b'Next-Generation Sequencing (NGS) | CE-IVD AssaysTRB Assay Next-Generation Sequencing (NGS) | CE-IVD AssaysLymphoTrack Dx TRB AssayAssay Description 1.Detection of initial clonal populations. 2.dentification of sequence information required to track clonal The LymphoTrackDx TRB Assay is an in vitro diagnostic productIintended for next-generation sequencing (NGS) for the Illuminarearrangements in subsequent samples.MiSeq instrument. The assay will determine the frequency distributionAnalysis of the rearranged TRB locus increases the probability of of TRB V-J gene rearrangements in patients suspected of havingidentifying T cell receptor gene rearrangements, as compared to lymphoproliferative disease. This assay aids in the identification oftesting for TRG gene rearrangements only. As a result, combining lymphoproliferative disorders. the analysis of TRB and TRG loci increases the sensitivity of clonality detection. Summary and Explanation of the Test The human T-cell receptor beta (TRB) gene locus on chromosome In contrast to the IdentiClone fragment analysis assays for clonality7 (7q34) includes 65 V (variable) gene segments, followed by two that utilizes three master mixes, this NGS-based assay contains aseparate clusters of genes each containing a D (diversity) gene, single multiplex master mix to target conserved V and J regions ofseveral J (joining) genes, and a C (constant) region spread over the TRB gene locus described in lymphoid malignancies, thereby,685 kilobases. The 2 C genes, TRBC1 and TRBC2, encode highly reducing sample DNA requirements and simplifying the testinghomologous products with no functional difference.workflow. The LymphoTrack Dx TRB master mix primers are designedDuring lymphoid cell development, antigen receptor genes undergo with Illumina adapters and 8 (Kit A) or 24 (Panel) different indices.somatic gene rearrangements. Specifically, during T-cell development This allows amplicons generated from different indexed TRB mastergenes encoding TRB molecules are assembled from multiple mixes to be pooled into a single library for loading onto one MiSeqpolymorphic gene segments that generate V - D - J combinations flow cell. unique in both length and sequence.The associated LymphoTrack Dx Software is capable of sortingSince leukemias and lymphomas originate from the malignant complex NGS data by gene target, offering a second layer oftransformation of individual lymphoid cells, all leukemias and multiplexing. This provides users the ability to reduce per samplelymphomas generally share one or more cell-specific or clonal testing costs by sequencing amplicons from any LymphoTrack Dxantigen receptor gene rearrangements. Therefore, tests that detect MiSeq Assay (e.g. TRB, TRG, IGH, IGK) at the same time. In addition,TRB clonal rearrangements can be useful in the study of B- andthe LymphoTrack Dx Software provides an easy and streamlinedT-cell malignancies.method for visualization of data. Guidelines to interpret samples for evidence or no evidence of clonality are included in the instructionsfor use provided with each kit. Specimen RequirementPositive clonal and negative polyclonal DNA controls are included in kits. 50 ng of high-quality genomic DNA.Background ReferencesThe LymphoTrack Dx TRB Assays represent a significant improvement1.EP Rock et al., J Exp Med 179 (1): 323-8 (1994).over fragment analysis methods for clonality testing by providing two2. JE Miller et al., Molecular Genetic Pathology (2nd ed.). Springer important and complementary uses:Simplified Representation of the TRB GeneV D1 J1 C1 D2 J2 C2Depicted are the variable (V), diversity (D), and joining (J) gene regions involved in TRB gene rearrangements, in addition to the downstream consensus (C) gene regions. 30'