Instructions for Use

LymphoTrack Software – MiSeq Version 2.4.6®

TM

This software package is provided with each LymphoTrack Assay for the Illumina® MiSeq instrument to analyze FASTQ files. The
software is composed of three distinct parts: the LymphoTrack Data Analysis Application, a LymphoTrack Reporter plugin thatruns automatically after analysis, and an optional LymphoTrack Visualization method consisting of four Microsoft Excel® XLSM
spreadsheets. The software runs on supported Microsoft Windows® platforms.
This software package allows for clonality analysis of single or multiple target data (IGHV Leader [or “Leader”], IGH FR1, IGH FR2,
IGH FR3, IGK, TRB or TRG) and additionally provides the somatic hypermutation status of samples run with the Leader or IGH FR1
assays.
For guidance with minimal residual disease (MRD) studies, please refer to the technical bulletin entitled Study of MRD – Using
LymphoTrack Assays – MiSeq,
M-0036. This document can be found on the LymphoTrack MRD Software (75000008).
To request the technical bulletin, emailing marketing@invivoscribe.com.
This assay is for Research Use Only. Not for use in Diagnostic Procedures.

Product Compatibility
This LymphoTrack Software – MiSeq package (following Invivoscribe assays:
Catalog #75000009) was developed specifically for and is compatible with only the

Description

Quantity

71210059

LymphoTrack IGHV Leader Somatic Hypermutation Assay Kit A – MiSeq

8 indices – 5 reactions each71210069

LymphoTrack IGHV Leader Somatic Hypermutation Assay Panel – MiSeq

24 indices – 5 reactions each71210009

LymphoTrack IGH FR1 Assay Kit A – MiSeq

8 indices – 5 reactions each71210039

LymphoTrack IGH FR1 Assay Panel – MiSeq

24 indices – 5 reactions each71210149

LymphoTrack IGH FR1 Assay Panel B – MiSeq

24 indices – 5 reactions each71210089

LymphoTrack IGH FR2 Assay Kit A – MiSeq

8 indices – 5 reactions each71210099

LymphoTrack IGH FR2 Assay Panel – MiSeq

24 indices – 5 reactions each71210109

LymphoTrack IGH FR3 Assay Kit A – MiSeq

8 indices – 5 reactions each71210119

LymphoTrack IGH FR3 Assay Panel – MiSeq

24 indices – 5 reactions each71210129

LymphoTrack IGH FR1/2/3 Assay Kit A – MiSeq

8 indices per FR – 5 reactions each71210139

LymphoTrack IGH FR1/2/3 Assay Panel – MiSeq

24 indices per FR – 5 reactions each71220009

LymphoTrack IGK Assay Kit A – MiSeq

8 indices – 5 reactions each71220019

LymphoTrack IGK Assay Panel – MiSeq

24 indices – 5 reactions each72250009

LymphoTrack TRB Assay Kit A – MiSeq

8 indices – 5 reactions each72250019

LymphoTrack TRB Assay Panel – MiSeq

24 indices – 5 reactions each72270019

LymphoTrack TRG Assay Kit A – MiSeq

8 indices – 5 reactions each72270009

LymphoTrack TRG Assay Panel – MiSeq

24 indices – 5 reactions each

75000008

LymphoTrack Software - MiSeq

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Table of Contents
1.

PRODUCT USE .............................................................................................................................................................................. 3

2.

GLOSSARY AND ABBREVIATIONS ....................................................................................................................................................... 3
2.1.

Glossary ....................................................................................................................................................................................................... 3

2.2.

Abbreviations .............................................................................................................................................................................................. 33.

MINIMUM SYSTEM REQUIREMENTS .................................................................................................................................................. 44.

WARNINGS AND PRECAUTIONS ........................................................................................................................................................ 4

5.

INSTALLING THE SOFTWARE ............................................................................................................................................................. 5

6.

RUNNING THE DATA ANALYSIS SOFTWARE..........................................................................................................................................6

7.

DATA VISUALIZATION USING LYMPHOTRACK REPORTER (AUTOMATED, RECOMMENDED) ........................................................................... 10

8.

DATA VISUALIZATION (MANUAL): RUNNING THE EXCEL DATA VISUALIZATION SPREADSHEETS- DISCOMMENDED ............................................. 11

9.

TECHNICAL AND CUSTOMER SERVICE ............................................................................................................................................... 1410.

SYMBOLS .................................................................................................................................................................................. 14

12.

SOFTWARE FREQUENTLY ASKED QUESTIONS ..................................................................................................................................... 15

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1. Product Use
The RUO LymphoTrack Software – MiSeq package is intended to be used for bioinformatics data analysis and visualization of
raw FASTQ files from the Illumina MiSeq instrument when used with LymphoTrack Assays.

2. Glossary and Abbreviations
2.1.

Glossary
Table 1. Glossary Terms

2.2.

Term
ClonalDefinition
The aggregate of genetically identical cells or organisms produced from a single progenitor cell.

Invalid

A sample or control result which does not meet the validity criteria.

Semantic versioning

A software version scheme consisting of 3 numbers (Major.Minor.Patch), aligned with the risk factor of
the update.

Software

LymphoTrack Software - MiSeq

SystemThe entire package of software, hardware and assay (as applicable) that make up the medical device.

Abbreviations
Table 2. Abbreviations definedAbbreviation

Definitions

CDR3

Complementarity-determining region, formed by the joining of V-(D)-J segments and establishes thediversity of the cellular immune response

CSV

Comma separated value file format

FASTQ

A text-based format for storing both a biological sequence (usually nucleotide sequence) and its
corresponding quality scores

FFPE

Formalin-fixed paraffin-embedded

IFU

Instructions for use

IGH FR1

Immunoglobulin heavy chain Framework 1

IGH FR2

Immunoglobulin heavy chain Framework 2

IGH FR3

Immunoglobulin heavy chain Framework 3

IGK

Immunoglobulin kappa light chain

PCR

Polymerase chain reaction

PDF

Portable document format

SHM

Somatic hypermutation

TRB

T-cell receptor beta

TRG

T-cell receptor gamma

TSV

Tab-separated value file format

USB

Universal Serial Bus; allows data exchange and delivery of power between many various types ofelectronics

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3. Minimum System 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 10 Pro or Windows 11 Pro (64-bit) is required.
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 – MiSeq v2.4.6 has been validated for use with Windows 10 Pro and Windows 11 Pro,
configured for English (United States) language and default display size settings, and Microsoft Excel 2007/2010/2013 foralternate data visualization. Use of other Windows/Excel versions and/or language/display size settings may not becompatible.

4. Warnings and Precautions


Default screen size. Optimal screen resolution for LymphoTrack Software – MiSeq 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 thedisplay of field text in the user interface.

Compatible files. LymphoTrack Software – MiSeq is compatible with FASTQ.GZ files generated by the MiSeq Reporter
Software. LymphoTrack Software – MiSeq is not compatible with FASTQ.GZ files generated by the Bcl2fastq software.

Adapter trimming. Adapter trimming must be selected  when creating MiSeq sample sheets so the resulting FASTQ.GZ
files generated are adaptor trimmed.



Sleep or hibernate settings. Consider disabling the sleep and/or sleep or hibernate mode features before executing theLymphoTrack 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 alphanumeric and hyphen characters (A-Z, a-z, 0-9, , - [hyphen]). If the software encounters a character not within
this set, it may fail. Please make sure to follow this sample naming nomenclature when setting up the Sample Sheet for the
MiSeq. 3) Do not exceed 255 characters for a pathname. Sample names that cause the output filenames to approach thislimit are difficult to copy to other locations.



Duplicate samples must have a unique sample name. Provide a unique name and identifier for each sample when creating
sample names. If samples are run in duplicate, they can be named “Sample1a” and “Sample1b”. Failure to provide unique
names to samples that will be run together on the same flow cell will result in only one sample being analyzed by thesoftware during the analysis process.

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5. Installing the Software
The LymphoTrack Software – MiSeq package (copied from the provided CD.

Install software only on a local drive and not a network drive.
The software may not function properly when running across a network connection.
Note:

5.1.
5.2.

Administrator permission is required to Install the software

Load the LymphoTrack Software – MiSeq CD into the disk drive; OR
Download the LymphoTrack Software – MiSeq package from the Invivoscribe Software Portal.
5.2.1.
5.2.2.

Using any web browser, navigate to https://catalog.invivoscribe.com/softwareportal/
Complete the following text fields:

5.2.2.1.
5.2.2.2.
5.2.2.3.
5.2.3.
5.2.4.

5.2.5.

Email: Enter a valid email address. A link to the software download will be sent to this address.
Customer Account Number: Enter your unique ID used when placing orders with Invivoscribe.
Software Code: Enter the software code found at the bottom of the associated sales order.

Check the Terms and Conditions box to proceed.
Click the Request Software icon.



5.3.

75000009) can downloaded through the invivoscribe Software Portal or

Following valid input text into the above fields, a link to the software download will be sent to the provided
email address.

Click the link or copy+paste into a web browser; the software will automatically download.

Install the LymphoTrack Software - MiSeq
5.3.1.

Double click the LymphoTrackMiSeq_2.4.6.RUO.msi file.
If a message appears, stating Windows protected your PC, click More info and verify the publisher isInvivoscribe, Inc., then click Run anyway.

5.3.1.1.


5.3.2.

Follow the Windows Setup Wizard installation prompts, select the directory file path to save the software andclick Install.



Install software only on a local drive (not a network drive); the software might not function properly if run
across a network connection.
If the User Account Control dialog appears, click Yes.

5.3.2.1.
5.3.3.

The software installer will open and allow the software to be installed to a local drive.
The default install location is C:\Invivoscribe\LymphoTrackMiSeq-2.4.6.RUO.

Once installation is complete, click Finish.

The software is now ready to use and can be accessed by navigating to the Start Menu or the directory file
path selected in step 5.3.2.

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6. Running the Data Analysis Software
6.1.

Identify the paired-end, adapter-trimmed FASTQ.GZ files to be analyzed.



6.2.

Copy the FASTQ.GZ files identified above to an empty folder on the computer. Download these files to the same local
computer which has the software installed.



6.3.

These files are located within the MiSeq run folder and are the FASTQ.GZ files that will be used as input for the
LymphoTrackMiSeq data analysis application.
The Undetermined_*.FASTQ.GZ files are not required for analysis.

The drive containing this folder requires at least 80 GB of available space.
This folder will be the <input_folder> referenced below for the software.

Double-click on the LymphoTrackDxMiSeq-2.4.6.IVD application file located in the Invivoscribe folder created during
installation.

 The default location is Local Disk (C:) > Invivoscribe.
6.4.
6.5.

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.


6.6.

The analysis mode must correspond to the type of data in the run (Leader, IGH FR1, IGH FR2, IGH FR3, IGK, TRB
and/or TRG).

Select the desired decimal mark notation from the Choose decimal mark options.

This option impacts the use of the Microsoft Excel data visualization macro later and is a required input.
The choices are to use a decimal point (“.”) or a decimal comma (“,”) for the decimal mark.

IMPORTANT!

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

6.7.

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

IMPORTANT!

6.8.

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 the folder that wasprovided as input to the analysis functionality, the program will freeze and analysis runtime will increasesignificantly (i.e,. it will take 30 minutes to process a small sample that takes seconds to process normally.

Once the <input_folder> and analysis mode are selected, click the Launch Program button.

IMPORTANT!

LymphoTrack Software – MiSeq

The <input_folder> selected must contain only FASTQ or FASTQ.GZ files. If neither file type nor a mix
of file types are present, the program will display an error message and request a different folder to bechosen. If the FASTQ files are generated by Bcl2fastq (the filenames contain the barcode sequence
instead of the “S1, S2, S3” etc… nomenclature) the program will display an error message and request thedata to be re-analyzed using the MiSeq Reporter Software.

For Research Use Only. Not for use in Diagnostic Procedures

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6.9.

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 the message
Analysis is complete… and show the location of the output folder as well as provide a prompt to open the appropriate
Excel Data Visualization spreadsheet.



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 mode andthe computer’s hardware will determine the amount of time the program takes to run. A typical read
distribution for a 24-sample run is 200,000 to 1 million reads per sample. These numbers may changewith future releases of the MiSeq platform.
Analysis of a typical single IGHV Leader, IGH FR1, IGH FR2, IGH FR3, IGK, TRB or TRG sample requires
approximately 1-2 minutes to complete. A typical 24-sample Leader-only, IGH FR1-only, IGH FR2only, IGH FR3-only, IGK-only, TRB-only or TRG-only run requires approximately 25-50 minutes tocomplete. Analysis of a typical 24-sample combined Leader, IGH FR1, IGH FR2, IGH FR3, IGK, TRB
and/or TRG run multiplies the analysis time by the number of targets being analyzed.

6.10.

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 3 outlines the relationship between the analysis mode and the name of the output folder(s).
Table 3. Output folder name according to analysis mode

6.11.

Analysis Mode

Output folder name(s)Leader
IGH FR1

LEADER_output
IGH_FR1_output

IGH FR2
IGH FR3
TRB

IGH_FR2_output
IGH_FR3_output
TRB_output

TRG
IGK

TRG_output
IGK_output

Any combination of:
Leader, IGH FR1, IGH FR2, IGH FR3, IGK,TRB and TRG

Two to seven of the output folders listed above

After the program has finished running successfully, 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., samplename_S1_L001_001_combined).
For Leader, IGH FR1, IGH FR2, IGH FR3, TRB and IGK analysis, please see Table 4 for descriptions of the nine files
provided for each sample.

Table 4. Description of IGH, TRB and IGK output files.

Leader, IGH FR1, IGH FR2, IGH FR3, TRB and IGK Analysis*.fastq_read_summary_merged_top10_searc
htop500.tsv*.fastq_unique_reads.tsv*.fastq_CDR3_unique_reads.tsv

LymphoTrack Software – MiSeq

Same format as the read_summary file, but only includes the top 10 reads from theread_summary merged with reads in the top 500. A read will merge with another if they
are only 1 or 2 nucleotides (nts) different, with the read with the highest count maintaining
its sequence identity, and the read with the 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.

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Table 4. Description of IGH, TRB and IGK output files.

Leader, IGH FR1, IGH FR2, IGH FR3, TRB and IGK Analysis*.fastq_VJ_sequence_frequency_family.tsv*.fastq_VJ_usage.tsv

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 canbe found such as V1-69 and V1-8. This file contains a list of all possible V family – J family
recombinations, each with a sorted list of reads (from highest to lowest %). This file is bestused in a “stack” graph in Excel.
A count matrix and % matrix of the different V-J recombination events.
Contains a list of the top 200 unique sequences sorted by count (number of reads),
specifying:







*.fastq_read_summary.tsv

Which V-J recombination event it best fits
LengthActual % of total reads
Cumulative % of total reads
How well the read covers the identified V-gene targeted by the primers (%)(IGH/IGK)
Putative CDR3 sequence

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 genesegment usage cannot be predicted due to the quality of the alignment, thesoftware will not be able to predict if the gene is in-frame and the result will ben/a.
If the rearrangement is predicted to not contain a stop codon (Y/N). When an inframe prediction cannot be made a prediction for whether a stop codon is present
cannot be accurately made.

Contains a sorted list of unique sequences, by count, specifying:





*.fastq_read_summary_family.tsv

Which V-J family recombination event it best fits
LengthActual % of total reads
Cumulative % of total reads
How well the read covers the identified V-gene targeted by the primers (%)(IGH/IGK)
Putative CDR3 sequence

Leader and IGH FR1 also contains:

Mutation rate to partial V-gene (%)

Leader, IGH FR1, IGH FR2, IGH FR3 also contains:*.fastq_VJ_usage_family.tsv*<timestamp>.pdf

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)

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
sequence frequency graph. See section 3 Data Visualization using LymphoTrack Reporter
for more information.

Note: IGK analysis additionally includes information for other elements in the IGK locus involved in recombinations (IGKINTR and IGKDEL).
Note: TRB analysis additionally includes information for other elements in the TRB locus involved in recombinations of D – J region
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”

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 For TRG analysis, please see Table 5 for descriptions of the seven files provided for each sample.
Table 5. Description of TRG output files.

TRG Analysis
A list of unique sequences sorted by count, specifying:






*.fastq_read_summary.tsv*.fastq_read_summary_merged_top10_sear
chtop500.tsv*.fastq_unique_reads.tsv*.fastq_CDR3_unique_reads.tsv*.fastq_VJ_sequence_frequency.tsv

Which V-J recombination event it best fits
LengthActual % of total reads
Cumulative % of total reads
Putative CDR3 sequence

Same format as the read_summary file, but only includes the top 10 reads from theread_summary merged with reads in the top 500. A read will merge with another if they
are only 1 or 2 nts different, with the read with the higher count maintaining its sequence
identity, and the read with the 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 readaccounted 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
the “merged 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
for more information.

Note: “Putative CDR3 sequence” is determined by matching an exact definition of a CDR3 sequence. Any mutation of anchor points or disruption of thereading frame will result in a value of “not found”.

6.12.

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).

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7. Data Visualization Using LymphoTrack Reporter (Automated, Recommended)
7.1.

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!

7.2.

The first page of the PDF report will contain an Index Q30 value, calculated as the percentage of bases that have aquality score of 30 or higher, but only from reads that are assigned to an index after trimming. This value can also be
used to determine if the index passed the standard Illumina specification.



7.3.

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

In some cases, the run Q30 score provided by the sequencer will underestimate the quality of a run because ofcounting undetermined sequences and untrimmed regions towards the Q30.
Undetermined and untrimmed regions are typically lower quality and do not contribute to the downstream
analysis by LymphoTrack.

The first page of the PDF report will contain a table of the merged read summary results, followed by the graphs of theV – J Sequence Frequency and V – J Usage results, and the remaining pages contain a table of the unmerged readsummary results.



The contents of these are detailed in Table 6:

Table 6. 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 withthe top 500 if reads are only 1 or 2 nts different from each other. Additionally, the index Q30
score is displayed under the Total Read Count.

V – J Sequence Frequency Graph

A histogram showing the top 200 sequence frequencies of each V family – J family (for IGH/IGK
and TRB) 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/IGK and
TRB) or V gene – J gene usage for all sequences (for TRG).
Contains a sorted list of the top 200 unique sequences by count, specifying:

Read Summary








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 theidentified V-gene targeted by the primers (%). (IGH/IGK)
Putative CDR3 sequence

Leader and IGH FR1 also contains:

LymphoTrack Software – MiSeq

Mutation rate to partial V-gene (%)

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Table 6. Pages included in the PDF output file.

PDF Output for LymphoTrack Reporter
Leader, IGH FR1, IGH FR2, IGH FR3 also contains:
Read Summary

If the rearrangement is predicted to be in-frame (Y/N/n/a). If either the V or J genesegment 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.

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

8. Data Visualization (Manual): Running the Excel Data Visualization
Spreadsheets- Discommended
8.1.

Check the Security settings within Microsoft Excel to make sure 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 Disable all macros with notification option.
Click Ok and Ok again.
Close Excel.

IMPORTANT!
8.2.

Double-click the appropriate Data Visualization spreadsheet file to open it.



The visualization files are located in the visualizations sub-folder previously located in the installation folder of thesoftware. The following visualization spreadsheet files are available:





8.3.

Because the visualization files can be saved as a workbook (which would remove the ability of the Macroto function), copy the visualization files to a different file path before beginning analyses.

LymphoTrackIGHVisualization.xlsm for Leader, IGH FR1, IGH FR2 and IGH FR3
LymphoTrackIGKVisualization.xlsm for IGK
LymphoTrackTRBVisualization.xlsm for TRB
LymphoTrackTRGVisualization.xlsm for TRG

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.

8.4.

The License Agreement will be displayed; this must be accepted to use the software.


Note:
8.5.

After accepting the License Agreement, a dialog box will open with the prompt: Please select the Read Summary
file for the sample you want.
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 and selectthe FASTQ_READ_SUMMARY file for the sample to be examined.

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8.6.

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 the computer’s hardware.

IMPORTANT!

Due to a limitation in Microsoft Excel, the Visualization software is not capable of displaying more than
1,048,576 rows of data per sheet. Only the first 1,048,576 rows of data will be shown if any of 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 by thislimitation. 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.
1) This option can be set from either Office → Excel Options in Excel 2007, or File → Options in
Excel 2010/2013.
2) Select Advanced, then scroll down to the Display settings.
3) 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 theVisualization software will generate charts correctly.

If this situation occurs and further assistance is required, please contact our Technical Support
department at +1 (858) 224-6600, Monday through Friday 7:00AM – 5:00PM PST/PDT, or via email
at support@invivoscribe.com.
8.6.1.
8.7.

If a message regarding Complex Formatting appears, click Yes.

Save the visualization file. When processing is complete, a message will appear stating the program has completed.
Saving the file at this point is strongly recommended.

IMPORTANT!

8.7.1.
8.8.

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

If a warning message regarding saving as a macro-free workbook appears, click the Yes button.

The completed workbook for Leader, IGH FR1, IGH FR2, IGH FR3, IGK, TRB and TRG will include the following eightworksheets described in Table 7.

Table 7. Worksheets included in Excel output file.

Completed Workbook for Leader, IGH FR1, IGH FR2, IGH FR3, IGK TRB and TRG
Contains a sorted list of the top 200 unique sequences by count, specifying:

Read Summary

LymphoTrack Software – MiSeq










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 and TRB)
How well the read covers the identified V-gene targeted by the primers (%) (IGH/IGK)
Putative CDR3 sequence

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Table 5. Worksheets included in Excel output file.

Completed Workbook for Leader, IGH FR1, IGH FR2, IGH FR3, IGK TRB and TRG
Leader and IGH FR1 also contains:

Mutation rate to partial V-gene (%)

Leader, IGH FR1, IGH FR2, IGH FR3 also contains:
Read Summary

If the rearrangement is predicted to be in-frame (Y/N/n/a). If either the V or J genesegment 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 therearrangement 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 nts 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 and
TRB) 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 and TRB) 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 and
TRB) 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 and TRB) or V
gene – J gene (for TRG) recombination.

VJ Sequence Frequency

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

Unique Reads
Note:
Note:
Note:
Note:
Note:

A FASTA file of unique sequences, sorted by count.

IGH listed in the table above refers to Leader, IGH FR1, IGH FR2 and IGH FR3 analysis. The frequency graphs for these gene targets only specify
IGH in the title. The data output folder specifies whether Leader or one of the framework (FR) regions was analyzed by the software and thisinformation 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).
TRB analysis additionally includes information for other elements in the TRB locus involved in recombinations of the D – J region
Top 200: When specified, the worksheets will show the top sequences (up to 200) for Leader, IGH FR1, IGH FR2, IGH FR3, IGK and TRB. All 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 thereading frame will result in a value of not found.

IMPORTANT!

LymphoTrack Software – MiSeq

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

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IMPORTANT!

The Mutation rate to partial V-gene (%) will display “n/a” for IGH FR2 and FR3. There is limited Vgene coverage due to the shorter amplicon sizes of these targets which results in inaccurate
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 thesereads. 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 non-clonal reads in these cases. Please note that Invivoscribe uses IgBLAST(http://www.ncbi.nlm.nih.gov/igblast/showGermline.cgi) for reference sequences and results
may differ slightly from the IMGT database. In addition, many pseudogenes and nonfunctional genes are not included in the analysis. Interpret rearrangements with somatic
hypermutation statuses at or near the 2% cutoff with caution, particularly when running theIGH FR1 assay which only targets a portion of the IGHV region as the sequence upstream ofthe primer binding site will not be assessed.

9. Technical and Customer Service
We appreciate your business. We are happy to assist you with understanding this software, and will provide ongoing
technical assistance 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

10. 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

11. Legal Notice
Please visit https://invivoscribe.com/legal-notices/ for the most up-to-date legal notices.

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12. Software Frequently Asked Questions
Q1:

When I click “Launch Program”, I get the following (or similar) error:

Application terminated with error: Cannot run program ".\resources\Parse_TRG_miseq.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 “Installing the Software” section) 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, IGK or TRB analysis have fewer than 200 reads?

A2:

The LymphoTrack Software – MiSeq 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 left out of
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 read cannot
be assigned none for both V and J genes, as these would not be reported, per the condition discussed in question 2.

Q4:

The V – J Sequence Frequency Graph has strange values for the y-axis. The % total reads is in the millions, how isthis possible?

A4:

This is likely due to running the LymphoTrack program using the incorrect decimal type for your region. See section
6. Running the Data Analysis Software, 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 left-toright 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 work correctly.

Note:
Q6:

This will permanently change the direction until it is changed back. Closing Excel will not revert this change.
The program stopped after clicking Launch Program and produced this error:
Application terminated with error: Filename not compatible. Please use only MiSeq MSR software generated FASTQ files.

For help with this error, please contact Invivoscribe Technical Support.
A6:

The LymphoTrack software – Miseq expects paired-end FASTQ files generated by the MiSeq Reporter (MSR) software
contained within the MiSeq machine. If the MSR software was not used to generate sequence files, the base-callingmust be performed using the MSR software. Please refer to the MiSeq User Guide for more information.

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Performing the base-calling using the MSR software is important because, while other programs can generate very
similar results as the MSR software (for example, Bcl2fastq), these programs can also generate very different results,
depending on the specific parameters. The LymphoTrack Assay is only valid when analyzing FASTQ files generated
from the MSR software.
Q7:

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

A7:

If there are very few or zero total reads analyzed according to the Read Summary, make sure the correct analysis
type is selected (Leader, IGH FR1, IGH FR2, IGH FR3, IGK, TRB or TRG) when running the LymphoTrack software –Miseq . 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 MiSeq instrument. If the MiSeq instrument has larger files, then retransfer the sequence data to ensure analysis of the full dataset.
The MiSeq sequencer displays different messages when processing the sequencing data. One message may state
Sequencing Complete, and another may later state Analysis Complete. Transferring the FASTQ data in between these
two messages may result in an incomplete LymphoTrack analysis.

Q8:

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

A8: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 the reference
and are possibly in the anti-sense orientation compared to the reference gene. This happens only when thealignment is so poor and when a sense or anti-sense alignment is nearly equivalent, and the anti-sense alignment
was slightly better. In cases such as these, it is best to ignore the read entirely.

Q9:

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

A9:

The log file provides a breakdown of how long each step is taking. Following the line INFO: Decompressing files,
there is another line that states how many files and how much time the decompression used. This number will be inmilliseconds. 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 tothe local computer, or decompress the files directly before running this software. Make sure that the compressedand decompressed files do not both exist in the same folder. The software will not proceed if both FASTQ andFASTQ.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 would include when less than 24 indices are included on a single MiSeq run and as a result, thesamples sequenced have a larger depth of coverage. A second example would be if the library is unbalanced andcontains 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 24-index run with balanced libraries.

Q10:

The Run Q30 reported by the MiSeq instrument fails the Illumina specification. If the LymphoTrack analysis is run
and the controls look like they performed as expected, did the run really fail?

A10:

The Run Q30 is calculated in real time during the base-calling process. At this point, no other data processing hastaken place, such as de-multiplexing and adapter trimming. This means that any sequence from a poor quality index
or occurring outside of the adapter sequence is contributing to the Q30 score. Sequence found outside of theadapter sequence is typically low quality, and this scenario happens often with primer dimers. If a library has asufficient level of primer dimers, this can cause a drop in Run Q30. As a secondary check, the LymphoTrack Reporter
output displays the index Q30 score as calculated from all trimmed reads in that index. If an index Q30 score also
fails the Illumina specification, consider that index to have failed.

LymphoTrack Software – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280358 Rev. S1 | August 2024