24 Next-Generation Sequencing (NGS) | CE-IVD Assays Invivoscribe 2019 | 25 NGS CE-IVD Assays LymphoTrackDx TRG Assay Assay Description The LymphoTrack Dx TRG Assays for the Illumina® MiSeq® , Thermo Fisher Scientific® Ion PGM™ or Ion S5™ instruments are in vitro diagnostic products intended for next-generation sequencing (NGS) based determination of the frequency distribution of TRG gene rearrangements in patients suspected with having lymphoproliferative disease. These assays aid in the identification of lymphoproliferative disorders. Summary and Explanation of the Test These assays utilize a single multiplex master mix to target conserved V and J regions of the human T-cell receptor gamma (TRG) gene that are described in lymphoid malignancies. Primers are designed with Illumina® or Thermo Fisher Scientific adapters and up to 24 different indices; thereby allowing amplicons generated from different TRG master mixes to be pooled together for sequencing on a single MiSeq® flow cell, or Ion PGM™ or Ion S5™ chip. The associated LymphoTrack Dx Software sorts complex NGS data by gene target, providing users the ability to reduce per sample testing costs by sequencing amplicons generated with 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 data visualization 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 TRG 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 TRG gene locus on chromosome 7 (7q14) includes 14 V (variable region) genes (Group I, II, III, and IV), 5 J (joining region) gene segments, and 2 C (constant region) genes spread over 200 kilobases. During development of lymphoid cells, antigen receptor genes undergo somatic gene rearrangements. Specifically during T-cell development, genes encoding TRG molecules are assembled from multiple polymorphic gene segments that undergo rearrangement generating V-J combinations 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 rearrangement. Therefore, tests that detect TRG clonal rearrangements 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. Simplified Representation of the TRG Gene Depicted are the variable region (V) genes and downstream consensus joining region genes (J) that are involved in TRG gene rearrangements. Kit A Components Master Mix Name Index # TRG MiSeq 01 A001 TRG MiSeq 02 A002 TRG MiSeq 03 A003 TRG MiSeq04 A004 TRG MiSeq 05 A005 TRG MiSeq 06 A006 TRG MiSeq 07 A007 TRG MiSeq 08 A008 Controls TRG 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 # TRG MiSeq 09 A009 TRG MiSeq 18 A018 TRG MiSeq 10 A010 TRG MiSeq 19 A019 TRG MiSeq 11 A011 TRG MiSeq 20 A020 TRG MiSeq 12 A012 TRG MiSeq 21 A021 TRG MiSeq 13 A013 TRG MiSeq 22 A022 TRG MiSeq 14 A014 TRG MiSeq 23 A023 TRG MiSeq 15 A015 TRG MiSeq 25 A025 TRG MiSeq 16 A016 TRG MiSeq 27 A027 Controls TRG POS (+) Qty. 3 NGS NEG (-) Qty. 3 Assay components Master Mix Name Index # Master Mix Name Index # Master Mix Name Index # TRG S5/PGM 01 IonXpress_001 TRG S5/PGM 07 IonXpress_007 TRG S5/PGM 11 IonXpress_011 TRG S5/PGM 02 IonXpress_002 TRG S5/PGM 08 IonXpress_008 TRG S5/PGM 12 IonXpress_012 TRG S5/PGM 03 IonXpress_003 TRG S5/PGM 09 IonXpress_009 TRG S5/PGM 13 IonXpress_013 TRG S5/PGM 04 IonXpress_004 TRG S5/PGM 10 IonXpress_010 TRG S5/PGM 14 IonXpress_014 Controls TRG POS (+) Qty. 2 NGS NEG (-) Qty. 2 V-J Sequence Frequencies. The LymphoTrack Dx bioinformatics software provides PDF reports which include Top 10 Merged Read Summary as well as a stacked bar graph depicting the relative frequencies of the V-J rearrangements identified in the sample. To learn more about the LymphoTrack Dx software, please refer to the LymphoTrack Dx Bioinformatics Software section. Ordering Information Catalog # Products Quantity Components 9-227-0019 LymphoTrack® Dx TRG Assay Kit A - MiSeq® 8 indices - 5 sequencing reactions each 9-227-0009 LymphoTrack® Dx TRG Assay Panel - MiSeq® 24 indices - 5 sequencing reactions each 9-500-0009 LymphoTrack® Dx Software - MiSeq® 1 CD complimentary with purchase 9-227-0007 LymphoTrack® Dx TRG Assay - S5/PGM™ 12 indices - 5 sequencing reactions each 9-500-0007 LymphoTrack® Dx Software - S5/PGM™ 1 CD complimentary with purchase These products are CE-IVD assays for in vitro diagnostic use. Reagents - MiSeq® Detection Reagents - Ion S5™ /PGM™ Detection