Instructions for Use

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM™
®

To identify and track B cell immunoglobulin heavy chain (IGH) gene rearrangements using next-generation sequencing with
the Thermo Fisher Scientific® Ion S5™ or Ion PGM™.
This assay is for Research Use Only. Not for use in diagnostic procedures.
Schematic depiction of the IGH gene locus and framework (FR) regions targeted:

Storage Conditions: -85 ºC to -65 ºC(DNA controls may be separated from assay kits and stored at 2 °C to 8 °C)

Catalog #71210007
71210037
71210047
71210057

Products

LymphoTrack IGH FR1 Assay – S5/PGM
LymphoTrack IGH FR2 Assay – S5/PGM
LymphoTrack IGH FR3 Assay – S5/PGM
LymphoTrack IGH FR1/2/3 Assay – S5/PGM

Quantity

12 indices – 5 reactions each
12 indices – 5 reactions each
12 indices – 5 reactions each
12 indices per target – 5 reactions eachPage 2 of 30

Table of Contents
1.

ASSAY USES ..................................................................................................................................................................................................... 32.

SUMMARY AND EXPLANATION OF THE TEST .............................................................................................................................................................. 3
2.1.
2.2.

3.

PRINCIPLES OF THE PROCEDURE ............................................................................................................................................................................. 4
3.1.
3.2.
3.3.
3.4.
3.5.
3.6.
3.7.

4.

Background....................................................................................................................................................................................................... 3
Summary .......................................................................................................................................................................................................... 4
Polymerase Chain Reaction (PCR)..................................................................................................................................................................... 4
Amplicon Purification ....................................................................................................................................................................................... 4
Amplicon Quantification ................................................................................................................................................................................... 5
Next-Generation Sequencing (NGS) ................................................................................................................................................................. 5
Multiplexing Amplicons .................................................................................................................................................................................... 5
IGHV Somatic Hypermutation (SHM) Evaluation .............................................................................................................................................. 5
Minimal Residual Disease (MRD) Testing ......................................................................................................................................................... 6

REAGENTS ....................................................................................................................................................................................................... 7
4.1.
4.2.
4.3.

Reagent Components ....................................................................................................................................................................................... 7
Warnings and Precautions .............................................................................................................................................................................. 10
Storage and Handling ..................................................................................................................................................................................... 10

5.

INSTRUMENTS ..................................................................................................................................................................................................11

6.

SPECIMEN COLLECTION AND PREPARATION ..............................................................................................................................................................12
6.1.
6.2.
6.3.
6.4.

7.

Precautions ..................................................................................................................................................................................................... 12Interfering Substances .................................................................................................................................................................................... 12
Specimen Requirements and Handling........................................................................................................................................................... 12
Sample Storage............................................................................................................................................................................................... 12

ASSAY PROCEDURE ...........................................................................................................................................................................................13
7.1.
7.2.
7.3.
7.4.
7.5.
7.6.
7.7.
7.8.
7.9.
7.10.
7.11.
7.12.

Materials Provided ......................................................................................................................................................................................... 13
Materials Required (not provided) ................................................................................................................................................................. 13
Reagent Preparation....................................................................................................................................................................................... 14
Amplification .................................................................................................................................................................................................. 15
AMPure XP Purification .................................................................................................................................................................................. 15
Quantification of Amplicons ........................................................................................................................................................................... 17
Pooling and Quantification of Library ............................................................................................................................................................. 18
Dilution of the Pooled Library......................................................................................................................................................................... 19
Template Preparation and Sequencing using the Ion Chef and Ion S5 ........................................................................................................... 19
Template Preparation and Sequencing using the Ion OT2 and Ion S5 ............................................................................................................ 19
Template Preparation and Sequencing using the Ion OT2 and Ion PGM ........................................................................................................ 19
Create a Planned Run. .................................................................................................................................................................................... 20

8.

DATA ANALYSIS................................................................................................................................................................................................21

9.

EXPECTED VALUES.............................................................................................................................................................................................22

10.

LIMITATIONS OF PROCEDURE ...............................................................................................................................................................................22

11.

SAMPLE DATA .................................................................................................................................................................................................22

12.

TROUBLESHOOTING GUIDE ..................................................................................................................................................................................23

13.

TECHNICAL AND CUSTOMER SERVICE ......................................................................................................................................................................23

14.

REFERENCES ....................................................................................................................................................................................................24

15.

SYMBOLS ........................................................................................................................................................................................................24

16.

LEGAL NOTICE ..................................................................................................................................................................................................25

17.

LYMPHOTRACK IGH (FR1/FR2/FR3)ASSAY – ION S5: SINGLE PAGE GUIDE ..................................................................................................................26

18.

LYMPHOTRACK IGH ASSAY – ION PGM: SINGLE PAGE GUIDE .....................................................................................................................................27

19.

APPENDIX A: CONFIGURE THE PLUGIN FILEEXPORTER AND LOAD CUSTOM BARCODES.........................................................................................................28

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023

Page 3 of 28

1. Assay Uses
LymphoTrack IGH FR1 Assay – S5/PGM
The LymphoTrack IGH FR1 Assay - S5/PGM targets the conserved framework 1 (FR1) region within the VH segments of the IGH
gene to identify clonal IGH VH – JH rearrangements, the associated VH – JH region DNA sequences, provides the distribution
frequency of VH region and JH region segment utilization, and the degree of somatic hypermutation of rearranged genes using
the Thermo Fisher Scientific Ion S5 or Ion PGM platform.
LymphoTrack IGH FR1/2/3 Assay – S5/PGM
The LymphoTrack IGH FR1 Assay – S5/PGM targets the conserved framework 1 (FR1) region within the VH segments of the IGH
gene to identify clonal IGH VH – JH rearrangements, the associated VH – JH region DNA sequences, provides the distribution
frequency of VH region and JH region segment utilization, and the degree of somatic hypermutation of rearranged genes using
the Thermo Fisher Scientific Ion S5 or Ion PGM platform.
The LymphoTrack IGH FR2 Assay – S5/PGM targets the conserved framework 2 (FR2) region within the VH segments of the IGH
gene to identify clonal IGH VH – JH rearrangements, the associated VH – JH region DNA sequences, and provides the distribution
frequency of VH region and JH region segment utilization using the Thermo Fisher Scientific Ion S5 or Ion PGM platform.
The LymphoTrack IGH FR3 Assay – S5/PGM targets the conserved framework 3 (FR3) region within the VH segments of the IGH
gene to identify clonal IGH VH – JH rearrangements, the associated VH – JH region DNA sequences, and provides the distribution
frequency of VH region and JH region segment utilization using the Thermo Fisher Scientific Ion S5 or Ion PGM platform.

2. Summary and Explanation of the Test
2.1.

Background
The immunoglobulin heavy chain (IGH) gene locus on chromosome 14 (14q32.3) includes 46-52 functional and 30 nonfunctional variable (VH) gene segments, 27 functional diversity (DH) gene segments, and 6 functional joining (JH) genesegments spread over 1,250 kilobases. The VH gene segments contain three conserved framework (FR) and twovariable complementarity-determining regions (CDRs).
Lymphoid cells are different from other somatic cells in the body; during development, the antigen receptor genes inlymphoid cells undergo somatic gene rearrangement.1 For example, during B-cell development, genes encoding theIGH molecules are assembled from multiple polymorphic gene segments that undergo rearrangements and selection,
generating VH – DH – JH combinations that are unique in both length and sequence. Since leukemias and lymphomas
originate from the malignant transformation of individual lymphoid cells, an individual’s leukemic or lymphoma cells
generally share one or more cell-specific or “clonal” antigen receptor gene rearrangements. Therefore, tests that
detect IGH clonal rearrangements can be useful in the study of B- and T-cell malignancies.
In addition, immunoglobulin heavy chain variable region (IGHV) gene hypermutation status provides important
prognostic information for patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL).
The presence of IGHV somatic hypermutation (SHM) is defined as greater or equal to 2% difference from the germline
VH gene sequence, whereas less than 2% difference is considered evidence of no SHM. The status of SHM for clone(s)has clinical relevance for B-CLL, as there is a clear distinction in the median survival of patients with and without SHM.
Hypermutation of the IGHV region is strongly predictive of a good prognosis while lack of mutation predicts a poor
prognosis.2
Initially, clonal rearrangements were identified using Restriction Fragment, Southern Blot Hybridization (RF-SBH)
techniques. However, these tests proved cumbersome, labor-intensive, required large amounts of DNA and were notsuitable for analysis of many of the less diverse antigen receptor loci.
During the last several decades, the use of RF-SBH assays has been supplanted by PCR-based clonality tests developedby Alexander Morley3, and are considered the current gold standard method. These assays identify clonality on thebasis of over-representation of amplified VH – DH – JH (or incomplete DH – JH products) gene rearrangement following
their separation using gel or capillary electrophoresis. Though sensitive and suitable for testing small amounts of DNA,these assays cannot readily differentiate between clonal populations and multiple rearrangements that might lie
beneath a single-sized peak, and are not designed to identify the specific VH – JH DNA sequence that is required to track

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023

Page 4 of 28

clonal populations in subsequent analyses. This second limitation can be of particular importance, as once the uniqueclone-specific DNA sequence is identified, it can be used in subsequent tests to track and follow these clonal cell
populations.
2.2.

Summary
The LymphoTrack IGH (FR1/FR2/FR3) Assay - S5/PGM (sold separately and as a set) represent a significant
improvement over existing clonality assays using fragment analysis as they efficiently detect IGH gene rearrangements
and, at the same time, identify the DNA sequence specific for each clonal gene rearrangement. Therefore, these assays
have two important and complementary uses: they provide critical information on the existence of clonality andidentify sequence information required to track those clones in subsequent samples. The LymphoTrack IGH FR1 Assay
additionally provides detailed sequence information on the degree of SHM.
Each single multiplex master mix targets one of the conserved IGH framework regions (FR1, FR2 or FR3) within the VH
and the JH regions described in lymphoid malignancies. Targeting all three framework regions significantly reduces
the risk of not being able to detect the presence of clonality, as somatic hypermutations in the primer binding sites of
the involved VH gene segments can impede DNA amplification.4
Primers included in the master mixes are designed with Thermo Fisher Scientific adapters and 12 different indices.
These assays allow for a one-step PCR and pooling of amplicons from several different samples and targets (generated
with other LymphoTrack Assays for the Ion S5 or Ion PGM, sold separately) onto one Ion S5 or PGM chip, allowing up to
12 samples per target to be analyzed in parallel in a single sequencing run.
The associated LymphoTrack Software – S5/PGM provides a simple and streamlined method of analysis andvisualization of data.
Positive and negative controls for clonality are included in the kit. An additional positive control specific for somatic
hypermutation is optional and can be purchased separately (40880008).
Note: For a more thorough explanation of the locus and the targeted sequencing strategy, please refer to (Miller J.E., 2013).5

3. Principles of the Procedure
3.1.

Polymerase Chain Reaction (PCR)
PCR assays are routinely used for the identification of clonal B- and T-cell populations. These assays amplify the DNA
between primers that target the conserved framework of the V and J regions of antigen receptor genes. The primers
lie on either side of an area where programmed genetic rearrangements occur during the maturation of all B and T
lymphocytes. Different populations of B and T lymphocytes arise as a result of these genetic rearrangements.
The antigen receptor genes that undergo rearrangements are the immunoglobulin heavy chain (IGH) and light chainloci (IGK and IGL) in B cells, and the T-cell receptor gene loci (TRA, TRB, TRG, and TRD) in T-cells. Each B- and T-cell has
one or two productive V-J rearrangements that are unique in both length and sequence. Therefore, when DNA from anormal or polyclonal population is amplified using DNA primers that flank the V–J region, amplicons unique in bothsequence and length are generated, reflecting the heterogeneous population. In some cases, where lymphocyte DNA
is absent, no amplicons will be generated. Samples containing IGH clonal populations, yield one or two prominent
amplified products of the same length and sequence which are detected with significant frequency within a diminishedpolyclonal background.

3.2.

Amplicon Purification
PCR amplicons are purified to remove excess primers, nucleotides, salts, and enzymes using solid-phase reversible
immobilization (SPRI) paramagnetic bead technology for high-throughput purification of PCR amplicons. Using an
optimized buffer, PCR products 100 bp or larger are selectively bound to paramagnetic beads while contaminants suchas excess primers, primer dimers, salts, and unincorporated dNTPs are washed away. Amplicons can then be eluted
and separated from the paramagnetic beads resulting in a purified PCR product for downstream analysis and ampliconquantification.

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023

Page 5 of 28

3.3.

Amplicon Quantification
Purified amplicons are quantified utilizing capillary electrophoresis, which applies the principles of traditional gel
electrophoresis to separate and quantify DNA on a chip based platform. Quantification is achieved by running a marker
of known concentration alongside the amplicons and then extrapolating the concentration of the amplicons.
Calculating the concentration of PCR amplicons allows equal amplicon representation in the final pooled library that is
loaded onto the Ion S5 cartridge or Ion PGM chip for sequencing.

3.4.

Next-Generation Sequencing (NGS)
Sanger sequencing methods represent the most popular in a range of ‘first-generation’ nucleic acid sequencing
technologies. Newer methods, which leverage tremendously parallel sequencing approaches, are often referred to as
NGS. These technologies can use various combination strategies of template preparation, sequencing, imaging, andbioinformatics for genome alignment and assembly.
NGS technologies used in this assay rely on the amplification of genetic sequences using a series of consensus forward
and reverse primers that include adapter and index tags. Amplicons generated with LymphoTrack Master Mixes arequantified, pooled, and loaded onto a chip for sequencing with the Thermo Fisher Scientific Ion S5 or Ion PGM
platform. These platforms require the pooled library of DNA fragments to be bound to individual beads prior tosequencing, one unique sequence per bead. Once bound to the beads the DNA fragments are amplified via emulsion
PCR until they cover the surface of the bead. The beads are then loaded onto a semi-conductor chip, where each beadoccupies an individual well and sequencing occurs.
Sequencing is conducted by flooding the chip with individual unincorporated nucleotides one base at a time (dATP,
dCTP, dGTP, dTTP), and the sequencing instruments detect the addition of nucleotides when hydrogen ions are
released during DNA polymerization causing a change in pH of the wells, measured as a change in voltage. The voltagechanges proportionally to the number of nucleotides added. After nucleotides are incorporated, unincorporated
nucleotides are washed away and the process begins again with a new dNTP.

3.5.

Multiplexing Amplicons
These products were designed to allow for two different levels of multiplexing in order to reduce costs and time forlaboratories. The first level of multiplexing originates from the multiple indices that are provided with the assays, up to
12. Each of these 12 indices can be considered to act as a unique barcode that allows amplicons from individual
samples to be pooled together after PCR amplification to generate the sequencing library. The resulting sequences are
sorted by the bioinformatics software to identify those that originated from an individual sample.
The second level of multiplexing originates from the ability of the accompanying software to sort sequencing data byboth index and target. This allows amplicons generated with targeted primers (even those tagged with the same index)
to be pooled together into a single library and sequenced on a single sequencing chip. An example would be tosequence products from several Invivoscribe LymphoTrack Assays together in the same run. However, it is important
to obtain a sufficient number of reads, or depth of coverage, for valid interpretation of each sample. Due to thecapacity of the Thermo Fisher Scientific Ion PGM Ion 316™ Chip v2 BC, which generates 2-3 million reads, it isrecommended to multiplex no more than three different gene targets together, such as IGH FR1, IGH FR2, and IGH FR3.
Up to five different gene targets can be multiplexed together on the Ion PGM Ion 318TM Chip v2 BC (4-5.5 million
reads), Ion S5 Ion 520™ Chip (3-6 million reads) and Ion S5 Ion 530™ Chip (15-20 million reads).
It is important to use the appropriate sequencing chemistry when multiplexing amplicons of different gene targets.
The number of sequencing cycles must be sufficient to sequence the largest amplicon in the multiplex. Two or moresequencing libraries generated from the same LymphoTrack gene target master mixes (e.g., two IGH FR1 sequencing
libraries, either from the same or different kit lots) can also be multiplexed together into a single sequencing library as
long as each index for that master mix is only included once per sequencing run.

3.6.

IGHV Somatic Hypermutation (SHM) Evaluation
For evaluation of the somatic hypermutation rate of the IGHV region, the LymphoTrack IGH FR1 Master Mixes can beused; however, this only targets a portion of the IGHV region as the sequence upstream of the primer binding site will
not be assessed. When analyzing the somatic hypermutation status of samples, the bioinformatics software will
provide the mutation rate based upon the percent mismatch of the clonal amplicons as compared to germline

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023

Page 6 of 28

reference genes, a prediction of whether the protein would be in or out of frame, a prediction of whether mutations orgene rearrangements result in a pre-mature stop codon, and the percentage of VH gene coverage for the region
targeted by the assay.
3.7.

Minimal Residual Disease (MRD) Testing
The correlation of MRD status with overall survival rate was initially demonstrated for subjects with chronic
lymphocytic leukemia using multi-parameter flow analysis (Montserrat, E). However, flow-based methods have provendifficult to standardize outside individual centers. Therefore, flow cytometry is not a suitable technology forinternational standardization, as it is difficult to compare results from different centers. Fortunately, a number ofinvestigators have described NGS-based approaches that have demonstrated success in detecting and monitoring MRD
in Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL) and other lymphoid malignancies.6,7
The LymphoTrack Assays are NGS-based deep sequencing assays that detect virtually all clonal rearrangements withintargeted T-cell receptor (TCR) or immunoglobulin (Ig) antigen receptor loci. This performance suggests that a tumorspecific biomarker target can be readily identified in all subjects. Once a specific rearrangement (the clonotype) has
been identified, LymphoTrack assays can be used to track these clonotype populations to a sensitivity as low as 10-6.
Please refer to the MRD Software IFU and the MRD Technical Bulletin for further details on how to use the Project
Planner tool to successfully design an MRD experiment that meets or exceeds the required level of sensitivity andconfidence.

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023

Page 7 of 28

4. Reagents
4.1.

Reagent Components

Table 1. Available Kits

Catalog #Description# of Indexed Master Mixes

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

12 indices
5 sequencing runs each
12 indices
5 sequencing runs each
12 indices
5 sequencing runs each
12 indices
5 sequencing runs each

Total Reactions
60
60
60
60+60+60

Table 2. LymphoTrack IGH FR1 Assay Kit Components

Reagents

Master Mixesǂ

Positive
Control DNA

Unit
Quantity

71210007 #of Units

Reagent Components

Index Number

IGH FR1 S5/PGM 01

IonXpress_001

1

IGH FR1 S5/PGM 02

IonXpress_002

1

IGH FR1 S5/PGM 03

IonXpress_003

1

IGH FR1 S5/PGM 04

IonXpress_004

1

IGH FR1 S5/PGM 07

IonXpress_007

1

IGH FR1 S5/PGM 08

IonXpress_008

IGH FR1 S5/PGM 09

IonXpress_009

IGH FR1 S5/PGM 10

IonXpress_010

1

IGH FR1 S5/PGM 11

IonXpress_011

1

IGH FR1 S5/PGM 12

IonXpress_012

1

IGH FR1 S5/PGM 13

IonXpress_013

1

IGH FR1 S5/PGM 14

IonXpress_014

1

IGH POS (+)
40880009)

N/A(

250 µL

45 µL

Storage
Temperature

1

Notes

N/A

1

IGH V1-46_03 / IGH J4_02
DNA diluted in tonsil DNA

2
or

Negative
Control DNA

NGS NEG (-)(
40920018)

N/A

45 µL

2

Tonsil DNA, highest
sequence frequency canvary between lots

Note: There are no preservatives used in the manufacturing of these kits.
Noteǂ: IonXpress Indices 5 and 6 are not used in this kit.
.

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023

Page 8 of 28
Table 3. LymphoTrack IGH FR2 Assay Kit Components

Reagents

Master Mixesǂ

Unit
Quantity

71210037 #of Units

Reagent Components

Index Number

IGH FR2 S5/PGM 01

IonXpress_001

1

IGH FR2 S5/PGM 02

IonXpress_002

1

IGH FR2 S5/PGM 03

IonXpress_003

1

IGH FR2 S5/PGM 04

IonXpress_004

1

IGH FR2 S5/PGM 07

IonXpress_007

1
1

IGH FR2 S5/PGM 08

IonXpress_008

IGH FR2 S5/PGM 09

IonXpress_009

IGH FR2 S5/PGM 10

IonXpress_010

1

IGH FR2 S5/PGM 11

IonXpress_011

1

IGH FR2 S5/PGM 12

IonXpress_012

1

IGH FR2 S5/PGM 13

IonXpress_013

1

IGH FR2 S5/PGM 14

IonXpress_014

1

Positive
Control DNA(

IGH POS (+)
40880009)

N/A

Negative
Control DNA

NGS NEG (-)(
40920018)

N/A

250 µL

45 µL

Storage
Temperature

N/A

1

2
or

45 µL

2

Unit
Quantity

71210047 #of Units

Notes

IGH V1-46_03 / IGH J4_02
DNA diluted in tonsil DNA
Tonsil DNA, highest
sequence frequency canvary between lots

Note: There are no preservatives used in the manufacturing of these kits.
Noteǂ: IonXpress Indices 5 and 6 are not used in this kit.
.

Table 4. LymphoTrack IGH FR3 Assay Kit Components

Reagents

Master Mixesǂ

Reagent Components

Index Number

IGH FR3 S5/PGM 01

IonXpress_001

1

IGH FR3 S5/PGM 02

IonXpress_002

1

IGH FR3 S5/PGM 03

IonXpress_003

1

IGH FR3 S5/PGM 04

IonXpress_004

1

IGH FR3 S5/PGM 07

IonXpress_007

1

IGH FR3 S5/PGM 08

IonXpress_008

IGH FR3 S5/PGM 09

IonXpress_009

IGH FR3 S5/PGM 10

IonXpress_010

1

IGH FR3 S5/PGM 11

IonXpress_011

1

IGH FR3 S5/PGM 12

IonXpress_012

1

IGH FR3 S5/PGM 13

IonXpress_013

1

IGH FR3 S5/PGM 14

IonXpress_014

1

Positive
Control DNA(

IGH POS (+)
40880009)

N/A

Negative
Control DNA(

NGS NEG (-)
40920018)

N/A

250 µL

45 µL

Storage
Temperature

1

N/A

1

2
or

45 µL

Notes

2

IGH V1-46_03 / IGH J4_02
DNA diluted in tonsil DNA
Tonsil DNA, highest
sequence frequency canvary between lots

Note: There are no preservatives used in the manufacturing of these kits.
Noteǂ: IonXpress Indices 5 and 6 are not used in this kit.

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023

Page 9 of 28
Table 5. LymphoTrack IGH FR1/2/3 Assay Kit Components
FR1 Master Mixesǂ

# of
Units

FR2 Master
Mixesǂ# ofUnits

FR3 Master Mixesǂ

# of
Units

Index Number

IGH FR1 S5/PGM 01

1

IGH FR2 S5/PGM 01

1

IGH FR3 S5/PGM 01

1

IonXpress_001

IGH FR1 S5/PGM 02

1

IGH FR2 S5/PGM 02

1

IGH FR3 S5/PGM 02

1

IonXpress_002

IGH FR1 S5/PGM 03

1

IGH FR2 S5/PGM 03

1

IGH FR3 S5/PGM 03

1

IonXpress_003

IGH FR1 S5/PGM 04

1

IGH FR2 S5/PGM 04

1

IGH FR3 S5/PGM 04

1

IonXpress_004

IGH FR1 S5/PGM 07

1

IGH FR2 S5/PGM 07

1

IGH FR3 S5/PGM 07

1

IonXpress_007

IGH FR1 S5/PGM 08

1

IGH FR2 S5/PGM 08

1

IGH FR3 S5/PGM 08

1

IonXpress_008

IGH FR1 S5/PGM 09

1

IGH FR2 S5/PGM 09

1

IGH FR3 S5/PGM 09

1

IonXpress_009

IGH FR1 S5/PGM 10

1

IGH FR2 S5/PGM 10

1

IGH FR3 S5/PGM 10

1

IonXpress_010

IGH FR1 S5/PGM 11

1

IGH FR2 S5/PGM 11

1

IGH FR3 S5/PGM 11

1

IonXpress_011

IGH FR1 S5/PGM 121

IGH FR2 S5/PGM 121

IGH FR3 S5/PGM 12

1

IonXpress_012

IGH FR1 S5/PGM 13

1

IGH FR2 S5/PGM 13

1

IGH FR3 S5/PGM 13

1

IonXpress_013

IGH FR1 S5/PGM 141

IGH FR2 S5/PGM 141

IGH FR3 S5/PGM 14

1

IonXpress_014

Reagents
Positive Control DNA*

Negative Control DNA*

Unit Quantity

250 µL

Reagent Components

Notes# of Units

Unit Quantity(

IGH POS (+)
40880009)

IGH V1-46_03 / IGH J4_02 DNA
diluted in tonsil DNA

4

45 µL

(

NGS NEG (-)
40920018)

Tonsil DNA, highest sequencefrequency can vary between lots

4

45 µL

Noteǂ: The storage temperature for master mixes is -85ºC to -65ºC.
Note*: The storage temperature for controls is 2ºC to 8ºC or -85ºC to -65ºC.
Note: There are no preservatives used in the manufacturing of these kits.
Noteǂ: IonXpress Indices 5 and 6 are not used in this kit.

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023

Page 10 of 28

4.2.

Warnings and Precautions
Please read the Instructions for Use carefully prior to starting the assay procedure and follow each step closely.

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4.3.

Use this assay kit as a system; do not substitute other manufacturers’ reagents. Dilution, reducing amplification
reactions, or other deviations from this protocol may affect the performance of this test and/or nullify any limited
sublicense that come with the purchase of these kits.
Do not mix or combine reagents from kits with different lot numbers.
Materials are stable until the labelled expiration date when stored and handled as directed. Do not use kitsbeyond their expiration date.
Adherence to the protocol will assure optimal performance and reproducibility. Ensure correct thermal cycler
programs are used, as other programs may provide inaccurate/faulty data such as false-positive and false-negative
results.
Dispose of unused reagents and waste in accordance with country, federal, state and local regulations.
Perform all laboratory procedures with standard personal protective equipment (gloves, laboratory coats andprotective eye wear). Follow good laboratory practices and universal precautions when working with specimens.
Do not pipette by mouth. Do not eat, drink or smoke in laboratory work areas. Wash hands thoroughly after
handling specimens and assay reagents. Handle specimens in approved biological safety containment facilities andopen only in certified biological safety cabinets. Use molecular biology grade water for the preparation of
specimen DNA.
Due to the high analytical sensitivity of these tests, use extreme care to avoid any contamination of reagents oramplification mixtures with samples, controls, or amplified materials. Use fresh, aerosol-resistant pipette tips
between samples and between dispensing reagents.
Closely monitor all reagents for signs of contamination (e.g., negative controls giving positive signals). Discard any
reagents suspected of contamination.
To minimize contamination, wear clean gloves when handling samples and reagents and routinely clean work
areas and pipettes prior to setting up PCR.
Follow uni-directional work flow between separate work areas in the PCR laboratory: begin with master mix
preparation, move to specimen preparation, then to amplification, and finally to detection. Autoclaving does not
eliminate DNA contamination. Perform pre- and post-PCR steps in separate spaces. Avoid taking paper and othermaterials from post-PCR into the pre-PCR space.
Dedicate all pipettes, pipette tips, and any equipment used in a particular area to that area of the laboratory.
Decontaminate non-disposable items with 10% bleach and rinse with distilled water two separate times before
returning them to the starting areas.
Use sterile, disposable plastic ware whenever possible to avoid contamination.

Storage and Handling






Store the assay at -85ºC to -65ºC until ready to use.
The optimum storage temperature for DNA controls is 2ºC to 8ºC, but DNA can also be stored at -85ºC to -65ºC.
All reagents and controls must be thawed and vortexed or mixed thoroughly prior to use to ensure that they arecompletely resuspended.
Due to high salt concentrations, PCR master mixes are sensitive to freeze/thaw cycles. Limit the number of cycles
to a maximum of five times.

If you have any questions, please contact the Invivoscribe technical staff. We would be happy to help you determine
your optimal storage needs.

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5. Instruments
The instruments listed in Table 6 are recommended to use with the following validated platform combinations for the
LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM library preparation and sequencing:

Ion Chef™ and Ion S5
Ion OneTouch 2™ (OT2) and Ion S5
Ion OT2 and Ion PGM

Table 6. Recommended Instruments.

Instrument Function

Validated Platform Combination with Recommended Instrument / Specifications
Ion Chef and Ion S5

Ion OT2 and Ion S5

Ion OT2 and Ion PGM

Veriti™ Thermal Cycler* or equivalent
Minimum Thermal Range: 15ºC to 96ºC
Minimum Ramping Speed: 0.8ºC / sec

Amplification of DNA samples

See section 7.4: Amplification for the thermal cycler program.

Purification of PCR products

Ambion® Magnetic Stand 96* (AM10027),
Agencourt SPRIPlate® 96 Ring Super Magnet Plate* (A32782),
Thermo Fisher Scientific DynaMag™-96 Side Skirted Magnet* (12027) or equivalent
See section 7.5: AMPure XP Purification for PCR product purification methods.

Quantification of purified PCR
products

Template Preparation

Agilent 2100 Bioanalyzer* or Perkin Elmer LabChip GX*
See section 7.6: Quantification of Amplicons for further details.

Thermo Fisher Scientific Ion Chef
System*Thermo Fisher Scientific Ion OT2System*See section 7.9. Template Preparationand Sequencing using the Ion Chef and
Ion S5 for further details.

See section 7.10. TemplatePreparation and Sequencing using the
Ion OT2 and Ion S5 for further details.

Thermo Fisher Scientific Ion S5 Instrument*
Sequencing

See sections 7.9. Template Preparation and Sequencing using the Ion Chef and
Ion S5 and 7.10. Template Preparation and Sequencing using the Ion OT2 andIon S5 for further details.

Thermo Fisher Scientific Ion OT2System*See section 7.11. TemplatePreparation and Sequencing using the
Ion OT2 and Ion PGM for further
details.

Thermo Fisher Scientific Ion PGM
Instrument*
See section7.11. Template Preparationand Sequencing using the Ion OT2 andIon PGM for further details.

*Follow manufacturer’s installation, operation, calibration and maintenance procedures.

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6. Specimen Collection and Preparation
6.1.

Precautions
Biological specimens from humans may contain potentially infectious materials. Handle all specimens in accordancewith your institute’s Bloodborne Pathogen program and/or Biosafety Level 2.

6.2.

Interfering Substances
The following substances are known to interfere with PCR:





6.3.

Specimen Requirements and Handling




6.4.

Divalent cation chelators
Low retention pipette tips
EDTA (not significant at low concentrations)
Heparin

The minimum input quantity is 50 ng of high-quality DNA (5 µL of sample DNA at a minimum concentration of 10
ng/µL).
This assay tests extracted and purified genomic DNA. DNA must be quantified with a method specific for doublestranded DNA (dsDNA) and free of inhibitors of PCR amplification.
Resuspend DNA in an appropriate solution such as 0.1X TE (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0, prepared withmolecular biology grade water) or molecular biology grade water alone.

Sample Storage
Store samples using a method that prevents degradation of DNA.

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7. Assay Procedure
7.1.

Materials Provided
See Table 2 - Table 5 for materials provided in each kit.

7.2.

Materials Required (not provided)

Table 7. Materials Required (not provided)

Reagent/Material

DNA Polymerase

Required and/or Recommended
Reagents/Suppliers
Roche®:• EagleTaq™ DNA PolymeraseorInvivoscribe:• FalconTaq DNA Polymeraseor equivalent

Catalog #05206944190
or

Notes

5 U/μL60970130

Molecular Biology Grade Water

N/A

N/A

DNase / RNase free

18 MΩ water

N/A

N/A

Required for PGM run

Calibrated Pipettes

N/A

N/A

Must be able to accurately measure
volumes between 0.2 μL and 1000 μL

Vortex Mixer

N/A

N/A

N/A

PCR Plates or Tubes

N/A

N/A

N/A

Filter Barrier Pipette Tips

N/A

N/A

Sterile, RNase/DNase/Pyrogen-free

Microcentrifuge Tubes

N/A

N/A

SterilePCR Purification Kit

Beckman Coulter®, Inc:
• Agencourt AMPure XP

A63880

N/A

Amplicon & Library
Quantification

Agilent® Technologies:• Agilent DNA 1000 Kit
orPerkin Elmer:• HT DNA 1K/12K/Hi Sens Labchip&• HT DNA HiSens Reagents

Ion PGM Sequencing

Ion S5 Sequencing

5067-1504
or
760517&
CLS760672

N/A

Thermo Fisher Scientific:• Ion PGM Hi-Q View OT2 Kit

A29900

N/A

Thermo Fisher Scientific:• Ion PGM Enrichment Beads

4478525

N/A

Thermo Fisher Scientific:• Ion PGM Hi-Q View Sequencing Kit• & Ion PGM Wash 2 Bottle Kit

A30044& A25591

N/A

Thermo Fisher Scientific:• Ion 316 Chip v2 BCor• Ion 318 Chip v2 BC

4488149or
4488146

Thermo Fisher Scientific:• Ion 520 & Ion 530 Kit – OT2
or• Ion 510 & Ion 520 & Ion 530 Kit – Chef

A27751
orA34019LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

N/A

N/A

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Table 7. Materials Required (not provided)

Required and/or Recommended
Reagents/Suppliers

Reagent/Material

Thermo Fisher Scientific:• Ion 520 Chip Kit
or• Ion 530 Chip Kit

Ion S5 Sequencing

The Torrent SuiteTM Software for
Ion PGM Systemor
Ion S5 System••

Version 5.0.4 or 5.2.2 for PGM*
or
Version 5.6 for S5*

Catalog #Notes

A27761orA27764

N/A

N/A

N/A

*Note: These software versions were used for assay verification on the specified instruments

7.3.

Reagent Preparation
To ensure DNA samples contain no PCR inhibitors and are of sufficient quality and quantity to generate a valid result
samples may be tested with the Specimen Control Size Ladder Master Mix from Invivoscribe (20960021 for ABI
detection or
20960020 for gel detection). The Specimen Control Size Ladder targets multiple genes and generates
a series of amplicons of 100, 200, 300, 400 and 600 bp; product sizes may vary ±5 bp due to size standard andinstrument variability. This is especially important for DNA from challenging specimens e.g., FFPE tissue.
Always use positive and negative controls to ensure the assay has been performed correctly.
Always set-up a no template control (NTC) to check for contamination during the PCR set-up.
7.3.1.
7.3.2.

Using gloved hands, remove the Master Mixes from the freezer. Allow the tubes to thaw; then gently vortex tomix followed by a very brief centrifugation.
In a containment hood or dead air box pipette 45 µL from each Master Mix tube into a clean PCR plate (one wellfor each Master Mix and one Master Mix per sample).


7.3.3.
7.3.4.

Include two controls in each run (one positive and one negative) as well as one NTC.
For the NTC, use molecular biology grade water as template instead of DNA.

Add 0.2 µL of Taq DNA Polymerase (@ 5 U/µL) to each well containing aliquoted Master Mixes.
Add 5 µL of sample DNA (at a minimum concentration of 10 ng/µL), control DNA, or molecular biology grade
water (NTC) to the individual wells containing the respective Master Mix reactions.


Pipette up and down 5-10 times to mix.
Seal the plate, briefly centrifuge and place in the PCR thermal cycler.
Table 8. Reaction Setup

Reagent

VolumeMaster Mix

45.0 μL

Taq DNA polymerase

0.2 μL

Sample or Control DNA

5.0 μLTotal

50.2 μL

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7.4.

Amplification
7.4.1.

Amplify the samples using the PCR program from Table 9.
Table 9. PCR Program

7.4.2.
7.5.

Step

Temperature

Time

Cycle

1

95 °C

7 minutes

1

2

95 °C

45 seconds

3

60 °C

45 seconds

4

72 °C

90 seconds

5

72 °C

10 minutes

1

6

15 °C

∞

1

29x

Once the amplification program has completed, remove the amplified PCR plate from the thermal cycler. If not
immediately continuing to the next steps, store the PCR products at 4 °C for 1 day.

AMPure XP Purification
Purification of the PCR products from samples, positive and negative controls and no template controls was performed
during assay verification using the Agencourt AMPure XP PCR Purification system.

Preparation:
7.5.1.
7.5.2.

Remove the AMPure XP reagent from storage and allow it to equilibrate to room temperature before use.
Gently shake the Agencourt AMPure XP bottle to resuspend any magnetic particles that may have settled.
Transfer the appropriate volume of Agencourt AMPure XP reagent needed for the plate to a new 2 mL tube tominimize the risk of contamination by pipette tips.


7.5.3.

The required volume of Agencourt AMPure XP reagent = n × 90 µL (n is number of samples to be purified).

Prepare a fresh of 70% ethanol using sterile water stock (0.5 mL for each sample to be purified).

Binding of Amplicons to Magnetic Particles:
7.5.4.

Add 90 μL of the aliquoted, room temperature Agencourt AMPure XP reagent to each sample to be purified.


7.5.5.

Place the mixed samples on an Ambion Magnetic Stand 96 and incubate at room temperature for 5 minutes toallow the magnetic particles to separate from the solution.


7.5.6.

Mix by pipetting up and down 10 times.
The color of the mixture should appear homogenous after mixing.
Incubate 5 minutes at room temperature.

Keep the plate on the magnetic stand at all times during this procedure, until step 7.5.10 below.

Using a P200 (or equivalent multichannel pipette) set to 135 μL, aspirate the clear supernatant and discard.


Use a P10 pipette (or equivalent multichannel pipette) set to 10 μL to remove any excess supernatant.
Avoid removing any magnetic particles.

Washing:
7.5.7.

Keeping the plate on the magnetic stand, add 200 μL of 70% ethanol to each sample. Incubate for 30 seconds atroom temperature.


7.5.8.
7.5.9.

Using a P200 (or equivalent multichannel pipette) set to 195 μL; aspirate the ethanol and discard.
Use a P10 pipette (or a multichannel pipette) set to 10 μL to remove excess ethanol.
Avoid removing any magnetic particles.

Repeat step 7.5.7 for a total of two washes.
With the plate still on the magnetic stand, allow the magnetic particles to air-dry for 5 minutes.

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Elution:
7.5.10.

Remove the plate from the magnetic stand. Add 40 μL of 1X TE buffer. Mix by pipetting until homogeneous.


7.5.11.
7.5.12.
7.5.13.

Make sure all magnetic particles are in solution.

Incubate at room temperature for 2 minutes.
Place the plate on the magnetic stand for at least 5 minutes or until the supernatant has cleared.
Transfer 35 μL of the eluate to a fresh plate and seal with cap strips. Label the plate and briefly centrifuge toensure the supernatant has completely settled to the bottom of the well.
Store at -20oC or proceed to the next step.

The gel images in Figure 1 - Figure 3 illustrate the effectiveness of a typical purification (showing amplicons
before and after purification) using IGH FR1, IGH FR2 and IGH FR3 Master Mixes respectively.

Figure 1: Example of a purification result for amplicons from the LymphoTrack IGH FR1 Master Mix. This image was generated byrunning unpurified and purified products on the LabChip GX.

Figure 2: Example of a purification result for amplicons from the LymphoTrack IGH FR2 Master Mix. This image was generated byrunning unpurified and purified products on the LabChip GX.

Figure 3: Example of a purification result for amplicons from the LymphoTrack IGH FR3 Master Mix. This image was generated byrunning unpurified and purified products on the LabChip GX.
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7.6.

Quantification of Amplicons
The following steps were performed during assay verification to analyze data generated from samples using either the
Agilent 2100 Bioanalyzer, starting with step 7.6.1 or the Perkin Elmer LabChip GX starting at step 7.6.3.

If quantifying purified PCR amplicons from different LymphoTrack Assays be sure to analyze separately (including the different frameworks)
due to the different size ranges of each assay target.

Agilent 2100 Bioanalyzer Quantification
Prepare an Agilent DNA 1000 Chip for use (please refer to the Agilent DNA 1000 kit instructions for more details).
Result Interpretation:
7.6.1.

The electropherogram of the ladder well is expected to resemble the electropherogram in Figure 4.
Major features of a successful run are:


13 peaks for the DNA 1000 ladder
All peaks are well resolved
Flat baseline
Correct identification of both markers

Figure 4: Example ofan electropherogram of theladder well showing
all 13 peaks.

7.6.2.

Determine the molar concentration (nmol/L) of each sample using the Bioanalyzer software. If necessary, use
manual integration to place the entire range of library fragments within a single peak.

Perkin Elmer LabChip GX Quantification
Prepare the LabChip GX Chip for use (please refer to the Perkin Elmer LabChip GX instructions for more details).
Data Analysis:
7.6.3.
7.6.4.
7.6.5.
7.6.6.
7.6.7.

By default, each time an experiment is run, the data file (*.gxd) is saved in a new folder (named with the currentdate) that can be accessed by the Data folder shortcut on the Desktop.
Transfer the folder containing the data file (*.gxd) to the computer for analysis.
Open the LabChip GX software, go to the menu bar and select File  Import Data file to open the transferreddata file (*.gxd).
In the upper left hand corner of the screen is a plate diagram. Select the wells used in the experiment to display
the associated data in the data tables below. The wells will appear blue when selected.
Go to the menu bar and select Analysis  Analysis Settings.


Select the Smear Analysis tab and add the applicable information from Table 10 - Table 12.
Once all information has been edited, click on the Apply button.

Table 10. Settings for the Smear Analysis with IGH FR1Start Size (bp)

End Size (bp)

Color

Name

Property Displayed in Well Table

Apply to Wells

350

600

Red

Region [350-600]

Size at Maximum [bp]

<all>

350

600

Red

Region [350-600]

Molarity (nmol/L)<all>

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Table 11. Settings for the Smear Analysis with IGH FR2Start Size (bp)

End Size (bp)

Color

Name

Property Displayed in Well Table

Apply to Wells

300

500

Red

Region [300-500]

Size at Maximum [bp]

<all>

300

500

Red

Region [300-500]

Molarity (nmol/L)<all>

Table 12. Settings for the Smear Analysis with IGH FR3Start Size (bp)

End Size (bp)

Color

Name

Property Displayed in Well Table

Apply to Wells

150

300

Red

Region [150-300]

Size at Maximum [bp]

<all>

150

300

Red

Region [150-300]

Molarity (nmol/L)<all>

7.6.8.
7.6.9.

Back at the main screen, go to the menu bar and select File  Export.
Check the Well Table in the pop up window (entitled LabChip GX – Export).

Figure 5:
Example of
LabChip GX –Export pop up
window.

7.6.10.
7.6.11.
7.7.

Click OK to export a *.csv file.
Calculate undiluted amplicon concentration by multiplying the dilution factor (50) with the concentration given
from the LabChip GX (nmol/L).

Pooling and Quantification of Library
The quantity of library DNA loaded into the Ion S5 or Ion PGM emulsion PCR is critical for generating high-quality data
in a sequencing run.
Amplicons generated from one or multiple LymphoTrack Assays can be pooled together into one library for sequencing using the instructions
listed below.

Based on the amplicon concentration calculated by the Bioanalyzer or LabChip GX, add an equal amount of amplicons(with the exception of the NTC sample).



e.g., combine 10 µL of amplicons at 4 nM each in a tube, using 1X TE buffer as a diluent.
Vortex the library tube for 5-15 seconds and then centrifuge for 3-5 seconds.

If there are any samples with concentrations considerably lower or higher than 4 nM adjust the sample/TE buffer
volumes added to the library, ensuring that equal amount of amplicons per sample are pipetted.
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7.8.

Dilution of the Pooled Library
Determine the template dilution factor that gives a final concentration of ~20 pM (~12 x 106 molecules per µL) using
the following formula:Template Dilution Factor = (Library Concentration in pM) / 20 pM
Example:The library concentration is 4 nM (4000 pM)
Template Dilution Factor = 4000 pM / 20 pM = 200
Thus, 1 µL of library mixed with 199 µL of 1X TE buffer or the Nuclease-free water provided in the Ion PGM Hi-Q View
OT2 Kit (1:200 dilution) yields approximately 20 pM (~12 x 106 molecules per µL).
Use the diluted library within 48 hours of preparation.

7.9.

Template Preparation and Sequencing using the Ion Chef and Ion S5
Prepare and enrich template-positive Ion Sphere Particles followed by sequencing on the Ion S5 adhering to thefollowing Thermo Fisher Scientific User Guide:Ion 510 & Ion 520 & Ion 530 Kit – Chef (7.9.1.

MAN0016854)

Create planned run following section 7.12.

All steps including installation, operation, calibration, cleaning and maintenance procedures are performed according to the manufacturer’s
instructions.

7.10.

Template Preparation and Sequencing using the Ion OT2 and Ion S5
Prepare and enrich template-positive Ion Sphere Particles using the OT2 followed by sequencing with the Ion S5,
adhering to the following Thermo Fisher Scientific User Guide:Ion 520 & Ion 530 Kit – OT2 (7.10.1.

MAN0010844)

Create planned run following section 7.12.

All steps including installation, operation, calibration, cleaning and maintenance procedures are performed according to the manufacturer’s
instructions.

7.11.

Template Preparation and Sequencing using the Ion OT2 and Ion PGM
Prepare and enrich template-positive Ion Sphere Particles using the OT2 followed by sequencing with the Ion PGM,
adhering to the following Thermo Fisher Scientific User Guides:

Ion PGM Hi-Q View OT2 Kit (MAN0014579)
Ion PGM Hi-Q View Sequencing Kit (MAN0014583)

All steps including installation, operation, calibration, cleaning and maintenance procedures are performed according to the manufacturer’s
instructions.

Note:
7.11.1.

Do not use the Ion PGM Calibration Standard.
Prepare template using the Ion OT2


7.11.2.

Select PGM: Ion PGM Hi-Q View OT2 Kit - 400 from the drop-down menu

Sequence the library using the Ion PGM

Follow section 7.11.3 for Chip Loading.
Create planned run following section 7.12.

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7.11.3.

Ion PGM Chip Loading
Use the chip loading instructions below for optimal chip loading.
7.11.3.1.
7.11.3.2.

Following Chip Check, prepare the chip for loading following the manufacturer’s instructions.
After dialing the pipette down to load the ISPs (~30 µL) into the chip at a rate of ~1 µL per second,transfer the chip in the bucket to the minifuge with the chip tab pointing in (toward the center of theminifuge).


7.11.3.3.
7.11.3.4.
7.11.3.5.
7.11.3.6.

Note:

Use 30 second minifuge spins with the chip tab pointing in and then turned to point out.

Firmly tap the point of the chip tab on the bench top 2-3 times. To avoid the generation of bubbles,
do not pipet the sample out and then back into the chip.
Tilt the chip at a 45° angle and slowly remove as much liquid as possible from the loading port bydialing up the pipette. Discard the liquid.
If some liquid remains in the chip, perform a 5-second quick spin with the chip-tab pointing out andremove and discard any additional liquid. Do not spin the chip upside-down.
If some liquid remains in the chip after the quick spin, lightly and rapidly tap the point of the chip tab
against the benchtop a few times and remove and discard any collected liquid. Do not flush the chip.
Immediately proceed to selecting the Planned Run and Performing the Run (section 7.12).

Validations were performed following the Thermo Fisher Scientific User Guides listed in section 14: References along with theweighted bucket chip loading procedure. After the sequencing primer annealing step, the reactions remained in the thermal
cycler at 15°C instead of at room temperature.

If using Torrent Suite Software v5.2.2 or v5.6, follow the procedure in Appendix A to verify the configuration of the FileExporter plugin andload the custom barcodes using the LymphoTrack_IonXpress.csv file included on the provided CD (75000007). If using TSS v5.0.4, please
continue to step 7.12.

7.12.

Create a Planned Run.

7.12.1.
7.12.2.
7.12.3.
7.12.4.

Create a Planned Run for Ion S5 or Ion PGM. Log into the Torrent Browser for the Torrent Server connected tothe system.
Click the Plan tab and then click on Generic Sequencing under Templates, then select Plan New Run on the top
right.
In the Plan Run Wizard, review each screen and make the appropriate selection following Table 8.
Select the Planned Run and perform the run.

CAUTION! If using TSS v5.2.2 or v5.6 please contact Thermo Fisher Tech Support for assistance with uploading barcodes.
Characters in file name:••••Create a unique name or identifier when naming each 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 sequenced on the same chip will result in multiple sample results beingcombined by the LymphoTrack Software – PGM/S5 during the analysis process.
It is important that the filenames only contain the following characters (A-Z, a-z, 0-9, ., _ (underscore), - (hyphen)).
If the software encounters a character not within this set or more than one consecutive space, it may fail.

Sample name when multiplexing:
Each index can only be listed in the planned run once; therefore, any necessary tracking information for samples sequenced with multiple
targets using the same index must be included within one Sample Name field (which is incorporated into the FASTQ file name).
It is recommended to keep track of all samples and targets in an Ion PGM or Ion S5 run that are sequenced using the same index. This set ofsamples/targets should be given a unique identifier to include in the Sample Name field in the Planned Run.
Examples of Sample Names that can be used for tracking purposes are listed below:•
S1_FR1_FR2_FR3_IGK (one sample sequenced with multiple assays using the same index)•
S1_FR1_S4_TRG (multiple samples sequenced with multiple assays using the same index)•
Pool02_IX002 (Pool 02 refers to all samples/targets sequenced with IonXpress_002 and tracked elsewhere)

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Indices when multiplexing:
Please note that the IonXpress Indices 5 and 6 are not used in the LymphoTrack IGH (FR1, FR2, FR3) and TRG Assay kits and Indices 3, 5, 6, 7
and 15 are not used in the LymphoTrack IGK Assay kits for the Ion PGM and Ion S5.

Table 13. Run Plan Wizard settings by platform combination.

Validated Platform Combination

Setting

Ion Chef and Ion S5

OT2 and Ion S5

OT2 and Ion PGM

Ion Reporter

Ion Reporter - None
Sample Grouping - Blank

Ion Reporter - None
Sample Grouping - Blank

Ion Reporter - None
Sample Grouping - Blank(Research)
Application

Application – DNA
Target Technique – Other

Application – DNA
Target Technique – Other

Application – DNA
Target Technique – Other

Sample Preparation Kit – leave blank

Sample Preparation Kit – leave blank

Sample Preparation Kit – leave blank

Library Kit Type – leave blank

Library Kit Type – leave blank

Library Kit Type – leave blank

Template Kit:
 Select Instrument: Chef
 Ion 510 & Ion 520 & Ion 530 Kit – Chef

Template Kit:
 Select Instrument: OneTouch
 Ion 520 & Ion 530 Kit – OT2

Template Kit:
 Select Instrument: OneTouch
 Ion PGM Hi-Q View OT2 Kit –400

Sequencing Kit
 Ion 510 & Ion 520 & Ion 530 Kit – Chef

Sequencing Kit
 Ion 520 & Ion 530 Kit – OT2

Sequencing Kit
 Ion PGM Hi-Q View Sequencing Kit

Kits

Templating Size – 400bp

Templating Size – 400bp

Flows – 850

Flows – 850

Flows – 850

Control Sequence – leave blank

Control Sequence – leave blank

Control Sequence – leave blank

Chip Type: Ion 520 Chip or Ion 530 Chip

Chip Type: Ion 520 Chip or Ion 530 Chip

Chip Type: Ion 316 Chip v2 BC or Ion 318 Chip v2 BC

Barcode Set – IonXpress or
LymphoTrack_IonXpress3

Barcode Set – IonXpress or
LymphoTrack_IonXpress3

Barcode Set – IonXpress or
LymphoTrack_IonXpress3

Plug-ins

Select FileExporter1,2

Select FileExporter1,2

Select FileExporter1,2

Projects

Select the appropriate Project folder forsaving the run or add a new project

Select the appropriate Project folder forsaving the run or add a new project

Select the appropriate Project folder forsaving the run or add a new projectEnter a Template Name and add any
notesEnter a Template Name and add any
notesEnter a Template Name and add any
notes

Reference

Leave all sections blank

Leave all sections blank

Leave all sections blank

Monitoring

Bead Loading (%) ≤ 30
Key Signal (1-100) ≤ 30
Usable Sequence (%) ≤ 30

Bead Loading (%) ≤ 30
Key Signal (1-100) ≤ 30
Usable Sequence (%) ≤ 30

Bead Loading (%) ≤ 30
Key Signal (1-100) ≤ 30
Usable Sequence (%) ≤ 30

Template Namen/a

Note1: The Torrent Suite™ Software version 5.0.4 PlugIn FileExporter may not generate FASTQ Files. Contact Thermo Fisher Tech Support for further assistance if
a “DOCSTRING ERROR” message is received.
Note2: Avoid long file names as they may interfere with the PlugIn FileExporter.
75000007)
Note3: If using TSS v5.2 or v5.6, please refer to the LymphoTrack_IonXpress.csv file located in the provided CD (8. Data Analysis

The LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM were designed to produce sequencing data that can be analyzed using
the LymphoTrack Software – S5/PGM package provided on the associated CD (75000007). This CD includes detailed
instructions for installation and use of the software.

Samples prepared with the LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM generate FASTQ files that can be easily
processed with the LymphoTrack Data analysis application.

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023

Page 22 of 28

8. Data Analysis
9. Expected Values
The calculations generated by the software are rounded to the nearest tenth to determine assay results.
S5 and PGM Run Validity


o
o
o

Loading > 50%
Enrichment > 50%
Clonal > 50%
IGH Positive Control: top % reads ≥ 2.5%
NGS Negative Control: top % reads < 1.0%
IGH SHM Positive Control (40880008, can be purchased separately)




o
o

top % reads ≥ 2.5%
Mutation rate ≥ 2.0%

10. Limitations of ProcedureThis assay does not identify 100% of clonal cell populations.
PCR-based assays are subject to interference by degradation of DNA or inhibition of PCR amplification due to heparin orother agents that might be present in the analyzed sample.
The IGH FR1 forward primers are not located at the immediate 5’ end of the IGHV region. Any mutations in nucleotides
upstream of the primer binding site will not be included in the calculated mutation rate.
A higher level of variance at or near the analytical limit of detection (LOD) is inherent to most technologies; including, but
not limited to next generation sequencing. Follow-up testing is suggested when a result presents near the assay
analytical LOD.

10. Limitations of Procedure
11. Sample Data
Figure 6: This table,
generated via the
LymphoTrack
Reporter, shows the
top 10 reads fromthe read summary
merged with the top
500 reads; a read will
merge with another ifthey are only one or
two bp different.
Sequences weregenerated using theLymphoTrack IGH FR1Assay – MiSeq andanalyzed using theLymphoTrack
Software – S5/PGM(
75000007).

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023

Page 23 of 28

12. Troubleshooting Guide
Table 14. Troubleshooting

Occurs During

Error

ActionSample and reagent preparation

Sample DNA quantity
is less than 50 ng
by a dsDNA-based method

Do not test the sample

Sample and reagent preparation

Sample DNA integrity is low

Test the sample using theSpecimen Control Size Ladder available from
Invivoscribe(
20960021 for ABI detection or
20960020 for gel detection)

Library creation by ampliconquantification and pooling

Amplicon concentrationis less than 1 nM

Check the Bioanalyzer or LabChip GX ladder andrepeat PCR if less than 1 nM.

Template preparation and S5 or PGM
initialization

N/A

Call Thermo Fisher Scientific Tech Support+1-800-831-6844

CD installation

LymphoTrack Software does not install
properly

Call Invivoscribe Tech Support+1-858-224-6600

Data analysis

LymphoTrack Software stops running

Call Invivoscribe Tech Support+1-858-224-6600

Data analysis

Excel Macro cannot be executed

Call Invivoscribe Tech Support+1-858-224-6600

Data analysis

No clonal sequence is detected for the Positive
Control

Call Invivoscribe Tech Support+1-858-224-6600

No Template Control (NTC)

NTC shows amplicons after PCR

Repeat the assay

13. Technical and Customer Service
Thank you for purchasing our LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM. We appreciate your business. We are
happy to assist you with understanding this assay, and will provide ongoing technical assistance Monday through Friday tokeep 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

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023

Page 24 of 28

14. References
1.
2.
3.
4.
5.
6.
7.













Tonegawa, S. (1983). Somatic Generation of Antibody Diversity. Nature 302, 575–581.
Ghia, P. et al., (2007). ERIC recommendations on IGHV gene mutational status analysis in chronic lymphocytic leukemia.
Leukemia 21, 1–3.
Trainor, KJ. et al., (1990). Monoclonality in B-lymphoproliferative disorders detected at the DNA level. Blood 75, 2220–
2222.
Miller, JE. (2013). Principle of Immunoglobulin and T Cell Receptor Gene Rearrangement. In Cheng, L., Zhang, D., Eble, J.
N. (Eds), Molecular Genetic Pathology (2nd Ed., sections 30.2.7.13 and 30.2.7.18). New York, USA: Springer Science& Business Media.
Montserrat, E. (2005). Treatment of Chronic Lymphocytic Leukemia: Achieving Minimal Residual Disease–Negative Status
As a Goal. Journal of Clinical Oncology, 23(13), pp. 2884–2885.
Miklos, DB. et al., (2013). Minimal residual disease quantification using consensus primers and high-throughput IGH
sequencing predicts post-transplant relapse in chronic lymphocytic leukemia. Leukemia 27(8): 1659–1665.
Campana, D. et al., (2012). Deep-sequencing approach for minimal residual disease detection in acute lymphoblastic
leukemia. Blood 120:5173-5180.
LymphoTrack Software – S5/PGM (75000007) Instructions for Use
User Guide: Ion 510 & Ion 520 & Ion 530 Kit – Chef (Man0016854, Rev. C.0)
User Guide: Ion 520 & Ion 530 Kit – OT2 (Man0010844, Rev. D.0)
User Guide: Ion PGM Hi-Q View OT2 Kit (Man0014579 Rev A.0)
User Guide: Ion PGM Hi-Q View Sequencing Kit (Man0014583, Rev A.0)
Agilent DNA 1000 Kit Guide
LabChip GX/GX II User Manual
HT DNA High Sensitivity LabChip Kit LabChip GX/GXII User Guide
http://www.thermofisher.com
http://ioncommunity.thermofisher.com
http://www.agilent.com
http://www.perkinelmer.com

15. 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 IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023

Page 25 of 28

16. Legal NoticeThis product is covered by one or more of the following patents and patent applications owned by or exclusively licensed to
Invivoscribe, Inc. (IVS). United States Patent Number 7,785,783, United States Patent Number 8,859,748 (together withdivisional application claims relating to the same original application), European Patent Number EP 1549764B1 (validated in
16 countries, and augmented by related European Patents Numbered EP2418287A3 and EP 2460889A3), Japanese Patent
Number JP04708029B2, Japanese Patent Application Number 2006-529437, Brazil Patent Application Number PI0410283.5,
Canadian Patent Number CA2525122, Indian Patent Number IN243620, Mexican Patent Number MX286493, Chinese Patent
Number CN1806051, and Korean Patent Number 101215194.
Use of this product may require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). Any necessary
license to practice amplification methods or to use reagents, amplification enzymes or equipment covered by third party
patents is the responsibility of the user and no such license is granted by Invivoscribe, Inc., expressly or by implication. This
product is sold FOR RESEARCH USE ONLY; not for use in diagnostic procedures.
©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 of others used herein) their respective owners.
Thermo Fisher Scientific® and Verti® are registered trademarks of Thermo Fisher Scientific.

Ion S5™, PGM™, Hi-Q™, Torrent Suite ™, OneTouch™, Ion 316™ and Ion 318™ are trademarks of Thermo Fisher
Scientific or its subsidiaries.
Agilent® and Bioanalyzer® are registered trademarks of Agilent Technologies, Inc.
Beckman Coulter®, Agencourt®, Ampure® and SPRIPLATE® are registered trademarks of Beckman Coulter, Inc.
Microsoft®, Windows® and Excel® are registered trademarks of Microsoft Corporation.
Perkin Elmer® and LabChip® GX are registered trademark of Perkin Elmer.
Roche® is a registered trademark and EagleTaq™ is a trademark of Roche.

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023

Page 26 of 28

17. LymphoTrack IGH (FR1/FR2/FR3) Assays – S5: Single Page Guide
17.1

Using gloved hands, remove the Master Mixes from the freezer. Use a different indexed Master Mix for each sample
and controls. Allow the Master Mix tubes to thaw; then gently vortex to mix.

17.2

In a containment hood or dead air box pipette 45 µL of Master Mix into individual wells of a PCR plate for the samples,
positive, negative, and no template controls (one well for each indexed Master Mix).

17.3

Add 0.2 µL Taq DNA polymerase (@5 U/µL) to each Master Mix.

17.4

Add 5 µL sample DNA (at a minimum concentration of 10 ng/µL) and 5 µL of control samples to wells containing therespective Master Mix reactions, and pipette up and down 5-10 times to mix.

17.5

Add 5 µL molecular biology grade water to the well containing the respective Master Mix for no template control, andpipette up and down 5-10 times to mix.

17.6

Seal the plate and amplify target DNA using the standard thermal cycler program:
Step

Temperature

Time

Cycle

1

95 °C

7 minutes

1

2

95 °C

45 seconds

3

60 °C

45 seconds

4

72 °C

90 seconds

5

72 °C

10 minutes

1

6

15 °C

∞

1

29x

17.7

Remove the amplification plate from the thermal cycler.

17.8

Purify the PCR products using the Agencourt AMPure XP PCR Purification system. Add 90 μL of particles to each 50 µL
reaction then elute purified DNA in 40 µL TE buffer.

17.9

Quantify amplicons with an appropriate method (e.g., Agilent 2100 Bioanalyzer or LabChip GX).

17.10

Based on the quantification, combine an equal amount of each amplicon in a tube (do not include the no templatecontrol); use TE buffer to make up a total volume of 10 µL per Master Mix. Gently vortex to mix followed by a brief
centrifugation.

17.11

Dilute the library to 20 pM with Nuclease-free water.

17.12

Perform emulsion PCR to prepare the template by using either the Ion Chef or the Ion OT2 paired with the Ion ES
instruments.
a.
b.

Using the Ion Chef instrument with the Ion 510 & Ion 520 & Ion 530 Kit – Chef orUsing the Ion OneTouch instrument with Ion 520 & Ion 530 Kit – OT2

17.13

Initialize the Ion S5 and load the Ion 520 or Ion 530 chip with the ISPs.

17.14

Create a Planned Run using the Torrent Browser.

17.15

Start the Ion S5 run.

17.16

Analyze and visualize the acquired data using the associated LymphoTrack Software – S5/PGM package.

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023

Page 27 of 28

18. LymphoTrack IGH (FR1/FR2/FR3) Assays – PGM: Single Page Guide
18.1.

Using gloved hands, remove the Master Mixes from the freezer. Use a different indexed Master Mix for each sample
and controls. Allow the Master Mix tubes to thaw; then gently vortex to mix.

18.2.

In a containment hood or dead air box pipette 45 µL of Master Mix into individual wells of a PCR plate for the samples,
positive, negative, and no template controls (one well for each indexed Master Mix).

18.3.

Add 0.2 µL Taq DNA polymerase (@5 U/µL) to each Master Mix.

18.4.

Add 5 µL sample DNA (at a minimum concentration of 10 ng/µL) and 5 µL of control samples to wells containing therespective Master Mix reactions, and pipette up and down 5-10 times to mix.

18.5.

Add 5 µL molecular biology grade water to the well containing the respective Master Mix for no template control, andpipette up and down 5-10 times to mix.

18.6.

Seal the plate and amplify target DNA using the standard thermal cycler program:
Step

Temperature

Time

Cycle

1

95 °C

7 minutes

1

2

95 °C

45 seconds

3

60 °C

45 seconds

4

72 °C

90 seconds

5

72 °C

10 minutes

1

6

15 °C

∞

1

29x

18.7.

Remove the amplification plate from the thermal cycler.

18.8.

Purify the PCR products using the Agencourt AMPure XP PCR Purification system. Add 90 μL of particles to each 50 µL
reaction then elute purified DNA in 40 µL TE buffer.

18.9.

Quantify amplicons with an appropriate method (e.g., Agilent 2100 Bioanalyzer or LabChip GX).

18.10.

Based on the quantification, combine an equal amount of each amplicon in a tube (do not include the no templatecontrol); use TE buffer to make up a total volume of 10 µL per Master Mix. Gently vortex to mix followed by a brief
centrifugation.

18.11.

Dilute the library to 20 pM with 1X TE buffer or the Nuclease-free water provided with the Hi-Q View OT2 Kit.

18.12.

Using the Ion OneTouch 2 instrument with the Ion PGM Hi-Q View OT2 Kit, perform an emulsion PCR to create templatepositive ion sphere particles (ISPs).

18.13.

Enrich the template-positive ISPs with the Ion OneTouch ES.

18.14.

Initialize the Ion PGM and load the Ion 316 Chip v2 BC (or Ion 318 Chip v2 BC) with the ISPs.

18.15.

Create a Planned Run using the Torrent Browser.

18.16.

Start the Ion PGM run.

18.17.

Analyze and visualize the acquired data using the associated LymphoTrack Software – S5/PGM package.

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023

Page 28 of 28

19. Appendix A: Configure the Plugin FileExporter and Load Custom Barcodes
If using Torrent Suite Software v5.2.2 or v5.6, verify the configuration of the FileExporter plugin and load the custom barcodes
using the LymphoTrack_IonXpress.csv file included on the provided CD (75000007).
19.1.

Verify the configuration of plugin FileExporter.

19.1.1.
19.1.2.

Log in to Torrent Suite Software as admin.
Confirm the correct plugin configuration for FileExporter.







19.1.3.

Verify the Change Plugin Configuration for the FileExporter plugin.






19.2.

From the gear icon () on the Torrent Suite Server Home Screen, select Plugins from the dropdown menu.
Find the FileExporter plugin and click on the gear icon, then select Configure (the configuration window willopen).
Under File > Options: check the FASTQ boxes.
For Archive Type, select Zip.
Select the preferred Naming Option.
Save the configuration.

Click on the gear icon () and select Configure.
Scroll down and click Admin Interface.
Scroll down and select Plugins on the left-hand side, then select FileExporter.
Verify the sections for Status and Userinputfields display { }, if not, delete the contents until { } is displayed.
Do not change any other parameters (not mentioned here).
Select Save.

Load Custom barcodes.

19.2.1.

Log on to Torrent Suite Software as admin.







19.2.2.

Select the gear icon () the select References from the dropdown menu.
Select Barcodes then select Add new DNA Barcodes.
Select Choose File and upload the LymphoTrack IonXpress.csv file
Name Barcode set name (e.g. LymphoTrack_IonXpress) and click Upload.
This name of barcodes will be used for all LymphoTrack assays in the run set up.
Verify the newly added barcodes are shown in the Barcodes menu.

Proceed to step 12.1 Create a Planned Run.

LymphoTrack IGH (FR1/FR2/FR3) Assays – S5/PGM
For Research Use Only. Not for use in diagnostic procedures

280376 Rev. D | December 2023