34 Next-Generation Sequencing (NGS) | Research Use Only (RUO) Assays Invivoscribe 2019 | 35 NGS RUO Assays LymphoTrack IGH FR1/FR2/FR3 Assays Assay Uses These research use only (RUO) assays for next-generation sequencing (NGS), identify clonal IGH VH-JH rearrangements, the associated VH-JH region DNA sequences, the frequency distribution of VH region and JH region segment utilization, as well as the degree of somatic hypermutation (SHM) of rearranged genes using the Illumina® MiSeq® , Thermo Fisher Scientific® Ion PGM™ or Ion S5™ . The LymphoTrack IGH FR1, IGH FR2, and IGH FR3 Assays contain primers that target the conserved framework 1 (FR1), framework 2 (FR2), and framework 3 (FR3) regions, respectively. The LymphoTrack IGH FR1/2/3 Assay kits contain the master mixes of all three frameworks. Summary and Explanation of the Test The LymphoTrack IGH Assays represent a significant improvement over clonality assays that utilize fragment analysis by providing four important and complementary uses: 1. Detection of clonal populations. 2.  Identification of sequence information and VH-JH segment utilization. 3. The LymphoTrack IGH FR1 Assays provide the degree of SHM of the immunoglobulin variable heavy chain (IGHV). 4.  The ability to track sequences in subsequent samples with the Invivoscribe LymphoTrack MRD Software. Primers included in the master mixes are designed with Illumina® adapters and indices (8 included in Kit A, 24 included in the Panel, and an independent 24 included in the Panel B) or Thermo Fisher adaptors and indices (12 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® cell, Ion S5 or Ion PGM chip. 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. Positive clonal (SHM negative) and negative polyclonal DNA controls are included in kits. A clonal SHM positive control can be purchased separately (4-088-0008). 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 development of lymphoid cells, the antigen receptor genes go through somatic gene rearrangements. 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. 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 populations can be useful in the study of B- and T-cell malignancies. Specimen Requirement 50 ng of high-quality genomic DNA. References 1. S Tonegawa. Nature 302: 575-581 (1983). 2.  JE Miller et al., Molecular Genetic Pathology (2nd ed.). Springer Science & Business Media. 2013: 30.2.7.13 and 30.2.7.18. 3. KJ Trainor et al., Blood 75: 2220-2222 (1990). 4. P Ghia. Leukemia 21: 1-3 (2007). Simple representation of the organization of the immunoglobulin heavy chain (IGH) gene locus on chromosome 14. Depicted are the variable region (VH) genes and downstream consensus joining region genes segments (JH) that are involved in rearrangements. VH family primers VL VH-FR1 VH-FR2 VH-FR3 JH primers JH DH The LymphoTrack IGH FR1/2/3 Assays contain components from respective individual FR Kit A's or Panels. LymphoTrack IGH FR1 Components LymphoTrack IGH FR2 Components LymphoTrack IGH FR3 Components Master Mix Name Index # Master Mix Name Index # Master Mix Name Index # IGH FR1 MiSeq 01 A001 IGH FR2 MiSeq 01 A001 IGH FR3 MiSeq 01 A001 IGH FR1 MiSeq 02 A002 IGH FR2 MiSeq 02 A002 IGH FR3 MiSeq 02 A002 IGH FR1 MiSeq 03 A003 IGH FR2 MiSeq 03 A003 IGH FR3 MiSeq 03 A003 IGH FR1 MiSeq 04 A004 IGH FR2 MiSeq 04 A004 IGH FR3 MiSeq 04 A004 IGH FR1 MiSeq 05 A005 IGH FR2 MiSeq 05 A005 IGH FR3 MiSeq 05 A005 IGH FR1 MiSeq 06 A006 IGH FR2 MiSeq 06 A006 IGH FR3 MiSeq 06 A006 IGH FR1 MiSeq 07 A007 IGH FR2 MiSeq 07 A007 IGH FR3 MiSeq 07 A007 IGH FR1 MiSeq 08 A008 IGH FR2 MiSeq 08 A008 IGH FR3 MiSeq 08 A008 IGH FR1 MiSeq 09 A009 IGH FR2 MiSeq 09 A009 IGH FR3 MiSeq 09 A009 IGH FR1 MiSeq 10 A010 IGH FR2 MiSeq 10 A010 IGH FR3 MiSeq 10 A010 IGH FR1 MiSeq 11 A011 IGH FR2 MiSeq 11 A011 IGH FR3 MiSeq 11 A011 IGH FR1 MiSeq 12 A012 IGH FR2 MiSeq 12 A012 IGH FR3 MiSeq 12 A012 IGH FR1 MiSeq 13 A013 IGH FR2 MiSeq 13 A013 IGH FR3 MiSeq 13 A013 IGH FR1 MiSeq 14 A014 IGH FR2 MiSeq 14 A014 IGH FR3 MiSeq 14 A014 IGH FR1 MiSeq 15 A015 IGH FR2 MiSeq 15 A015 IGH FR3 MiSeq 15 A015 IGH FR1 MiSeq 16 A016 IGH FR2 MiSeq 16 A016 IGH FR3 MiSeq 16 A016 IGH FR1 MiSeq 18 A018 IGH FR2 MiSeq 18 A018 IGH FR3 MiSeq 18 A018 IGH FR1 MiSeq 19 A019 IGH FR2 MiSeq 19 A019 IGH FR3 MiSeq 19 A019 IGH FR1 MiSeq 20 A020 IGH FR2 MiSeq 20 A020 IGH FR3 MiSeq 20 A020 IGH FR1 MiSeq 21 A021 IGH FR2 MiSeq 21 A021 IGH FR3 MiSeq 21 A021 IGH FR1 MiSeq 22 A022 IGH FR2 MiSeq 22 A022 IGH FR3 MiSeq 22 A022 IGH FR1 MiSeq 23 A023 IGH FR2 MiSeq 23 A023 IGH FR3 MiSeq 23 A023 IGH FR1 MiSeq 25 A025 IGH FR2 MiSeq 25 A025 IGH FR3 MiSeq 25 A025 IGH FR1 MiSeq 27 A027 IGH FR2 MiSeq 27 A027 IGH FR3 MiSeq 27 A027 IGH FR1 MiSeq 17 A017 IGH FR1 MiSeq 24 A024 IGH FR1 MiSeq 26 A026 IGH FR1 MiSeq 28 A028 IGH FR1 MiSeq 29 A029 IGH FR1 MiSeq 30 A030 IGH FR1 MiSeq 31 A031 IGH FR1 MiSeq 32 A032 IGH FR1 MiSeq 33 A033 IGH FR1 MiSeq 34 A034 IGH FR1 MiSeq 35 A035 IGH FR1 MiSeq 36 A036 IGH FR1 MiSeq 37 A037 IGH FR1 MiSeq 38 A038 IGH FR1 MiSeq 39 A039 IGH FR1 MiSeq 40 A040 KIT A PANEL B KIT A KIT A Reagents - MiSeq® Detection PANEL A PANEL A PANEL A