 functional diversity (DH) gene segments, and 6 functional joining (JH) gene segments spread over 1,250 kilobases. During B-cell development, genes encoding the IGH protein are assembled from multiple polymorphic gene segments that undergo rearrangements and selection, generating cell specific VH-DH-JH rearrangements 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- cell malignancies. An additional level of diversity is further generated in the antigen receptors by introducing point mutations in the variable regions, also named SHM. In instances where there is a high degree of SHM, there is the risk that primers located within the variable region will not be able to bind and clonal products will not amplify. In these cases, the leader primers located upstream of the variable region can be beneficial for the detection of clonal products, due to the conserved nature of the VHL region. In addition, the SHM rate of the entire variable gene can be determined using the VHL primers. Determining the immunoglobulin variable heavy chain gene (IGHV) hypermutation rate is considered a gold standard for determining the prognosis of patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). In addition, NGS methods can improve disease stratification. Specimen Requirement 50 ng of high-quality genomic DNA. References 1. B Stamatopoulous et al., Leukemia 4:837-845 (2017). 2. F Davi et al., Leukemia 22: 212-214 (2008). 3.  JE Miller et al., Molecular Genetic Pathology (2nd ed.). Springer Science & Business Media. 2013: 302.2.7.13 and 30.2.7.18. 4. P Ghia et al., Leukemia 21: 2-3 (2007). 5. P Ghia et al., Blood 105: 1678-1685 (2005). 6. S Tonegawa. Nature 302: 575-581 (1983). Simplified representation of the immunoglobulin heavy chain (IGH) gene locus on chromosome 14. Depicted are the variable (VH) and downstream consensus joining (JH) region genes involved in rearrangements. Upstream of the variable gene segments, the leader sequence (VHL) is also depicted. Diversity region genes are not depicted. VH1 VHL VH2 VH3 VH4 VH5 VH6 VH7 JH Panel Components (includes all master mixes from Kit A plus the items below) Master Mix Name Index # Master Mix Name Index # IGH Leader MiSeq 09 A009 IGH Leader MiSeq 18 A018 IGH Leader MiSeq 10 A010 IGH Leader MiSeq 19 A019 IGH Leader MiSeq 11 A011 IGH Leader MiSeq 20 A020 IGH Leader MiSeq 12 A012 IGH Leader MiSeq 21 A021 IGH Leader MiSeq 13 A013 IGH Leader MiSeq 22 A022 IGH Leader MiSeq 14 A014 IGH Leader MiSeq 23 A023 IGH Leader MiSeq 15 A015 IGH Leader MiSeq 25 A025 IGH Leader MiSeq 16 A016 IGH Leader MiSeq 27 A027 Controls IGH SHM POS (+) Qty. 3 IGH POS (+) Qty. 3 NGS NEG (-) Qty. 3 Ordering Information Catalog # Products Quantity Components 9-121-0059 LymphoTrack® Dx IGHV Leader Somatic Hypermutation Assay Kit A - MiSeq® 8 indices - 5 sequencing reactions each 9-121-0069 LymphoTrack® Dx IGHV Leader Somatic Hypermutation Assay Panel - MiSeq® 24 indices - 5 sequencing reactions each 9-500-0009 LymphoTrack® Dx Software - MiSeq® 1 CD complimentary with purchase Kit A Components Master Mix Name Index # IGH Leader MiSeq 01 A001 IGH Leader MiSeq 02 A002 IGH Leader MiSeq 03 A003 IGH Leader MiSeq 04 A004 IGH Leader MiSeq 05 A005 IGH Leader MiSeq 06 A006 IGH Leader MiSeq 07 A007 IGH Leader MiSeq 08 A008 Controls IGH SHM POS (+) Qty. 1 IGH POS (+) Qty. 1 NGS NEG (-) Qty. 1 These products are CE-IVD assays for in vitro diagnostic use. Example Data. The top 10 sequences from a read summary generated by the LymphoTrack Dx Software - MiSeq® with the SHM mutation rate and predictions pertaining to whether a sequence is in-frame or contains a premature stop codon are depicted. To learn more about the LymphoTrack Dx software, please refer to the LymphoTrack Dx Bioinformatics Software section. Rank Sequence Length Merge count V-gene J-gene % Total reads Cumulative % Mutation rate partial V-gene (%) In-frame (Y/N) No stop codon (Y/N) V-coverage 1 TTCTCGTGGTG 455 29603 IGHV4-59_08 IGHJ4_02 9.93 9.93 11.26 Y Y 98.63 2 CTCGCCCTCCT 463 205 IGHV5-51_01 IGHJ4_02 0.07 9.99 0.00 Y Y 99.66 3 GGTTTTCCTTG 484 201 IGHV3-7_01 IGHJ4_02 0.07 10.06 7.77 Y Y 100.00 4 CTCGCCCTCCT 463 185 IGHV5-51_01 IGHJ5_02 0.06 10.12 6.08 Y Y 99.32 5 CTCGCCCTCCT 469 170 IGHV5-51_01 IGHJ4_02 0.06 10.18 0.00 Y Y 99.32 6 CTCGCCCTCCT 466 160 IGHV5-51_01 IGHJ4_02 0.05 10.23 0.00 Y Y 99.66 7 CTGCTGCTGAC 460 159 IGHV2-5_10 IGHJ5_02 0.05 10.29 8.08 Y Y 97.64 8 GGTTTTCCTTG 493 156 IGHV3-48_02 IGHJ6_02 0.05 10.34 3.72 Y Y 98.99 9 CTCGCCCTCCT 334 153 IGHV5-51_02 IGHJ2_01 0.05 10.39 3.72 Y N 27.70 10 CTCGCCCTCCT 334 152 IGHV5-51_02 IGHJ2_01 0.05 10.44 3.38 Y N 26.01 Reagents - MiSeq® Detection