Instructions for Use

LymphoTrack Software – S5/PGM™ Version 2.4.7®

This software package is provided with each LymphoTrack Assay for the Thermo Fisher Scientific Ion S5™ and Ion PGM™
instruments to analyze raw FASTQ files. The software is composed of three distinct parts: the LymphoTrack Data Analysis
application, a LymphoTrack Reporter plugin that runs automatically after analysis, and an optional LymphoTrack Visualization
method consisting of three Microsoft Excel® *.xlsm spreadsheets. The software runs on supported Microsoft Windows®
platforms.
This software package allows for clonality analysis of single target data (IGH FR1, IGH FR2, IGH FR3, IGK, or TRG) as well as for
simultaneous analysis of IGH FR1, IGH FR2, IGH FR3 IGK, and TRG data.
For guidance with minimal residual disease (MRD) studies, please refer to the technical bulletin entitled “Study of MRD – Using
LymphoTrack Assays”. This document can be found on the RUO LymphoTrack MRD Software CD (75000008). To request
the technical bulletin, please email marketing@invivoscribe.com.
For Research Use Only. Not for use in Diagnostic Procedures.

Product Compatibility
This LymphoTrack Software – S5/PGM (following Invivoscribe assays:

75000007) was developed specifically for and is compatible with only theCatalog #Description

71210007

LymphoTrack IGH FR1 Assay – S5/PGM

71210037

LymphoTrack IGH FR2 Assay – S5/PGM

71210047

LymphoTrack IGH FR3 Assay – S5/PGM

71210057

LymphoTrack IGH FR1/2/3 Assay – S5/PGM

71220007

LymphoTrack IGK Assay – S5/PGM

72270007

LymphoTrack TRG Assay – S5/PGM

75000007

LymphoTrack Software – S5/PGM

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Minimum System Requirements
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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 8 for 64-bit operating systems or higher (Java configured for 32-bit operating systems is not compatible with theLymphoTrack Software – S5/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 deprecated data visualization.
A CD-ROM drive.
The LymphoTrack Software – S5/PGM v2.4.7 has been validated for use with Windows 7 or Windows 10 configured for
the English (United States) language and default display size settings and Microsoft Excel 2007/2010/2013 for alternate
data visualization. Use of other Excel/Windows versions and/or language/display size settings may not be compatible.

Warnings and Precautions
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Software version. Ensure the most current version of the LymphoTrack Software – S5/PGM is installed and used for data
analysis.
Default screen size. Optimal screen resolution for LymphoTrack Software – S5/PGM is 100%. Increasing the monitor
default screen size above 100% may affect the display of field text in the user interface.
System font. User interface is designed to use the default system font settings on a Windows computer using the English(US) language locale. Changes to the system font settings or using a language locale other than English (US) may impact
the display of field text in the user interface.
Decompressing data. The Ion S5 or Ion PGM run data may be inside a compressed (*.zip) folder. If so, the data must be
extracted to a folder by right-clicking on the file and choosing Extract All from the contextual menu.
Do not rename the S5 or PGM files before analyzing them with this software. Changing the default names might lead toerror messages and unsuccessful processing of data files due to a potential failure of the application to recognize thecorrect input file(s).
Compatible files. LymphoTrack Software – S5/PGM is compatible with fastq.gz files generated by the Ion S5 or Ion PGM
Reporter software.
Sleep or hibernate settings. Consider disabling the sleep and/or hibernate modes before executing the LymphoTrack
Data Analysis Application. If the computer enters sleep or hibernate mode, the software may stop running.
Characters in pathname and file name. 1) Avoid spaces in the pathname for the data files or software (pathnames
include file folders and file names); more than one consecutive space is not permitted. 2) It is important that the
filenames only contain the following characters (A-Z, a-z, 0-9, ., _ (underscore), - (hyphen)). If the software encounters acharacter not within this set it may fail. Please make sure to follow this sample naming nomenclature when setting up the
Sample Sheet run for the Ion S5 or Ion PGM. 3) Do not exceed 255 characters for pathnames. Sample names that cause
the output filenames to approach this limit are difficult to copy to other locations.
Duplicate samples must have a unique sample name. Give each sample a unique name or identifier when naming
samples. If duplicate samples are run, a similar name can be used (i.e., Sample1a and Sample1b). Failure to provide
unique names to samples that will be run together on the same cartridge will result in only one sample being analyzed by
the software during the analysis process.
Data Cybersecurity. Invivoscribe strongly recommends installation of user-supplied anti-virus software to protect thecomputer against viruses. Invivoscribe strongly recommends installing the software to one user only and ensuring theoutput reports are kept in a secure location with network password protection.
Permissions. Ensure that the user has write permissions to the input folder. In cases where a different user transferred
the data than the current user, both users must have access and write permissions to the input folder. In cases where thisis not true, the current user will either need to move the data, or gain permissions to the input folder for the analysis toproceed.

LymphoTrack Software – S5/PGM

For Research Use Only. Not for use in diagnostic procedures.

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1. Installing the Software
1.1.
1.2.

Copy the LymphoTrackS5-PGM_2.4.7.zip archive from the software CD to a local drive on the computer. Install
software only on a local drive and not a network drive. The software may not function properly if running across a
network connection.
Extract the archive file that was copied to the local computer in the previous step. This can typically be accomplishedby right clicking on the file and choosing Extract All from the contextual menu.

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A folder named LymphoTrackS5-PGM_2.4.7 is created once the archive is extracted.

2. Running the Data Analysis Application
2.1.

Identify the *.fastq.gz files to be analyzed.

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2.2.

The nomatch*.fastq files are not required for analysis.

Download these files from the Ion Torrent Server into a folder on the local drive, as these will be used as input for the
LymphoTrack S5/PGM Data Analysis application.

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Download these files to the same computer that has the software installed.
The drive containing the folder with the files to be analyzed requires at least 80 GB of available space.
Ensure enough space is available for a full analysis. If the drive does not have enough space, please remove
unnecessary files from the drive.
This folder will be the <input_folder> as referenced below for the software.

IMPORTANT!

2.3.
2.4.
2.5.
2.6.

Save all files to the local computer that the user has permission to use to prevent risk to patient
data integrity. When transferring the input folder, ensure the Ion S5 or Ion PGM has completed its
analysis. Keep all data files in the same folder; do not split up data to multiple folders.

Double-click on the startLymphoTrackS5-PGM file located in the LymphoTrackS5-PGM_2.4.7 folder copied to thecomputer during installation.
Read the license agreement. To accept the terms, click the Accept button to proceed.
Check the desired analysis mode(s) from the Choose analysis mode(s) checkbox options. The analysis mode mustcorrespond to the assay used to generate the run data (IGH FR1, IGH FR2, IGH FR3, IGK and/or TRG).
Select the desired decimal mark notation from the Choose decimal mark options. This option impacts the use of theMicrosoft Excel data visualization macro later and is a required input. The choices are to use a decimal point (“.”) or adecimal comma (“,”) for the decimal mark.
IMPORTANT!

Decimal mark. To determine which option to choose, open a new Excel worksheet and type‘=10000’ into cell A1. Right-click this cell, and select Format Cells.
 From the Number tab, look in the Category list, and select Number. To the right, theDecimal places value is set to 2.
 Below this value is a Use 1000 Separator checkbox. Checked this box, and the sample
number above will contain the original number entered, formatted according to the Excel
defaults for a number with a decimal mark.
 If this number appears as “10,000.00”, then select the decimal point option. If thisnumber appears as “10.000,00”, then select the decimal comma option.

2.7.

Click on the Browse button to choose an input folder. This folder is the <input_folder> referenced above and contains
the files to be analyzed. When the input folder is selected, it will appear empty.
IMPORTANT!

LymphoTrack Software – S5/PGM

The <input_folder> selected must not contain any sample files with the same name as the
input folder. If the program was run with a sample that shared the same filename as thefolder that was provided as input to the analysis functionality, the program will freeze andanalysis runtime will increase significantly (i.e,. it will take 30 minutes to process a small sample
that takes seconds to process normally.

For Research Use Only. Not for use in diagnostic procedures.

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2.8.

Once the <input_folder> and analysis mode are selected, click the Launch Program button.
The <input_folder> selected must contain only *.fastq or *.fastq.gz files. If neither file typeis present or a mix of file types are present, the program will display an error message andrequest a different folder to be chosen.

IMPORTANT!

2.9.

Status of analysis. After clicking the Launch Program button, the main program window will display the program’s
status. When the program is finished analyzing the input data files, the main program window will display themessage Analysis complete… and show the location of the output folder(s) in addition to generating a prompt toopen the appropriate Excel Data Visualization spreadsheet(s). A new analysis can be started from the main program
window as soon as the previous analysis is complete.
IMPORTANT!

Length of analysis. The size and number of samples being analyzed as well as the analysis modeand the computer hardware will determine the amount of time the program requires forprocessing the data. A typical sample size for a 12-sample run is 500,000 to 3 million reads.
These numbers will depend on the chip and chemistry used on the Ion S5 or Ion PGM platforms.
Analysis of a typical single IGH FR1, IGH FR2, IGH FR3, IGK, or TRG sample requires approximately
1 to 2 minutes to complete. A typical 12-sample IGH FR1-only, IGH FR2-only, IGH FR3-only, IGKonly, or TRG-only run requires approximately 8-20 minutes to complete. Analysis of a typical 12sample combined IGH FR1, IGH FR2, IGH FR3, IGK, and/or TRG run multiplies the analysis time by
the number of targets being analyzed.

2.10.

Output folder(s). Depending on the analysis mode chosen, one or more output folders will be created within the<input_folder> containing the source *.fastq or *.fastq.gz files. Table 1 outlines the relationship between theanalysis mode and the name of the output folder(s).

Table 1. Output folder name according to analysis mode

2.11.

Analysis Mode

Output folder name(s)IGH FR1

IGH_FR1_output

IGH FR2

IGH_FR2_output

IGH FR3

IGH_FR3_output

TRG

TRG_output

IGK

IGK_output

Any combination of:
IGH FR1, IGH FR2, IGH FR3, IGK, andTRG

Two to five of the output folders listed above

After the program has successfully finished running, the output folder will contain several *.tsv files and a *.pdf file foreach sample processed. Note that “*” in the tables below represents the sample name as entered into the sample
sheet, followed by platform specific information (e.g. index001_001.fastq).
 For IGH FR1, IGH FR2, IGH FR3 and IGK analyses, please see Table 2 for descriptions of the nine files provided foreach sample.

LymphoTrack Software – S5/PGM

For Research Use Only. Not for use in diagnostic procedures.

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Table 2. Description of IGH and IGK output files.

IGH FR1, IGH FR2, IGH FR3 and IGK Analysis*.fastq_read_summary_merged_top10_s
earchtop500.tsv*.fastq_unique_reads.tsv*.fastq_VJ_sequence_frequency_family.tsv

Same format as the read_summary file, but includes the top 10 reads from the read_summary mergedwith reads in the top 500. A read will merge with another if they are only 1 or 2 nucleotides (nts)different, the read with the highest count maintains its sequence identity, and the read with the lower
count is added to the higher one.
A FASTA file of unique sequences, sorted by count.
Each family is a grouping of all possible recombinations found in a particular V or J region such as a V1
family or a J4 family. Within the V1 family any number of recombinations can be found such as V169 and V1-8. This file contains a list of all possible V family – J family recombinations, each with asorted list of reads (from highest to lowest %). This file is best used in a “stack” graph in Excel.
Contains a list of the top 200 unique sequences sorted by count (# of reads), specifying:Which V-J recombination event it best fits
Length
Actual % of total readsCumulative % of total readsHow well the read covers the identified V-gene targeted by the primers (%)
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Putative CDR3 sequence*.fastq_read_summary.tsv

IGH FR1 also contains:
Mutation rate to partial V-gene (%)
IGH FR1, IGH FR2, IGH FR3 also contains:
If the rearrangement is predicted to be in-frame (Y/N/n/a).
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If either the V or J gene segment usage cannot be predicted due to the quality of thealignment, the software will not be able to predict if the gene is in-frame and the result
will be n/a.
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If the rearrangement is predicted to not contain a stop codon (Y/N). When an in-frame
prediction cannot be made a prediction for whether a stop codon is present cannot be
accurately made.

*.fastq_CDR3_unique_reads.tsv

A FASTA file of putative CDR3 sequences found in the *.fastq_unique_reads file.

*.fastq_read_summary_family.tsv

Contains a sorted list of unique sequences, by count, specifying:Which V-J family recombination event it best fits
Length
Actual % of total readsCumulative % of total readsHow well the read covers the identified V-gene targeted by the primers (%)
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Putative CDR3 sequence
IGH FR1 also contains:
Mutation rate to partial V-gene (%)
IGH FR1, IGH FR2, IGH FR3 also contains:
If the rearrangement is predicted to be in-frame (Y/N/n/a)
If the rearrangement is predicted to not contain a stop codon (Y/N)*.fastq_VJ_usage.tsv*.fastq_VJ_usage_family.tsv*<timestamp>.pdf

Note:

A count matrix and % matrix of the different V-J recombination events.
A count matrix and % matrix of the different V family – J family recombination events.
The * .pdf report generated by the LymphoTrack Reporter will contain similar information as
the merged read summary file, the read summary file, a V – J usage graph, and a V – J sequencefrequency graph. See section 3: Data Visualization Using LymphoTrack Reporter (Automated,
Recommended) for more information.

IGK analysis additionally includes information for other elements in the IGK locus involved in recombinations (IGKINTR and IGKDEL).

 For TRG analysis, please Table 3 for descriptions of the seven files provided for each sample.

LymphoTrack Software – S5/PGM

For Research Use Only. Not for use in diagnostic procedures.

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Table 3. Description of TRG output files.

TRG Analysis*.fastq_read_summary.tsv*.fastq_read_summary_merged_top10_s
earchtop500.tsv*.fastq_unique_reads.tsv*.fastq_CDR3_unique_reads.tsv*.fastq_VJ_sequence_frequency.tsv

Note:
Note:

2.12.

A list of unique sequences sorted by count, specifying:Which V-J recombination event it best fits
Length
Actual % of total readsCumulative % of total reads
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Putative CDR3 sequence
Same format as the read_summary file, but only includes the top 10 reads from the read_summary
merged with reads in the top 500. A read will merge with another if they are only 1 or 2 bp
different, with the read with the higher count maintaining its sequence identity, and the read withthe lower count added to the higher one.
A FASTA file of unique sequences, sorted by count.
A FASTA file of putative CDR3 sequences found in the *.fastq_unique_reads file.
A list of all possible V-J recombinations, each with a sorted list of reads (from highest to lowest %).
The first number represents the % of total reads that the first unique read accounted for out of all
reads sequenced. This file is best used in a "stack" graph in Excel.

*.fastq_VJ_usage.tsv

A count matrix and % matrix of the different V-J recombination events.

*<timestamp>.pdf

The *.pdf report generated by the LymphoTrack Reporter will contain similar information as themerged read summary file, the read summary file, a VJ usage graph, and a VJ sequence frequency
graph. See section 3 Data Visualization Using LymphoTrack Reporter (Automated, Recommended)
for more information.

“Putative CDR3 sequence” is determined by matching an exact definition of a CDR3 sequence. Any mutation of anchor points or disruption of
the reading frame will result in a value of “not found”.
IGK analysis additionally includes information for other elements in the IGK locus involved in recombinations (IGKINTR and IGKDEL).

The output folder(s) contain *.pdf reports and *.tsv output data files. The *.tsv files can be used as inputs for the Data
Visualization spreadsheet(s).

LymphoTrack Software – S5/PGM

For Research Use Only. Not for use in diagnostic procedures.

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3. Data Visualization Using LymphoTrack Reporter (Automated, Recommended)
3.1.

A *.pdf report for each sample is automatically generated at the completion of analysis. The report is named after
the sample name present in the sample sheet, followed by platform specific information, and ends with a timestamp(DDMMYYhhmmss).
IMPORTANT!

3.2.

Caution when saving the *.pdf report: Depending on the program used to open the *.pdf, one ormore of the columns can be edited. Do NOT make changes to any part of the *.pdf. If the user
suspects an edit has been made, do NOT save the *.pdf. If the user suspects an edit has beensaved, re-run the analysis to generate a new PDF report.

The first page of the PDF report will contain a table of the Merged Read Summary results, followed by the graphs of
the V – J Sequence Frequency and V – J Usage results, and the remaining pages contain a table of the unmerged Read
Summary results. The contents of these are detailed in Table 4.

Table 4. Pages included in the pdf output file.

PDF Output for LymphoTrack Reporter

Merged Read Summary

Same format as the Read Summary worksheet, but only for the top 10 reads after merging with the
top 500 if reads are only 1 or 2 bp different from each other.

V – J Sequence Frequency Graph

A histogram showing the top 200 sequence frequencies of each V family – J family (for IGH and IGK)
or V gene – J gene (for TRG) recombination.

V – J Usage Graph

A 3D bar chart showing the V family-J family usage of the top 200 sequences (for IGH and IGK) or V
gene – J gene usage for all sequences (for TRG).
Contains a sorted list of the top 200 unique sequences by count, specifying:Which V – J recombination event each sequence best fits
SequenceSequence lengthActual % of total readsCumulative % of total reads listed for top 200 reads - How well the read covers the identified
V-gene targeted by the primers (%). (IGH/IGK)
Putative CDR3 sequence

Read Summary

Leader and IGH FR1 also contains:
Mutation rate to partial V-gene (%)
Leader, IGH FR1, IGH FR2, IGH FR3 also contains:
If the rearrangement is predicted to be in-frame (Y/N/n/a). If either the V or J gene segment
usage cannot be predicted due to the quality of the alignment, the software will not be able
to predict if the gene is in-frame and the result will be n/a.
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If the rearrangement is predicted to not contain a stop codon (Y/N). When an in-frame
prediction cannot be made a prediction for whether a stop codon is present cannot be
accurately made.

Note:
Note:

“Putative CDR3 sequence” is determined by matching an exact definition of a CDR3 sequence. Any mutation of anchor points or disruption of
the reading frame will result in a value of “not found”.
IGK analysis additionally includes information for other elements in the IGK locus involved in recombinations (IGKINTR and IGKDEL).

LymphoTrack Software – S5/PGM

For Research Use Only. Not for use in diagnostic procedures.

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4. Data Visualization Running the Excel Data Visualization Spreadsheets(Manual, Discommended)
4.1.

Check the Security settings within Microsoft Excel to verify Macro support is properly enabled:
Excel 2007: Click the Office button and choose Excel Options.
Excel 2010/2013: Click the File menu and choose Options.
From the Options window, click on Trust Center and then the Trust Center Settings... button.
Click on Macro Settings and set the appropriate Macro security level. The Disable all macros with notification
option is the recommended setting.
 Click Ok and Ok again.
 Close Excel.

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4.2.

Open the appropriate Data Visualization spreadsheet file by double-clicking it. The visualization files are located in
the LymphoTrackS5-PGM_2.4.7 folder previously copied from the software CD to the computer: LymphoTrackIGHVisualization.xlsm for IGH FR1, IGH FR2 and IGH FR3
 LymphoTrackIGKVisualization.xlsm for IGK LymphoTrackTRGVisualization.xlsm for TRG

4.3.

When opening the file, the message: Security Warning - Macros have been disabled may appear.
 Excel 2007: Click the Options... button to the right of the warning and select Enable this content and click Ok.
 Excel 2010/2013: Click the Enable Content button.
 The data visualization macro will start automatically.

4.4.
4.5.

The License Agreement will be displayed. The license agreement must be accepted to use the software.
After accepting the License Agreement, a dialog box will open with the prompt: Please select the Read Summary filefor the sample you want.

Note:
4.6.
4.7.
4.8.

The macro can only perform data visualization on one sample at a time, so this procedure will need to be repeatedfor each sample to be visualized.
Browse to the output folder created by the Data Analysis application that contains the data to be visualized, andselect the *.fastq_read_summary file for the sample to be examined.
Click the Open button.
The macro will load the data files for the chosen sample and create several worksheets, each of which will present
different readable views of the data. This may take a few moments, depending on the size of the sample and thecomputer hardware.

IMPORTANT!

Due to a limitation in Microsoft Excel, the Visualization software is not capable of displaying morethan 1,048,576 rows of data per sheet. Only the first 1,048,576 rows of data will be shown if anyof the Data Analysis output files for the current sample contain more rows than this limit.
If this limit is reached, the program will display a message stating which sheet(s) are affected bythis limitation. Click OK to proceed.
Though not all data may be displayed, the graphs are not affected and will still be accurate.

IMPORTANT!

Graphs are generated assuming a “left-to-right” display direction. In countries where the normal
reading direction is “right-to-left”, the default Excel settings may be incompatible and graphs are
not generated correctly. To fix this, the Excel display option must be changed to “left-to-right”.
This option can be set from either Office → Excel Options in Excel 2007, or File → Options in
Excel 2010/2013. Select Advanced, then scroll down to the Display settings. Change the Default
direction option to left-to-right, then click OK. The next time Excel is opened, the worksheets
will read from left-to-right, and the Visualization software will generate charts correctly..
If this situation is encountered and further assistance is required, please contact Invivoscribe
Technical Support at +1 (858) 224-6600, Monday through Friday 7:00AM – 5:00PM PST/PDT, or
via email at support@invivoscribe.com.

LymphoTrack Software – S5/PGM

For Research Use Only. Not for use in diagnostic procedures.

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4.9.
4.10.

If a message regarding Complex Formatting appears, click Yes.
Saving the visualization file. When processing is complete, a message will appear stating the program hascompleted. Saving the file at this point is strongly recommended.

IMPORTANT!

4.11.
4.12.

When saving the file, make sure to choose Save As and choose an Excel Workbook (XLSX or XLS),
because the default behavior of choosing Save or clicking the disk button will be to replace the
XLSM Macro with the current workbook. This will require reinstallation of the Macro with a new
copy from the software CD for subsequent analyses.

If a warning message regarding saving as a macro-free workbook appears, click the Yes button.
The completed workbook for IGH FR1, IGH FR2, IGH FR3, IGK and TRG will include the following eight worksheets
described in Table 5.

Table 5. Worksheets included in Excel output file.

Completed Workbook for IGH FR1, IGH FR2, IGH FR3, IGK and TRG

Read Summary

Contains a sorted list of the top 200 unique sequences by count, specifying:Which V-J recombination event each sequence best fits
SequenceSequence lengthActual % of total readsCumulative % of total reads listed for all reads (TRG only)Cumulative % of total reads listed for top 200 reads (IGH/IGK)
How well the read covers the identified V-gene targeted by the primers (%) (IGH/IGK)
Putative CDR3 sequence
IGH FR1 also contains:
Mutation rate to partial V-gene (%)
IGH FR1, IGH FR2, IGH FR3 also contains:
If the rearrangement is predicted to be in-frame (Y/N/n/a). If either the V or J gene segment usage
cannot be predicted due to the quality of the alignment, the software will not be able to predict if
the gene is in-frame and the result will be n/a.

If the rearrangement is predicted to not contain a stop codon (Y/N). When an in-frame prediction
cannot be made a prediction for whether a stop codon is present cannot be accurately made.

Putative CDR3 sequence.

Merged Read Summary

Same format as the Read Summary worksheet, but only for the top 10 reads after merging with the top 500 if
reads are only 1 or 2 bp different from each other.

VJ Sequence Frequency Graph

A histogram showing the top 200 sequence frequencies of each V family – J family (for IGH/IGK) or V gene – J
gene (for TRG) recombination.

VJ Usage

Raw counts and frequencies of the top 200 V family - J family (for IGH/IGK) or V gene – J gene (for TRG)
recombinations.

VJ Usage Percent Graph

A 3-D graph showing the top 200 sequence frequencies of each V family – J family (for IGH/IGK) or V gene – J
gene (for TRG) recombination.

VJ Usage Raw Graph

A 3-D graph showing the top 200 raw counts of each V family – J family (for IGH/IGK) or V gene – J gene (forTRG) recombination.

VJ Sequence Frequency

A “stack” list of all possible V family – J family (for IGH/IGK) or V gene – J gene (for TRG) recombinations, eachwith a sorted list of the top 200 reads (from highest to lowest %).

Unique Reads
Note:

Note:
Note:
Note:

A FASTA file of unique sequences, sorted by count.

(IGH) listed in the table above refers to IGH FR1, IGH FR2 and IGH FR3 analyses. The frequency graphs for these gene targets only specify IGH in
the title. The data output folder specifies which of the framework (FR) regions was analyzed by the software and this information is included in
the data file location field at the top of the Read Summary worksheet.
IGK analysis additionally includes information for other elements in the IGK locus involved in recombinations (IGKINTR and IGKDEL).
Top 200: When specified above, the worksheets will show the top sequences (up to 200) for IGH FR1, IGH FR2, IGH FR3 and IGK. All of thesequences are shown for TRG in the Read Summary.
“Putative CDR3 sequence” is determined by matching an exact definition of a CDR3 sequence. Any mutation of anchor points or disruption of
the reading frame will result in a value of “not found”.

LymphoTrack Software – S5/PGM

For Research Use Only. Not for use in diagnostic procedures.

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IMPORTANT!

V/J gene assignments found in the Read Summary can sometimes be labeled “none”. These
assignments are based on the quality of the best alignment between the read and the reference
genes. If the best alignment falls below a quality threshold, the gene assignment cannot be
confidently made and is labeled “none”.

IMPORTANT!

The Mutation rate to partial V-gene (%) will display “n/a” for IGH FR2 and IGH FR3. There islimited V-gene coverage due to the shorter amplicon sizes of these targets which results ininaccurate mutation rate calculations.

 When analyzing the somatic hypermutation status of samples, focus on the top 4 reads (limit monoclonal or biclonal
specimens to the top 2 or 4 reads, respectively), as the somatic hypermutation rate (SHM) and in-frame/stop-codon
analyses are most informative for these reads. Due to the unbiased nature of the NGS platforms, many more reads
than just the top 4 are sequenced and analyzed, most of which are low abundance rearrangements, or can be
attributed to artifacts in amplification or sequencing. SHM and in-frame/stop-codon analyses are not useful in nonclonal reads in these cases.
 Please note that Invivoscribe uses GRCh37 for reference sequences and results may differ slightly from otherdatabases. In addition, many pseudogenes and non-functional genes are not included in the analysis. Interpret
rearrangements with somatic hypermutation statuses at or near the 2% cutoff with caution, particularly whenrunning the IGH FR1 assay which only targets a portion of the IGHV region as the sequence upstream of the primer
binding site will not be assessed.

5. Technical and Customer Service
We appreciate your business. We are happy to assist you with understanding this software, and will provide ongoing technicalassistance Monday through Friday to keep the assays performing efficiently in your laboratory.
Contact Information
Invivoscribe, Inc
10222 Barnes Canyon Road | Building 1 | San Diego | California 92121-2711 | USA
Phone: +1 858 224-6600 | Fax: +1 858 224-6601 | Business Hours: 7:00AM - 5:00 PM PST/PDT
Technical Service: support@invivoscribe.com | Customer Service: sales@invivoscribe.com | Website: www.invivoscribe.com

6. Symbols
The following symbols are used in Invivoscribe NGS product labeling.
Catalog Number

Expiration Date

Reagent Volume

Manufacturer

Lot Number

Consult Instructions for Use

Storage Conditions

Research Use Only

LymphoTrack Software – S5/PGM

For Research Use Only. Not for use in diagnostic procedures.

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7. Software Frequently Asked Questions
Q1:

When Launch Program is selected, the following (or similar) error appears:
Application terminated with error: Cannot run program ".\resources\Parse_TRG_PGM.exe":
CreateProcess error=2, The system cannot find the file specified.

For help with this error, please contact Invivoscribe Technical Support.
A1:

Was the compressed program file unzipped? (see section 1: Installing the Software) This error commonly occurs
when trying to run the program before decompressing the program file. Windows allows the ability to view thecontents of a zip file simply by double-clicking the file, and the contents appear to be complete and functional, but
it will not run properly from this state.

Q2:

Why does the Read Summary from the IGH and/or IGK analysis have fewer than 200 reads?

A2:

The LymphoTrack Software – S5/PGM aligns the top 200 reads during analysis, but the Read Summary only reports
reads that have aligned to a V or J gene found in the reference. If a read cannot align to the reference, it is
excluded from the Read Summary.
The reasons for a non-alignment to the reference are numerous, but usually focus on a problem with the sample,
leading to a large amount of non-specific amplification to occur. This event can stem from low numbers of target
cells in the sample, improper PCR conditions, or low quality sample types, such as fragmented FFPE. To reduce thechance of these events, please follow the Instructions for Use precisely.

Q3:

Why does the V-assignment or J-assignment say “none” in the Read Summary table?

A3:

To assign a read to a specific V and/or J gene, a minimum quality threshold must be attained during the alignment.
If the alignment is poor, a confident assignment cannot be made and is instead labeled “none”. Note that a readcannot be assigned “none” for both V and J genes, as these would not be reported, per the condition discussed inquestion 2.

Q4:

The “VJ Sequence Frequency Graph” has strange values for the y-axis. The “% total reads” is in the millions, howis this possible?

A4:

This is likely due to running the LymphoTrack program using the incorrect decimal type for your region. See section
2: Running the Data Analysis Application, specifically for selecting the decimal mark.

Q5:

All of the graph worksheets are empty, but the non-graph worksheets have data. Where did the graphs go?

A5:

This may be due to the default direction of the Excel program being “right-to-left” instead of “left-to-right”. If thecolumn labels on an empty worksheet start at “A” on the right-side of the worksheet, switch the direction to “leftto-right” before using the Data Visualization macro. To change this setting, go to Excel options from the Office
button in the upper left-hand corner, click on ‘Advanced’, and scroll down to the Display section. An option called
Default direction is listed here, and selecting ‘Left-to-right’ will allow subsequent data visualization runs to be work
correctly. Note: This will permanently change the direction until it is changed back. Closing Excel will not revert
this change.

Q6:

Why do the analysis results seem to include fewer reads expected?

A6:

If there are very few or zero total reads analyzed according to the Read Summary, verify the correct analysis typewas selected (IGH FR1, IGH FR2, IGH FR3, IGK or TRG) when running the LymphoTrack Software – S5/PGM. If there
seems to be a depressed number of total reads, compare the size of the FASTQ files being analyzed with size of the
FASTQ files present on the Ion S5 or Ion PGM instrument. If the Ion S5 or Ion PGM instrument has larger files, thenthe sequence data must be re-transferred to ensure analysis of the full dataset.
The Ion S5 and Ion PGM sequencers display different messages when processing the sequencing data. One
message may state “Sequencing Complete”, and another may later state “Analysis Complete”. Transferring theFASTQ data in between these two messages may result in an incomplete LymphoTrack analysis.

LymphoTrack Software – S5/PGM

For Research Use Only. Not for use in diagnostic procedures.

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Q7:

Why does the Read Summary contain a read in the top ten that is not in the Merged Read Summary?

A7:The “disappearing” read may have been merged with a read ranked above it, or it is possible the alignment of thisread was exceptionally poor. During the merging process, reads in the read summary are aligned with those in theunique reads file; however, reads in the read summary have been oriented to match the alignment to thereference, and are possibly in the anti-sense orientation compared to the reference gene. This happens only when
the alignment is so poor and when a sense or anti-sense alignment is nearly equivalent, and the anti-sensealignment was slightly better. In cases such as these, it is best to ignore the read entirely.

IMPORTANT:

Only reads from the Merged Read Summary can be used to determine evidence for clonality.

Q8:

Why is the analysis is taking a long time (over an hour per sample)?

A8:

In the log file, a breakdown of how long each step is taking can be observed. Following the line “INFO:
Decompressing files”, there is another line that states how many files and how much time the decompression used.
This number is in milliseconds. If decompression is taking several hours, check if the target files are on a fileserver.
Typically, running any I/O intensive process like decompression over a network will be much slower. If possible,
copy the files over to the local computer, or decompress the files directly before running this software. Make surethat the compressed and decompressed files do not both exist in the same folder. The software will not proceed if
both *.fastq and *.fastq.gz files are in the target folder.
If the subsequent parsing and analysis steps are taking a long time, the *.fastq files may be very large. Examples oflarge *.fastq files include when less than 12 indices are included on a single Ion S5 or Ion PGM run and as a result,the samples sequenced have a larger depth of coverage. A second example would be if the library is unbalanced
and contains a higher proportion of a sample with one index versus the proportion of samples with other indexes.
The predicted run-time stated in this IFU assumes a 12-index run with balanced libraries.

Q9:

Why is the analysis output folder missing the PDF reports?

A9:

The computer may have run out of hard drive space. Please ensure there is enough hard drive space (at least 80Gb) before starting the analysis. If this error occurs again, please contact Technical Support.

Q10:

What is the resolution for a failure when launching the LymphoTrack Software – S5/PGM by double-clicking thestartLymphoTrackS5-PGM.jar file?

A10:

This issue can occur when there is a problem with the current installation of Java. The correct version of Java (Java8 64-bit) must be installed on the system for the LymphoTrack Software – S5/PGM to run. Additionally, thestartLymphoTrackS5-PGM.jar file must be set to be opened with the correct version of Java.
The following troubleshooting steps can be taken to ensure that the correct version of Java is installed, and that the
JAR is opened with the correct version of Java:
1.

Verify the Java software is installed:
1.1. Open the Windows Menu and type Settings in the search bar, then select the first icon that displays.
1.2. In the search bar of the new window, type Add or remove programs, and select the first option thatappears.
1.3. Scroll through the list of installed programs.
••

2.

If a program with a name starting with the letters Java 8 and ending with the characters (64-bit) is in
the list, the correct version of Java is installed.
If not, proceed to section 3 for instructions to install Java 8 64-bit.

Uninstall old versions of Java:
2.1.
2.2.

Follow steps 1.1 and 1.2.
Scroll through the list of installed programs. Identify any program that begins with the letters Java andalso contains the letters Java 8 and 64-bit, click on the entry in the list, then click Uninstall.

LymphoTrack Software – S5/PGM

For Research Use Only. Not for use in diagnostic procedures.

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Uninstall Java and Reinstall Java:
If the correct version of Java is installed, and the software still does not open, uninstall and re-install Java.
3.1.
3.2.
3.3.
3.4.

4.

Follow steps 1.1 and 1.2.
Scroll through the list of installed programs and click on the entry with the name Java 8 and ending withthe characters (64-bit), then click Uninstall.
Navigate to the Java website to download the Java 8 64-bit software directly from Oracle at
http://www.java.com/.
Follow the instructions to install the software.

Point the JAR file to the correct Java executable:
4.1.
4.2.
4.3.
4.4.
4.5.

Right click on the startLymphoTrackS5-PGM.jar file, then select Properties.
In the dialog that appears, select the Change button next to the opens with text.
In the next dialog that appears, select Choose another app.
In the new window that opens, click on More apps, then select Look for another app on this PC.
In the browsing window that appears, navigate to the folder location of the Java 8 64-bit installation.
•

4.6.
4.7.

This will likely be: Local Disk (C:) > Program Files > Java > jre1.8.0_(version number) > bin.

Select the javaw.exe and click Open.
Once installation is confirmed, repeat section 4.1 through 4.3 and select the checkbox indicating Always
open .jar files with this app, then navigate to the same javaw.exe file from step 4.6. This will allow theLymphoTrack Software to open correctly.

LymphoTrack Software – S5/PGM

For Research Use Only. Not for use in diagnostic procedures.

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8. End User License Agreement
THIS END USER LICENSE AGREEMENT MUST BE ACCEPTED BY AN AUTHORIZED REPRESENTATIVE OF THE END USER OF THIS PRODUCT PRIOR TO USING THE
LYMPHOTRACK® SOFTWARE (the “Software”). BY USING THE LYMPHOTRACK® SOFTWARE, YOU ASSERT THAT YOU ARE AN AUTHORIZED REPRESENTATIVE OF THE
END USER WITH AUTHORITY TO ENTER INTO THIS AGREEMENT. PLEASE READ THIS AGREEMENT CAREFULLY. BY USING THE SOFTWARE, YOU ARE AGREEING TO
BE BOUND BY THE TERMS OF THIS AGREEMENT. IF YOU DO NOT AGREE TO THE TERMS OF THIS AGREEMENT, PLEASE DISCONTINUE USAGE. IF YOU DO AGREE
TO THE TERMS OF THIS AGREEMENT ON BEHALF OF YOURSELF AND/OR THE ENTITY YOU REPRESENT YOU MAY CONTINUE USAGE.
This End User License Agreement (“EULA”) is made and entered into by and between INVIVOSCRIBE, INC., a California corporation (“Licensor”), and You, as the userof the Software (either you as an individual or a legal entity), the “Licensee” for the licensing and usage of the Licensor’s Software. Licensee acknowledges andagrees that Licensee’s right to use the Software in any manner shall be controlled by this EULA and that such use shall be strictly in accordance with the terms andconditions of this EULA.
1.

GRANT OF LICENSE. Licensor grants to Licensee a non-exclusive, non-transferable, limited license, without the right to sublicense, to use the Software and any
accompanying written materials and any documents or other content embodying information contained within the Software (collectively, the “Product”) soley for
Licensee’s own internal use. Except as agreed in a document signed by Licensor, the Software license granted in the preceding sentence is limited to use with
Licensor's LymphoTrack® Assays purchased from Licensor or from an authorized distributor of Licensor. Licensor reserves all rights not expressly granted to Licensee
or to Licensee. The limited license granted by this EULA and Licensee’s payment of the license fee give Licensee the right to use the Product only in accordance withthe terms of this EULA. This license is not a sale of the original software or any copy thereof.
2. CONFIDENTIALITY. Licensee agrees that the Product is owned by Licensor and is based on and includes the intellectual property of Licensor, including but not limited
to one or more proprietary trade secrets, copyrights, patent applications, and/or granted patents (“Intellectual Property”) of Licensor. Licensee’s possession and useof the Product shall be strictly in accordance with this EULA, and receipt or possession does not convey any rights to divulge, reproduce, or allow others to use theProduct outside the terms of this EULA without specific written authorization of Licensor. Licensee agrees not to disclose, publish, translate, release, or distributecopies of the Product or any portion thereof to others outside the terms of this EULA. Licensee may not modify, adapt, translate, reverse engineer, decompile,
disassemble, or create derivative works based on the Product or any portion thereof, including any documents or other content produced using the software.
3. OWNERSHIP OF PRODUCT. Title to, ownership of, and all rights and interests in the Product including but not limited to the Software, including but not limited to theSoftware, all copies thereof, and “Intellectual Property” relating thereto, remains at all times vested in Licensor, and this EULA does not grant any ownership rights toLicensee.
4. COPY RESTRICTIONS. The Product is copyrighted. Unauthorized copying or modification of the Product, including but not limited to the Software that has been
modified, merged, or included with other software, is expressly forbidden. Licensee may be held legally responsible for any copyright infringement that is caused or
incurred by Licensee’s failure to abide by the terms of this EULA.
5. TRANSFER RESTRICTIONS. This Product is licensed to Licensee, and may not be transferred to anyone without the prior written consent of Licensor. In no event may
Licensee transfer, assign, rent, lease, sell, or otherwise dispose of the Product, or any portion thereof, on a temporary basis, except as expressly provided herein.
6. TERMINATION. This EULA will be terminated automatically without notice if Licensee fails to comply with any of its provisions.
7. LIMITED WARRANTY. THE PRODUCT, INCLUDING SOFTWARE AND ACCOMPANYING WRITTEN MATERIALS, INCLUDING INSTRUCTIONS FOR USE, ARE PROVIDED “ASIS,” WITHOUT WARRANTY OF ANY KIND, AND LICENSOR DISCLAIMS ALL OTHER WARRANTIES, WHETHER EXPRESS, IMPLIED, OR STATUTORY, INCLUDING ANY IMPLIEDWARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE, TITLE, NON-INFRINGEMENT AND ANY WARRANTIES ARISING FROM COURSE OF
DEALING OR COURSE OF PERFORMANCE REGARDING OR RELATING TO THE SOFTWARE, THE DOCUMENTATION OR ANY MATERIALS FURNISHED OR PROVIDED TO
YOU UNDER THIS EULA. LICENSOR DOES NOT WARRANT THAT THE SOFTWARE WILL OPERATE UNINTERRUPTED OR THAT IT WILL BE FREE FROM DEFECTS OR THATTHE SOFTWARE WILL MEET (OR IS DESIGNED TO MEET) YOUR BUSINESS REQUIREMENTS.
8. LIMITED LIABILITY. TO THE MAXIMUM EXTENT PERMITTED BY LAW, IN NO EVENT SHALL LICENSOR BE LIABLE FOR ANY LOST PROFITS OR BUSINESS OPPORTUNITIES,LOSS OF USE, LOSS OF REVENUE, LOSS OF GOODWILL, BUSINESS INTERRUPTION, LOSS OF DATA, OR ANY OTHER INDIRECT, SPECIAL, INCIDENTAL, OR CONSEQUENTIAL
DAMAGES UNDER ANY THEORY OF LIABILITY, WHETHER BASED IN CONTRACT, TORT, NEGLIGENCE, PRODUCT LIABILITY, OR OTHERWISE. BECAUSE SOMEJURISDICTIONS DO NOT ALLOW THE EXCLUSION OR LIMITATION OF LIABILITY FOR CONSEQUENTIAL OR INCIDENTAL DAMAGES, THE PRECEDING LIMITATION MAY
NOT APPLY TO LICENSEE. LICENSOR’S LIABILITY UNDER THIS EULA WILL NOT, IN ANY EVENT, REGARDLESS OF WHETHER THE CLAIM IS BASED IN CONTRACT, TORT,STRICT LIABILITY, OR OTHERWISE, EXCEED THE LICENSE FEES LICENSEE PAID FOR THE SOFTWARE, IF ANY. THE FOREGOING LIMITATIONS SHALL APPLY REGARDLESSOF WHETHER LICENSOR HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES AND REGARDLESS OF WHETHER ANY REMEDY FAILS OF ITS ESSENTIAL PURPOSE.
9. EXCLUSIVE REMEDY. Licensee’s exclusive remedy and Licensor’s entire liability for breach by Licensor of this EULA shall be limited, at Licensor’s sole and exclusive
discretion, to: (i) replacement of any defective Software or related Documentation; or (ii) refund of the consideration paid to Licensor for the Software
10. GOVERNING LAW. This EULA shall be governed by the laws of the State of California, irrespective of its conflicts of laws principles, and Licensee consents tojurisdiction by the state and federal courts sitting in the State of California.
11. COMPLETE AGREEMENT. This EULA is the entire agreement between Licensor and Licensee with respect to the specific terms set forth herein concerning license andwarranties of the Product and any other included term or obligation. This EULA replaces all prior understandings and agreements, whether written or oral. This EULA
may not be modified unless Licensor and Licensee both agree to such modification in writing.
12. SEVERABILITY. If for any reason a court of competent jurisdiction finds any provision or part of any provision of this EULA unenforceable, that part or provision shallbe enforced to the maximum extent permitted by law so as to affect the intent of the parties, and the remainder of the EULA shall continue in full force and effect.
©2023 Invivoscribe, Inc. All rights reserved. The trademarks mentioned herein are the property of Invivoscribe, Inc. and/or its affiliates, or (as to the trademarks ofothers used herein) their respective owners. PGM™ and Ion S5™ are trademarks of Thermo Fisher Scientific and its subsidiaries. MICROSOFT®, WINDOWS® and
EXCEL® are registered trademarks of Microsoft Corporation.

LymphoTrack Software – S5/PGM

For Research Use Only. Not for use in diagnostic procedures.

280359 Rev. M | December 2023