b'Next-Generation Sequencing (NGS) | Research Use Only (RUO) AssaysIGHV Leader Somatic Hypermutation Assay Next-Generation Sequencing (NGS) | RUO AssaysLymphoTrack IGHV LeaderSomatic Hypermutation AssayAssay Uses Background This research use only (RUO) assay for next-generation sequencingThe human immunoglobulin heavy chain (IGH) gene locus on (NGS), identifies clonal IGH V H -J Hrearrangements, the associatedchromosome 14 (14q32.3) includes 46 - 52 functional and 30 V H -J HDNA sequences, and the frequency distribution of V Hregion andnonfunctional variable (V H ) gene segments, 27 functional diversity J Hregion segment utilization. The assay also uses the Illumina MiSeq(D H ) gene segments, and 6 functional joining (J H ) gene segments platform to define the extent of somatic hypermutation (SHM) presentspread over 1250 kilobases.in the IGHV gene of analyzed samples. If you would like to test for IGHV somatic hypermutation using the Thermo Fisher Ion PGM or Ion S5During B-cell development, genes encoding the IGH molecules are platform please refer to the LymphoTrack IGH FR1 Assay (7-121-0007).assembled from multiple polymorphic gene segments that undergo rearrangements and selection, generating V H -D H -J HcombinationsSummary and Explanation of the Testthat are unique in both length and sequence. 1The LymphoTrack IGHV Leader Somatic Hypermutation Assay forSince leukemias and lymphomas originate from the malignant NGS provides significant improvements over clonality assays usingtransformation of individual lymphoid cells, all leukemias and fragment analysis and Sanger sequencing. The assay efficientlylymphomas generally share one or more cell-specific or "clonal" detects the majority of IGH gene rearrangements using a singleantigen receptor gene rearrangements. Therefore, tests that detect multiplex master mix, identifies the DNA sequence specific for eachIGH clonal rearrangements can be useful in the study of B-cell clonal gene rearrangement, and calculates the degree of SHM formalignancies. An additional level of diversity is generated in the each sample. antigen receptors by somatic point mutations in the variable regions and this mutation status provides important prognostic information The master mixes included in this assay target the Leader (V H L) andfor chronic lymphocytic leukemia (CLL)2and small lymphocytic the joining (J H ) gene regions of IGH and are designed with Illuminalymphoma (SLL). In addition, NGS methods can improve disease adapters and indices (8 included in Kit A and 24 included in Panels).stratification and elucidate subclone gene profiles.Including adapters and indices in the primer design allows for a one-step PCR approach to generate sequence-ready amplicons, followed by direct pooling of samples for sequencing on a Illumina MiSeqSpecimen Requirementflow cell. 50 ng of high-quality genomic DNA.The included LymphoTrack bioinformatics software enables simplified analysis and visualization of data generated from this assay. ReferencesPositive (clonal positive, SHM negative), negative (polyclonal), and1.Miller et al., Molecular Genetic Pathology (2nd ed.). Springer SHM positive (clonal positive, SHM positive) controls are includedScience & Business Media. 2013: 302.2.7.13 and 30.2.7.18. in the kit. Primers included in the master mixes are designed with Illumina adapters and indices (8 or 24 indices per framework kits). 2. Ghia et al., Leukemia 21: 2-3 (2007).This allows for a one-step PCR reaction to generate sequence-ready amplicons and pooling of several different samples on the same Illumina MiSeq flow cell. The LymphoTrack bioinformatics software enables easy analysis and visualization of data 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 [email protected] H L V H 1 V H 2 V H 3 V H 4 V H 5 V H 6 V H 7 J HSimple representation of the organization of the immunoglobulin heavy chain (IGH) gene on chromosome 14. Depicted are the variable region (V H ) genes and downstream consensus joining region genes (J H ) that are involved in rearrangements. Upstream of the variable gene segments, the leader sequence (V H L) is also depicted. Diversity region genes are not depicted.42'