28 Next-Generation Sequencing (NGS) | CE-IVD Assays Invivoscribe 2019 | 29 NGS CE-IVD Assays Sample Name Total reads = 32,458 LymphoTrackDx Bioinformatics Software Software Use The LymphoTrack Dx Bioinformatics Software package is provided with each LymphoTrack Dx Assay to analyze raw FASTQ files for clonality analysis of single or multiple target data sets (IGHV Leader, IGH FR1, IGH FR2, IGH FR3, IGK, TRB, or TRG). For data generated with LymphoTrack Dx IGHV Leader or IGH FR1 Assays, the software provides additional information, including the rate of somatic hypermutation and whether a clone would be functional based upon the presence of a premature stop codon. The software can also predict whether an open reading frame would be in- or out-of-frame. The provided software is composed of two distinct parts: 1. A bioinformatics Data Analysis Application 2. Microsoft Excel® Data Visualization spreadsheets and automated Sample-to-PDF Reports for streamlined data analysis. for each gene target 3. VJ Usage and VJ sequence frequency graphs. Minimum Software Requirements • Processor: Intel Core 2 Duo or newer CPU recommended. • Hard Drive: At least 80 GB of free disk space is required; 250 GB recommended. • RAM: 4 GB required; 8 GB or more recommended. • Operating System: Windows 7 (64-bit) is required. • Java 7 for 64-bit operating systems or higher (Java configured for 32-bit operating systems is not compatible with the LymphoTrack Dx Software – MiSeq or LymphoTrack Dx Software - PGM). The most recent version of Java can be downloaded directly from Oracle at http://www.java.com/. • A PDF reader, such as Adobe Acrobat Reader, to visualize data reports generated by the LymphoTrack Reporter. • Microsoft Office Excel 2007, 2010, or 2013 with Macro support enabled is required for data visualization. • A CD-ROM drive. • The LymphoTrack Dx Software – MiSeq v2.4.3 has been validated for use with Windows 7 configured for English (United States) language and default display size settings, and Microsoft Excel 2007/2010/2013 for alternate data visualization. Use of other Windows/Excel versions and/or language/display size settings may not be compatible. • The LymphoTrack Dx Software – PGM v2.3.1 has been validated for use with Microsoft Excel 2007/2010/2013 and Windows 7 configured for the English (United States) language and default display size settings. Use of other Microsoft Excel/Windows versions and/or language/display size settings may not be compatible. Sequencing Summary Using the merged read summary, along with the easy to follow flow charts in the accompanying LymphoTrack Dx Assay instructions for use (IFU), interpretation is quick and easy. Sequence Frequency Graph Ordering Information Catalog # Products Quantity Components 9-500-0009 LymphoTrack® Dx Software - MiSeq® 1 CD complimentary with kit purchase 9-500-0007 LymphoTrack® Dx Software - S5/PGM™ 1 CD complimentary with kit purchase These products are CE-IVD assays for in vitro diagnostic use. 0 1 2 3 4 5 6 % Total Reads LymphoTrack IGH Assay - V – J Sequence Frequencies LymphoTrack IGH Assay - V - J Sequence Frequencies % Total Reads The stacked bar graph depicts the top 200 sequencing reads for a sample. Each individual colored bar represents a unique population of cells. Different colors stacked at the same point on the x-axis represent unique sequences that utilize the same V and J gene families. The amplicons of these products vary in sequence and may also vary in product size. 1.  The sample name is clearly identified and the total number of reads (= Read Depth) generated for the sample is provided. Following the IFU, it is easy to determine whether the data generated for a sample can be assessed for the presence or absence of clonality. 2.  The sequence of clonal populations is provided and populations are ranked from most abundant to least prevalent. Sequences that differ by 1-2 basepairs are automatically merged to account for possible sequencing errors and to improve the accuracy and ease of sample interpretation. 3.  Sequences are aligned with reference genes to allow for easy identification of specific types of gene rearrangements such as IGHV3-21, which is characteristic of some CLL cases and correlates with a poor prognosis. 4.  The percentage that a unique sequence contributes to the total number of reads for a sample is calculated. Following the guidelines in the IFU, samples can be interpreted for the evidence indicating the presence or absence of clonality. 5.  For the LymphoTrack Dx IGHV Leader and IGH FR1 Assays, the somatic hypermutation status of a sequence is automatically calculated by comparing the identified sequence with a germline reference. In addition, predictions on whether the sequence would be functional can be drawn by the provided information regarding the presence of a premature stop codon or an open reading frame that is out-of-frame. Rank Sequence Length Merge count V-gene J-gene % Total reads Cumulative % Mutation rate partial V-gene (%) In-frame (Y/N) No stop codon (Y/N) V-coverage 1 TTCTCGTGGTG 455 29603 IGHV4-59_08 IGHJ4_02 9.93 9.93 11.26 Y Y 98.63 2 CTCGCCCTCCT 463 205 IGHV5-51_01 IGHJ4_02 0.07 9.99 0.00 Y Y 99.66 3 GGTTTTCCTTG 484 201 IGHV3-7_01 IGHJ4_02 0.07 10.06 7.77 Y Y 100.00 4 CTCGCCCTCCT 463 185 IGHV5-51_01 IGHJ5_02 0.06 10.12 6.08 Y Y 99.32 5 CTCGCCCTCCT 469 170 IGHV5-51_01 IGHJ4_02 0.06 10.18 0.00 Y Y 99.32 6 CTCGCCCTCCT 466 160 IGHV5-51_01 IGHJ4_02 0.05 10.23 0.00 Y Y 99.66 7 CTGCTGCTGAC 460 159 IGHV2-5_10 IGHJ5_02 0.05 10.29 8.08 Y Y 97.64 8 GGTTTTCCTTG 493 156 IGHV3-48_02 IGHJ6_02 0.05 10.34 3.72 Y Y 98.99 9 CTCGCCCTCCT 334 153 IGHV5-51_02 IGHJ2_01 0.05 10.39 3.72 Y N 27.70 10 CTCGCCCTCCT 334 152 IGHV5-51_02 IGHJ2_01 0.05 10.44 3.38 Y N 26.01 2 4 3 5 1 1 2 3 4 5 Unique sequence CATCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGG GCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCC AGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAG CCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCTAGAGATCTCACAGGTTGTATTAGT ACCAGCTGCTATCCTCCGAACTACTTTGACTACTGGGGCCAGGGAACCCT Gene rearrangement family IGHV1-46_03 - IGHJ4_02 * If a CD-ROM drive is not available, please contact us at: support@invivoscribe.com