42 Next-Generation Sequencing (NGS) | Research Use Only (RUO) Assays Invivoscribe 2019 | 43 NGS RUO Assays LymphoTrackTRB Assays Assay Uses This research use only (RUO) assay for next-generation sequencing (NGS) identifies clonal TRB Vβ-(Dβ-)Jβ rearrangements, the associated Vβ-(Dβ-)Jβ region DNA sequences, it provides the frequency distribution of Vβ, Dβ, and Jβ region segment utilization using the Illumina® MiSeq® platform. Analysis of the rearranged TRB locus increases the probability of identifying T-cell receptor gene rearrangements, as compared to testing for TRG gene rearrangements only. As a result, combining the analysis of TRB and TRG loci increases the sensitivity of clonality detection. Summary and Explanation of the Test In contrast to the conventional fragment analysis assays for clonality that utilize three master mixes, this NGS-based assay contains a single multiplex master mix to target conserved V, D, and J regions of the TRB gene locus described in lymphoid malignancies. This reduces DNA sample requirements and simplifies the testing workflow. The LymphoTrack TRB master mix primers are also designed with Illumina® adapters and 8 indices (Kit A) or 24 indices (Panel). This allows amplicons generated from different indexed TRB master mixes to be pooled into a single sequencing library. The associated LymphoTrack Software is capable of sorting complex NGS data by gene target. This offers a second layer of multiplexing to reduce per sample testing costs by allowing amplicons from any LymphoTrack Assay (e.g. IGH, IGK, TRB, TRG) to be sequenced on the same flow cell. In addition, the LymphoTrack Software provides easy visualization of data and the LymphoTrack Minimal Residual Disease Data Analysis Tool allows identified sequences to be tracked and monitored in subsequent samples. Positive clonal and negative polyclonal DNA controls are included in kits. 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 LymphoTrack TRB Assay represent a significant improvement over clonality assessment by fragment analysis 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 T-cell receptor beta (TRB) gene locus on chromosome 7 (7q34) includes 65 Vβ (variable) gene segments, followed by two separate clusters of genes each containing a Dβ (diversity) gene, several Jβ (joining) genes, and a Cβ (constant) region spread over 685 kilobases. The 2 Cβ genes, TRBC1 and TRBC2, encode highly homologous products with no functional difference. During lymphoid cell development, antigen receptor genes undergo somatic gene rearrangements. Specifically, during T-cell development, genes encoding TRB molecules are assembled from multiple polymorphic gene segments that generate Vβ-Dβ-Jβ 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 rearrangements. Therefore, tests that detect TRB clonal rearrangements can be useful in the study of B- and T-cell malignancies. Specimen Requirement 50 ng of high-quality genomic DNA. References 3. EP Rock et al., J Exp Med 179 (1): 323-8 (1994). 4.  JE Miller et al., Molecular Genetic Pathology (2nd Ed., sections 30.2.7.13 and 30.2.7.18). New York, USA: Springer Science & Business Media (2013). Simplified Representation of the TRB Gene Depicted are the variable (Vβ), diversity (Dβ), and joining (Jβ) gene regions involved in TRB gene rearrangements, in addition to the downstream consensus (Cβ) gene regions. TRB Assay LymphoTrack ® Vβ Dβ1 Dβ2 Cβ1 Cβ2 Jβ1 Jβ2 Kit A Components Master Mix Name Index # TRB MiSeq 01 A001 TRB MiSeq 02 A002 TRB MiSeq 03 A003 TRB MiSeq 04 A004 TRB MiSeq 05 A005 TRB MiSeq 06 A006 TRB MiSeq 07 A007 TRB MiSeq 08 A008 Controls TRB 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 # TRB MiSeq 09 A009 TRB MiSeq 18 A018 TRB MiSeq 10 A010 TRB MiSeq 19 A019 TRB MiSeq 11 A011 TRB MiSeq 20 A020 TRB MiSeq 12 A012 TRB MiSeq 21 A021 TRB MiSeq 13 A013 TRB MiSeq 22 A022 TRB MiSeq 14 A014 TRB MiSeq 23 A023 TRB MiSeq 15 A015 TRB MiSeq 25 A025 TRB MiSeq 16 A016 TRB MiSeq 27 A027 Controls TRB POS (+) Qty. 3 NGS NEG (-) Qty. 3 Reagents - MiSeq® Detection Ordering Information Catalog # Products Quantity Components 7-225-0009 LymphoTrack® TRB Assay Kit A - MiSeq® 8 indices - 5 sequencing reactions each 7-225-0019 LymphoTrack® TRB Assay Panel - MiSeq® 24 indices - 5 sequencing reactions each 7-500-0009 LymphoTrack® Software - MiSeq® 1 CD complimentary with purchase 7-500-0008 LymphoTrack® MRD Software* 1 CD complimentary with purchase V-J Sequence Frequencies. The LymphoTrack 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 200 most prevalent rearrangements sequenced and identified in the sample. To learn more about the LymphoTrack software, please refer to the LymphoTrack Bioinformatics Software section. 1. *MRD Software can be used to track sequences generated by either LymphoTrack Assays - MiSeq® or Ion S5/PGM™ MRD applications are for Research Use Only. To obtain a copy, please contact your local distributor or send an e-mail to MRD@invivoscribe.com