38 Next-Generation Sequencing (NGS) | Research Use Only (RUO) Assays Invivoscribe 2019 | 39 NGS RUO Assays LymphoTrackIGK Assays Assay Uses This research use only (RUO) assay for next-generation sequencing (NGS), identifies clonal IGK Vƙ-Jƙ, Vƙ-Kde, and intron-Kde (INTR-Kde) rearrangements, the corresponding DNA sequences, and provides the distribution frequency of Vƙ-Jƙ, Vƙ-Kde, and INTR-Kde segment utilization using the Illumina® MiSeq® or Thermo Fisher Scientific® Ion PGM™ and Ion S5™ platforms. Summary and Explanation of the Test The LymphoTrack IGK Assay represents a significant improvement over 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 LymphoTrack MRD Software. Unlike conventional fragment analysis assays, this NGS method utilizes a single multiplex master mix to target conserved regions of IGK that are described in lymphoid malignancies. 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. To reduce per sample testing costs, 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™ chips. The LymphoTrack bioinformatics software enables simplified analysis and visualization of data and the LymphoTrack MRD Software allows identified 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 immunoglobulin kappa (IGK) gene locus on chromosome 2 (2p11.2) includes 76 V (variable) region genes spanning 7 subgroups and 5 J (joining) region gene segments upstream of the Cƙ region. The Kde, approximately 24 kb downstream of the Jƙ-Cƙ region, can also rearrange with Vƙ gene segments and the isolated recombination signal sequence in the Jƙ-Cƙ intronic region. During development of lymphoid cells, antigen receptor genes undergo somatic gene rearrangements. Specifically during B-cell development, genes encoding IGK molecules are assembled from multiple polymorphic gene segments that undergo rearrangements generating V-J 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 IGK clonal populations can be useful in the study of B- and T-cell malignancies and complement IGH testing, as the IGK receptor is less susceptible to somatic mutations. 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 the IGK Gene Depicted are the variable region (Vƙ) genes or variable intragenic Jƙ-Cƙ intron (Jƙ-Cƙ INTR) and downstream consensus joining region genes (Jƙ) or kappa deleting element (Kde) that are involved in IGK gene rearrangements. Kde Kde Kit A Components Master Mix Name Index # IGK MiSeq 01 A001 IGK MiSeq 02 A002 IGK MiSeq 03 A003 IGK MiSeq 04 A004 IGK MiSeq 05 A005 IGK MiSeq 06 A006 IGK MiSeq 07 A007 IGK MiSeq 08 A008 Panel Components (includes all master mixes from Kit A plus the items below) Master Mix Name Index # Master Mix Name Index # IGK MiSeq 09 A009 IGK MiSeq 18 A018 IGK MiSeq 10 A010 IGK MiSeq 19 A019 IGK MiSeq 11 A011 IGK MiSeq 20 A020 IGK MiSeq 12 A012 IGK MiSeq 21 A021 IGK MiSeq 13 A013 IGK MiSeq 22 A022 IGK MiSeq 14 A014 IGK MiSeq 23 A023 IGK MiSeq 15 A015 IGK MiSeq 25 A025 IGK MiSeq 16 A016 IGK MiSeq 27 A027 Assay Components Master Mix Name Index # Master Mix Name Index # IGK S5/PGM 01 IonXpress_001 IGK S5/PGM 11 IonXpress_011 IGK S5/PGM 02 IonXpress_002 IGK S5/PGM 12 IonXpress_012 IGK S5/PGM 04 IonXpress_004 IGK S5/PGM 13 IonXpress_013 IGK S5/PGM 08 IonXpress_008 IGK S5/PGM 14 IonXpress_014 IGK S5/PGM 09 IonXpress_009 IGK S5/PGM 16 IonXpress_016 IGK S5/PGM 010 IonXpress_010 IGK S5/PGM 17 IonXpress_017 V-J Sequence Frequencies. The LymphoTrack bioinformatics software provides a stacked bar graph depicting the relative frequencies for 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. Ordering Information Catalog # Products Quantity Components 7-122-0009 LymphoTrack® IGK Assay Kit A - MiSeq® 8 indices - 5 sequencing reactions each 7-122-0019 LymphoTrack® IGK Assay Panel - MiSeq® 24 indices - 5 sequencing reactions each 7-500-0009 LymphoTrack® Software - MiSeq® 1 CD complimentary with purchase 7-122-0007 LymphoTrack® IGK 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 Controls IGK POS (+) Qty. 3 NGS NEG (-) Qty. 3 Controls IGK POS (+) Qty. 2 NGS NEG (-) Qty. 2 Controls IGK POS (+) Qty. 1 NGS NEG (-) Qty. 1 *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