32 Next-Generation Sequencing (NGS) | Research Use Only (RUO) Assays Invivoscribe 2019 | 33 NGS RUO Assays LymphoTrack IGHV Leader Somatic Hypermutation Assay Assay Uses This research use only (RUO) assay for next-generation sequencing (NGS), identifies clonal IGH VH-JH rearrangements, the associated VH-JH DNA sequences, and the frequency distribution of VH region and JH region segment utilization. The assay also uses the Illumina® MiSeq® platform to define the extent of somatic hypermutation (SHM) present in the IGHV gene of analyzed samples.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 IGH FR1 Assay (7-121-0007). Summary and Explanation of the Test The LymphoTrack IGHV Leader Somatic Hypermutation Assay for NGS provides significant improvements over clonality assays using fragment analysis and Sanger sequencing. The assay 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 calculates the degree of SHM for each sample. The master mixes included in this assay target the Leader (VHL) and the joining (JH) gene regions of IGH and are designed with Illumina® adapters and indices (8 included in Kit A and 24 included in Panels). Including adapters and indices in the primer design allows for a one- step PCR approach to generate sequence-ready amplicons, followed by direct pooling of samples for sequencing on a Illumina® MiSeq® flow cell. The included LymphoTrack bioinformatics software enables simplified analysis and visualization of data generated from this assay. Positive (clonal positive, SHM negative), negative (polyclonal), and SHM positive (clonal positive, SHM positive) controls are included in the kit. Primers included in the master mixes are designed with Illumina® adapters and indices (8 or 24 indices per framework kits). This allows for a one-step PCR reaction to generate sequence-ready amplicons and pooling of several different samples on the same Illumina® MiSeq® flow cell. The LymphoTrack bioinformatics software enables easy analysis and visualization of data and the LymphoTrack MRD Software allows sequences to be tracked in subsequent samples. Please see the LymphoTrack MRD software section to learn how the LymphoTrack Assays can be applied to MRD studies, or email marketing@invivoscribe.com. 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 1250 kilobases. 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 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. An additional level of diversity is generated in the antigen receptors by somatic point mutations in the variable regions and this mutation status provides important prognostic information for chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). In addition, NGS methods can improve disease stratification and elucidate subclone gene profiles. 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. KJ Trainor et al., Blood 75: 2220-2222 (1990). 5. P Ghia et al., Leukemia 21: 2-3 (2007). 6. P Ghia et al., Blood 105: 1678-685 (2005). 7. S Tonegawa. Nature 302: 575-581 (1983). Simple representation of the organization of the immunoglobulin heavy chain (IGH) gene on chromosome 14. Depicted are the variable region (VH) genes and downstream consensus joining region genes (JH) that are involved in rearrangements. Upstream of the variable gene segments, the leader sequence (VHL) is also depicted. Diversity region genes are not depicted. 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 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 Example Data. Depicted are the top 10 sequences from a read summary generated by the LymphoTrack Software - MiSeq® . Highlighted columns represent fields that are unique to SHM analysis and include the SHM mutation rate and predictions pertaining to whether a sequence is in-frame or contains a premature stop codon. To learn more about the LymphoTrack software, please refer to the LymphoTrack Bioinformatics Software section. Ordering Information Catalog # Products Quantity Components 7-121-0059 LymphoTrack® IGHV Leader Somatic Hypermutation Assay Kit A - MiSeq® 8 indices - 5 sequencing reactions each 7-121-0069 LymphoTrack® IGHV Leader Somatic Hypermutation Assay Panel - MiSeq® 24 indices - 5 sequencing reactions each 7-500-0009 LymphoTrack® Software - MiSeq® 1 CD complimentary with purchase VH1 VHL VH2 VH3 VH4 VH5 VH6 VH7 JH 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