22 Next-Generation Sequencing (NGS) | CE-IVD Assays Invivoscribe 2019 | 23 NGS CE-IVD Assays LymphoTrackDx IGK Assays Assay Description The LymphoTrack Dx IGK Assays for the Illumina® MiSeq® , or Thermo Fisher Scientific® Ion PGM™ and Ion S5™ instruments are in vitro diagnostic products intended for next-generation sequencing (NGS) based determination of the frequency distribution of IGK gene rearrangements in patients suspected of having lymphoproliferative disease. These assays aid in the identification of lymphoproliferative disorders. Summary and Explanation of the Test In contrast to the IdentiClone™ fragment analysis assays for clonality that utilize two master mixes, these NGS assays contain a single multiplex master mix to target conserved regions of the IGK gene locus described in lymphoid malignancies, thereby, reducing sample DNA requirements and simplifying the testing workflow. The LymphoTrack Dx IGK master mix primers are also designed with Illumina® or Thermo Fisher Scientific adapters and up to 24 different indices. This allows amplicons generated from different indexed IGK master mixes to be pooled into a single library for loading onto one MiSeq® flow cell, Ion PGM™ or Ion S5™ chips. The associated LymphoTrack Dx Software is capable of sorting complex NGS data by gene target, providing users the ability to reduce per sample testing costs by sequencing amplicons from any LymphoTrack Dx Assay (e.g. IGH, IGK, TRB, TRG) at the same time. In addition, the LymphoTrack Dx Software provides an easy and streamlined method for visualization of data and guidelines provided in the instructions for use allow samples to be interpreted for evidence or no evidence of clonality. Positive clonal and negative polyclonal DNA controls are included in kits. Background The LymphoTrack Dx IGK Assays represent a significant improvement over existing fragment analysis clonality assays by providing two important and complementary uses: 1. Detection of initial clonal populations. 2.  Identification of sequence information required to track clonal rearrangements in subsequent samples. The human immunoglobulin kappa (IGK) gene locus on chromosome 2 (2p11.2) includes 76 V (variable) region genes spanning 7 subgroups and 5 J (joining) region gene segments upstream of the (Cƙ) region. During lymphoid cell development, antigen receptor genes undergo somatic gene rearrangements. Specifically, during B-cell development, genes encoding IGK molecules are assembled from multiple polymorphic gene segments that generate V-J combinations unique in both length and sequence. In addition, the kappa deleting element (Kde), approximately 24 kb downstream of the Jƙ-Cƙ region can also rearrange with Vƙ gene segments and the isolated recombination signal sequence in the Jƙ-Cƙ intron (Jƙ-Cƙ INTR). 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 IGK clonal rearrangements can be useful in the study of B- and T-cell malignancies and complement IGH testing, as the IGK receptor is less susceptible to somatic mutations. Specimen Requirement 50 ng of high-quality genomic DNA. References 1. EP Rock et al., J Exp Med 179 (1): 323-8 (1994). 2. JE Miller et al., Molecular Diagnostic 4 (2): 102-117(1999). Simplified Representation of the IGK Gene Depicted are the variable region (Vƙ) or variable intragenic Jƙ-Cƙ intron (Jƙ-Cƙ INTR) genes involved in IGK gene rearrangements in addition to the downstream consensus joining region genes (Jƙ) or kappa deleting element (Kde). Kde Kde Kit A Components Master Mix Name Index # IGK MiSeq 01 A001 IGK MiSeq 02 A002 IGK MiSeq 03 A003 IGK MiSeq 04 A004 IGK MiSeq 05 A005 IGK MiSeq 06 A006 IGK MiSeq 07 A007 IGK MiSeq 08 A008 Panel Components (includes all master mixes from Kit A plus the items below) Master Mix Name Index # Master Mix Name Index # IGK MiSeq 09 A009 IGK MiSeq 18 A018 IGK MiSeq 10 A010 IGK MiSeq 19 A019 IGK MiSeq 11 A011 IGK MiSeq 20 A020 IGK MiSeq 12 A012 IGK MiSeq 21 A021 IGK MiSeq 13 A013 IGK MiSeq 22 A022 IGK MiSeq 14 A014 IGK MiSeq 23 A023 IGK MiSeq 15 A015 IGK MiSeq 25 A025 IGK MiSeq 16 A016 IGK MiSeq 27 A027 Assay Components Master Mix Name Index # Master Mix Name Index # IGK S5/PGM 01 IonXpress_001 IGK S5/PGM 11 IonXpress_011 IGK S5/PGM 02 IonXpress_002 IGK S5/PGM 12 IonXpress_012 IGK S5/PGM 04 IonXpress_004 IGK S5/PGM 13 IonXpress_013 IGK S5/PGM 08 IonXpress_008 IGK S5/PGM 14 IonXpress_014 IGK S5/PGM 09 IonXpress_009 IGK S5/PGM 16 IonXpress_016 IGK S5/PGM 010 IonXpress_010 IGK S5/PGM 17 IonXpress_017 V-J Sequence Frequencies. The LymphoTrack Dx Software provides a stacked bar graph depicting the relative frequencies for the most prevalent rearrangements identified in a sample. To learn more about the LymphoTrack Dx software, please refer to the LymphoTrack Dx Bioinformatics Software section. Ordering Information Catalog # Products Quantity Components 9-122-0009 LymphoTrack® Dx IGK Assay Kit A - MiSeq® 8 indices - 5 sequencing reactions each 9-122-0019 LymphoTrack® Dx IGK Assay Panel - MiSeq® 24 indices - 5 sequencing reactions each 9-500-0009 LymphoTrack® Dx Software - MiSeq® 1 CD complimentary with purchase 9-122-0007 LymphoTrack® Dx IGK Assay - S5/PGM™ 12 indices - 5 sequencing reactions each 9-500-0007 LymphoTrack® Dx Software - S5/PGM™ 1 CD complimentary with purchase Controls IGK POS (+) Qty. 3 NGS NEG (-) Qty. 3 Controls IGK POS (+) Qty. 2 NGS NEG (-) Qty. 2 Controls IGK POS (+) Qty. 1 NGS NEG (-) Qty. 1 These products are CE-IVD assays for in vitro diagnostic use. Reagents - MiSeq® Detection Reagents - Ion S5™ /PGM™ Detection