40 Next-Generation Sequencing (NGS) | Research Use Only (RUO) Assays Invivoscribe 2019 | 41 NGS RUO Assays LymphoTrackTRG Assays Assay Uses This research use only (RUO) assay for next-generation sequencing (NGS) identifies clonal TRG V-J rearrangements and the associated V-J region DNA sequences. It also provides the frequency distribution of V-J segment utilization using the Illumina® MiSeq® or Thermo Fisher Scientific® Ion S5™ and PGM™ instruments. Summary and Explanation of the Test The LymphoTrack TRG Assay represents a significant improvement over existing clonality assays that utilize fragment analysis by providing three important and complementary uses: 1. Detection of clonal populations. 2.  Identification of sequence information and gene segment utilization. 3.  Ability to track sequences in subsequent samples with the use of the Invivoscribe MRD Software. The single multiplex master mix targets all conserved regions within the variable (V) and the joining (J) genes described in lymphoid malignancies. The average PCR amplicon size of the TRG gene rearrangements generated using this assay was designed to be compatible with fragmented DNA isolated from challenging samples such as FFPE sections. Primers are designed with Illumina® adapters and indices (8-24) or Thermo Fisher Scientific adaptors and indices (12), thereby allowing for a one-step PCR reaction to generate sequence-ready amplicons. In addition, amplicons from different samples (amplified with different indexed master mixes) or LymphoTrack kits can be sequenced together on a single Illumina® MiSeq® flow cell Ion S5™ or PGM™ chip to reduce per sample testing costs. The LymphoTrack bioinformatics software enables simplified analysis and visualization of data generated from this assay 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) and negative (polyclonal) DNA controls are included in the kits. Background The human T-cell receptor gamma (TRG) gene locus on chromosome 7 (7q14) includes 14 variable region (Group I, II, III, and IV), 5 joining region (J) gene segments, and 2 constant (C) genes spread over 200 kilobases. During development of lymphoid cells, the antigen receptor genes (including gene segments within the TRG locus), undergo somatic gene rearrangement. These developmentally regulated gene rearrangements generate V-J combinations that are unique 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 TRG clonal populations can be useful in the study of T-cell and certain B-cell malignancies. Since the TRG locus rearranges before the TRB locus, and all unsuccessful or unproductive rearrangements are retained in the cells, examination of the TRG locus can identify both clonal d/g as well as clonal a/b T-cells. Clonal a/b T-cells would be expected to have biallelic TRG gene rearrangements. Specimen Requirement 50 ng of high-quality genomic DNA. References 1. S Tonegawa et al., 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). Simplified Representation of theTRG Gene Simple representation of the organization of the T-cell receptor gamma gene on chromosome 7. Depicted are the variable region genes (Vƴ 2–Vƴ 11) and downstream joining region genes (Jƴ 1/2–Jƴ P1/2) that are involved in rearrangements in T-cell lymphomas. Kit A Components Master Mix Name Index # TRG MiSeq 01 A001 TRG MiSeq 02 A002 TRG MiSeq 03 A003 TRG MiSeq 04 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 bioinformatics software provides a stacked bar graph depicting the relative frequencies for the V-J rearrangements identified and sequenced in a sample. To learn more about the LymphoTrack software, please refer to the LymphoTrack Bioinformatics Software section. Ordering Information Catalog # Products Quantity Components 7-227-0019 LymphoTrack® TRG Assay Kit A - MiSeq® 8 indices - 5 sequencing reactions each 7-227-0009 LymphoTrack® TRG Assay Panel - MiSeq® 24 indices - 5 sequencing reactions each 7-500-0009 LymphoTrack® Software - MiSeq® 1 CD complimentary with purchase 7-227-0007 LymphoTrack® TRG Assay - S5/PGM™ 12 indices - 5 sequencing reactions each 7-500-0007 LymphoTrack® Software - S5/PGM™ 1 CD complimentary with purchase 7-500-0008 LymphoTrack® MRD Software* 1 CD complimentary with purchase *MRD Software can be used to track sequences generated by either LymphoTrack Assays - MiSeq® or Ion S5/PGM™ . Reagents - MiSeq® Detection Reagents - Ion S5/PGM™ Detection