b'Next-Generation Sequencing (NGS) | Research Use Only (RUO) AssaysIGHV Leader Somatic Hypermutation Assay Next-Generation Sequencing (NGS) | RUO Assays IGHV Leader Somatic Hypermutation Assay Next-Generation Sequencing (NGS) | RUO AssaysLymphoTrack IGHV LeaderSomatic Hypermutation Assay Reagents - MiSeq DetectionKit A Components Panel Components(includes all master mixes from Kit A plus the items below)Master Mix Name Index # Master Mix Name Index # Master Mix Name Index #Assay Uses BackgroundIGH Leader MiSeq 01 A001 IGH Leader MiSeq 09 A009 IGH Leader MiSeq 18 A018This research use only (RUO) assay for next-generation sequencingThe human immunoglobulin heavy chain (IGH) gene locus onIGH Leader MiSeq 02 A002 IGH Leader MiSeq 10 A010 IGH Leader MiSeq 19A019(NGS), identifies clonal IGH V H -J Hrearrangements, the associatedchromosome 14 (14q32.3) includes 46 - 52 functional and 30IGH Leader MiSeq 03 A003 IGH Leader MiSeq 11 A011 IGH Leader MiSeq 20 A020V H -J HDNA sequences, and the frequency distribution of V Hregion andnonfunctional variable (V H ) gene segments, 27 functional diversityIGH Leader MiSeq 04 A004 IGH Leader MiSeq 12 A012 IGH Leader MiSeq 21 A021J 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. IGH Leader MiSeq 05 A005 IGH Leader MiSeq 13 A013 IGH Leader MiSeq 22 A022in the IGHV gene of analyzed samples. If you would like to test for IGHVIGH Leader MiSeq 06 A006 IGH Leader MiSeq 14 A014 IGH Leader MiSeq 23 A023somatic 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 undergoIGH Leader MiSeq 07 A007 IGH Leader MiSeq 15 A015 IGH Leader MiSeq 25 A025rearrangements and selection, generating V H -D H -J Hcombinations IGH Leader MiSeq 08 A008 IGH Leader MiSeq 16 A016 IGH Leader MiSeq 27 A027Summary and Explanation of the Testthat are unique in both length and sequence. 1 Controls ControlsThe LymphoTrack IGHV Leader Somatic Hypermutation Assay forSince leukemias and lymphomas originate from the malignantIGH SHM POS (+) Qty. 1 IGH SHM POS (+) Qty. 3NGS 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"IGH POS (+) Qty. 1 IGH POS (+) Qty. 3detects the majority of IGH gene rearrangements using a singleantigen receptor gene rearrangements. Therefore, tests that detectNGS NEG (-) Qty. 1 NGS NEG (-) Qty. 3multiplex 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 informationMutation rate No stop The master mixes included in this assay target the Leader (V H L) andfor chronic lymphocytic leukemia (CLL)2and small lymphocyticRank Sequence Length MergeV-gene J-gene % TotalCumulativepartial V-geneIn-framecodon V-coveragethe joining (J H ) gene regions of IGH and are designed with Illuminalymphoma (SLL). In addition, NGS methods can improve diseasecount reads % (%) (Y/N) (Y/N)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- 1 TTCTCGTGGTG 455 29603 IGHV4-59_08 IGHJ4_02 9.93 9.93 11.26 Y Y 98.63step PCR approach to generate sequence-ready amplicons, followed by direct pooling of samples for sequencing on a Illumina MiSeqSpecimen Requirement 2 CTCGCCCTCCT 463 205 IGHV5-51_01 IGHJ4_02 0.07 9.99 0.00 Y Y 99.66flow cell. 50 ng of high-quality genomic DNA. 3 GGTTTTCCTTG 484 201 IGHV3-7_01 IGHJ4_02 0.07 10.06 7.77 Y Y 100.00The included LymphoTrack bioinformatics software enables simplified4 CTCGCCCTCCT 463 185 IGHV5-51_01 IGHJ5_02 0.06 10.12 6.08 Y Y 99.32analysis and visualization of data generated from this assay. References 5 CTCGCCCTCCT 469 170 IGHV5-51_01 IGHJ4_02 0.06 10.18 0.00 Y Y 99.32Positive (clonal positive, SHM negative), negative (polyclonal), and1.Miller et al., Molecular Genetic Pathology (2nd ed.). Springer6 CTCGCCCTCCT 466 160 IGHV5-51_01 IGHJ4_02 0.05 10.23 0.00 Y Y 99.66SHM positive (clonal positive, SHM positive) controls are includedScience & Business Media. 2013: 302.2.7.13 and 30.2.7.18.7 CTGCTGCTGAC 460 159 IGHV2-5_10 IGHJ5_02 0.05 10.29 8.08 Y Y 97.64in 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). 8 GGTTTTCCTTG 493 156 IGHV3-48_02 IGHJ6_02 0.05 10.34 3.72 Y Y 98.99This allows for a one-step PCR reaction to generate sequence-ready amplicons and pooling of several different samples on the same9 CTCGCCCTCCT 334 153 IGHV5-51_02 IGHJ2_01 0.05 10.39 3.72 Y N 27.70Illumina MiSeq flow cell. The LymphoTrack bioinformatics software10 CTCGCCCTCCT 334 152 IGHV5-51_02 IGHJ2_01 0.05 10.44 3.38 Y N 26.01enables easy analysis and visualization of data and the LymphoTrack MRD Software allows sequences to be tracked in subsequent samples.Example Data. Depicted are the top 10 sequences from a read summary generated by the LymphoTrack Software - MiSeq. Highlighted columns represent Please see the LymphoTrack MRD software section to learn how thefields that are unique to SHM analysis and include the SHM mutation rate and predictions pertaining to whether a sequence is in-frame or contains a LymphoTrack Assays can be applied to MRD studies, or emailpremature stop codon. To learn more about the LymphoTrack software, please refer to the LymphoTrack Bioinformatics Software section.marketing@invivoscribe.com.Ordering InformationCatalog # Products Quantity ComponentsV H L V H 1 V H 2 V H 3 V H 4 V H 5 V H 6 V H 7 J H 7-121-0059 LymphoTrack IGHV Leader Somatic Hypermutation Assay Kit A - MiSeq8 indices - 5 sequencing reactions eachSimple representation of the organization of the immunoglobulin heavy chain (IGH) gene on chromosome 14. Depicted are the variable region (V H ) genes and7-121-0069 LymphoTrack IGHV Leader Somatic Hypermutation Assay Panel - MiSeq24 indices - 5 sequencing reactions eachdownstream 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. 7-500-0009 LymphoTrack Software - MiSeq1 CD complimentary with purchase40Invivoscribe 2021|41'