Instructions for Use

LymphoTrack® Dx TRG Assay – MiSeqTM
Identify and track clonal T cell receptor gamma (TRG) gene rearrangements using Next-Generation Sequencing (NGS)with the Illumina® MiSeq.
This assay is for In Vitro Diagnostic Use.
Schematic depiction of the TRG gene locus:

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

Catalog #92270019
92270009

Products
LymphoTrack Dx TRG Assay Kit A – MiSeq
LymphoTrack Dx TRG Assay Panel – MiSeq

Quantity
8 indices – 5 reactions each
24 indices – 5 reactions eachPage 2 of 27

Table of Contents
1.

INTENDED USE ......................................................................................................................................................................................... 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.

4.

Thermal cycler .......................................................................................................................................................................................... 8
Magnetic Stand ......................................................................................................................................................................................... 8
Real-time PCR Instrument......................................................................................................................................................................... 8
Illumina MiSeq Instrument* ..................................................................................................................................................................... 8

SPECIMEN COLLECTION AND PREPARATION ...................................................................................................................................................... 9
6.1.
6.2.
6.3.
6.4.

7.

Reagent Components ............................................................................................................................................................................... 6
Warnings and Precautions ........................................................................................................................................................................ 7
Storage and Handling................................................................................................................................................................................ 7

INSTRUMENTS .......................................................................................................................................................................................... 8
5.1.
5.2.
5.3.
5.4.

6.

Polymerase Chain Reaction (PCR) ............................................................................................................................................................. 4
Amplicon Purification ............................................................................................................................................................................... 4
Amplicon Quantification ........................................................................................................................................................................... 4
Next-Generation Sequencing (NGS).......................................................................................................................................................... 4
Multiplexing Amplicons ............................................................................................................................................................................ 5

REAGENTS ............................................................................................................................................................................................... 6
4.1.
4.2.
4.3.

5.

Background ............................................................................................................................................................................................... 3
Summary................................................................................................................................................................................................... 3

Precautions ............................................................................................................................................................................................... 9Interfering Substances .............................................................................................................................................................................. 9Specimen Requirements and Handling ..................................................................................................................................................... 9
Sample Storage ......................................................................................................................................................................................... 9

ASSAY PROCEDURE .................................................................................................................................................................................. 10
7.1.
7.2.
7.3.
7.4.
7.5.
7.6.
7.7.
7.8.
7.9.
7.10.
7.11.
7.12.
7.13.

Materials Provided.................................................................................................................................................................................. 10
Materials Required (not provided) ......................................................................................................................................................... 10
Reagent Preparation ............................................................................................................................................................................... 11
Amplification........................................................................................................................................................................................... 11
AMPure XP Purification........................................................................................................................................................................... 12
Quantification of Amplicons ................................................................................................................................................................... 13
Pooling and Quantification of Library ..................................................................................................................................................... 14
Dilution of the Pooled Library ................................................................................................................................................................. 14
Setup qPCR for Library Quantification .................................................................................................................................................... 14
Preparation of the Library for the MiSeq Sequencing Run ..................................................................................................................... 14
MiSeq Flow Cell Loading ......................................................................................................................................................................... 16
MiSeq Sample Sheet Setup .................................................................................................................................................................... 16
MiSeq Run Start ...................................................................................................................................................................................... 17

8.

DATA ANALYSIS ...................................................................................................................................................................................... 17

9.

EXPECTED VALUES ................................................................................................................................................................................... 17

10.

LIMITATIONS OF PROCEDURE ...................................................................................................................................................................... 17

11.

INTERPRETATION AND REPORTING ............................................................................................................................................................... 18

12.

SAMPLE DATA ........................................................................................................................................................................................ 20

13.

PERFORMANCE CHARACTERISTICS ................................................................................................................................................................ 20

14.

TROUBLESHOOTING GUIDE ........................................................................................................................................................................ 21

15.

TECHNICAL AND CUSTOMER SERVICE ............................................................................................................................................................ 22

16.

REFERENCES .......................................................................................................................................................................................... 2217.

SYMBOLS .............................................................................................................................................................................................. 22

18.

LEGAL NOTICE ........................................................................................................................................................................................ 23

19.

LYMPHOTRACK DX TRG ASSAY - MISEQ: SINGLE PAGE GUIDE ........................................................................................................................... 24

20.

APPENDIX A: BUILDING A SEQUENCING LIBRARY WITH MULTIPLE NGS TARGETS ..................................................................................................... 25

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1. Intended Use
The LymphoTrack Dx TRG Assay for the Illumina MiSeq is an in vitro diagnostic product intended for next-generation
sequencing (NGS) based determination of the frequency distribution of TRG gene rearrangements in patients suspectedwith having lymphoproliferative disease. This assay aids in the identification of lymphoproliferative disorders.

2. Summary and Explanation of the Test
2.1.

Background
The human T-cell receptor gamma (TRG, previously known as TCRG) gene locus on chromosome 7 (7q14) includes 14
Vγ (variable region) genes (Group I, II, III and IV), 5 Jγ (joining region) gene segments and 2 Cγ (constant region) genes
spread over 200 kilobases.
Lymphoid cells are different from the other somatic cells in the body; during development, the antigen receptor
genes in lymphoid cells undergo somatic gene rearrangement.1 For example, during T-cell development genes
encoding the TRG molecules are assembled from multiple polymorphic gene segments that undergo rearrangements
and selection, generating Vγ–Jγ combinations that are unique in both length and sequence. Since leukemias andlymphomas 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 TRG clonal rearrangements can be useful in the study of B- and T-cell malignancies.
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
developed by Alexander Morley2 and are considered the current gold-standard method. These assays identify
clonality on the basis of over-representation of amplified V–D–J (or incomplete D–J) products following theirseparation 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 V–J DNA sequence that is required to track
clonal populations in subsequent analyses. This second limitation can be of particular importance, as once theunique clone-specific DNA sequence is identified, the sequence can be used in subsequent tests to track and follow
the clonal cell populations.

2.2.

SummaryThis LymphoTrack Dx TRG Assay – MiSeq represents a significant improvement over existing clonality assays using
fragment analysis as it efficiently detects the majority of TRG gene rearrangements using a single multiplex mastermix and, at the same time, identifies the DNA sequence specific for each clonal gene rearrangement. Therefore, thisassay has two important and complementary uses, it both aids in the detection of initial clonal populations andidentifies sequence information required to track those clones in subsequent samples.
Each single multiplex master mix for TRG targets the conserved regions within the Vγ and the Jγ regions described inlymphoid malignancies. Primers included in the master mixes are designed with Illumina adapters and up to 24
different indices. This method allows for a one-step PCR and pooling of amplicons from several different samples andtargets (generated with other LymphoTrack Dx Assays for the Illumina MiSeq instrument, sold separately), onto one
MiSeq flow cell allowing up to 24 samples per target to be analyzed in parallel in a single run.
The associated LymphoTrack Dx Software – MiSeq provides a simple and streamlined method of analysis andvisualization of data. By following the guidelines provided in section 11: Interpretation and Reporting the sample
results summarized in the software can be easily interpreted for the presence or absence of clonality.
Always interpret the results of molecular clonality tests in the context of clinical, histological and immunophenotypic
data.
Positive and negative controls for clonality are included in the kit.
Note:

For a more thorough explanation of the locus and the targeted sequencing strategy, please refer to Miller J.E., 2013.3

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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 V and J regions of antigen receptor genes. These primers target theconserved regions and lie on either side of an area where programmed genetic rearrangements occur during thematuration 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 froma normal or polyclonal population is amplified using DNA primers that flank the V–J region, amplicons that are uniquein both sequence and length are generated, reflecting the heterogeneous population. In some cases, wherelymphocyte DNA is absent, no amplicons will be generated. Samples containing TRG clonal populations yield one or
two prominent amplified products of the same length and sequence which are detected with significant frequency,
within a diminished polyclonal 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
such as 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 more purified PCR product for downstream analysis
and amplicon quantification.

3.3.

Amplicon Quantification
Purified amplicons are quantified using the KAPA™ Library Quantification Kits for Illumina platforms. Purified anddiluted PCR amplicons and a set of six pre-diluted DNA standards are amplified by quantitative (qPCR) methods, using
the KAPA SYBR® FAST qPCR Master Mix and primers. The primers in the KAPA kit target Illumina P5 and P7 flow cell
adapter oligo sequences.
The average Ct score for the pre-diluted DNA Standards are plotted against log10 to generate a standard curve, which
can then be used to calculate the concentration (pM) of the PCR amplicons derived from sample DNA. Calculating
the concentration of PCR amplicons allows equal amplicon representation in the final pooled library that is loaded
onto the MiSeq 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 toas NGS. These technologies can use various combination strategies of template preparation, sequencing, imaging
and bioinformatics 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 the LymphoTrack Dx
Master Mixes are quantified, pooled and loaded onto a flow cell for sequencing with an Illumina MiSeq sequencing
platform. Specifically, the amplified products in the library are hybridized to oligonucleotides on a flow cell and areamplified to form local clonal colonies (bridge amplification). Four types of reversible terminator bases (RT-bases) areadded and the sequencing strand of DNA is extended one nucleotide at a time. To record the incorporation of
nucleotides, a CCD camera takes an image of the light emitted as each RT-base is added, and then cleaved to allow
incorporation of the next base.

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3.5.

Multiplexing Amplicons
This assay was 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.
Each of these 24 indices acts 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 thebioinformatics software which identifies 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 sameindex) to be pooled together to generate the library to be sequenced on a single flow cell. An example would be tosequence a combination of products from several Invivoscribe LymphoTrack Dx Assay kits for the MiSeq such as IGHV
Leader, IGH FR1, IGH FR2, IGH FR3, IGK, TRB and TRG together. When multiplexing amplicons of different genetargets, it is important to use the appropriate sequencing chemistry. The number of sequencing cycles must be
sufficient to sequence the largest amplicon in the multiplex. For example, when multiplexing a combination of IGH
FR1, IGH FR2, IGH FR3, IGK, TRB and TRG amplicons together, use the MiSeq v2 (500 cycle) sequencing kit for up to 4
targets or v3 (600 cycle) sequencing kit for up to 7 targets. When multiplexing any of these amplicons together with
IGHV Leader, use the MiSeq v3 (600 cycle) sequencing kit. If multiplexing only IGH FR3 and TRG amplicons together,which both have shorter amplicon sizes, use the MiSeq v2 (300 or 500 cycle) sequencing kits and adjust the cycle
settings in the sample sheet. For further instructions, please refer to Appendix A (section 20).
The number of samples that can be multiplexed onto a single flow cell is also dependent on the flow cell that is
utilized. Illumina’s standard flow cells (MiSeq v3) can generate 22 - 25 million reads. To determine the number ofreads per sample, divide the total number of reads for the flow cell by the number of samples that will be multiplexed
and the number of reads for each sample will be sufficient for valid interpretation. For more information, refer tosection 11: Interpretation and Reporting. Illumina also manufacturers other flow cells that utilize the same
sequencing chemistry, but generate fewer reads. When using these alternative flow cells one must consider thatfewer total reads either means less depth per sample or fewer samples can be run on the flow cell to achieve thesame depth per sample.

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4. Reagents
4.1.

Reagent Components

Table 1. Available Kits

Catalog #Description# of Indexed Master Mixes

Total Reactions

92270019

LymphoTrack Dx TRG Assay Kit A – MiSeq

8 indices – 5 sequencing runs each

4092270009

LymphoTrack Dx TRG Assay Panel – MiSeq

24 indices – 5 sequencing runs each

120

Table 2. Kit Components

Reagent

92270009# of Units

TRG MiSeq 01

1

1

TRG MiSeq 02

1

1

TRG MiSeq 03

1

1

TRG MiSeq 04

1

1

TRG MiSeq 05

1

1

TRG MiSeq 06

1

1

TRG MiSeq 07

1

1

TRG MiSeq 08

1

1

TRG MiSeq 09

01

TRG MiSeq 1001

TRG MiSeq 11

0

1

0

1

TRG MiSeq 12

Master Mixesǂ

Positive Control
DNA

92270019# of Units

Reagent Components

TRG MiSeq 13(

Unit
Quantity

225 µL

0

1

TRG MiSeq 14

01

TRG MiSeq 15

01

TRG MiSeq 1601

TRG MiSeq 18

01

TRG MiSeq 19

01

TRG MiSeq 20

01

TRG MiSeq 21

01

TRG MiSeq 22

01

TRG MiSeq 23

01

TRG MiSeq 25

01

TRG MiSeq 27

0

1

1

3

TRG POS (+)
42270019)

45 µL

Storage
Temperature

Notes
N/A

V11/ J1/2 DNA
diluted in tonsil DNA
or

Negative
Control DNA

NGS NEG (-)(
40920018)

45 µL

1

3

Tonsil DNA, highest
sequence frequency
can vary between lots

Note: There are no preservatives used in the manufacturing of these kits.
ǂNote: Indices 17, 24, and 26 are not used in these kits

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4.2.

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

▪
▪

▪
▪
▪

▪
▪

▪
▪

▪
▪
▪
4.3.

This product is for In Vitro Diagnostic Use.
Use the 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 comes with the purchase of these kits.
Materials are stable until the labeled expiration date when stored and handled as directed. Do not use kits beyond
their expiration date.
Close 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 falsenegative results.
Do not mix or combine reagents from kits with different lot numbers.
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
and open only in certified biological safety cabinets.
Use molecular biology grade water for the preparation of specimen DNA.
Due to the high analytical sensitivity of this test, 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 areasand pipettes prior to setting up PCR.
Follow uni-directional work flow in the PCR laboratory between the separate work areas: begin with master mix
preparation, move to specimen preparation, then to amplification and finally to detection. Autoclaving does not
eliminate DNA contamination. Do not bring amplified DNA into the areas designated for master mixes or specimen
preparation.
Dedicate all pipettes, pipette tips and any equipment used in a particular area to that area of the laboratory.
Decontaminate non-disposable items in 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▪
▪
▪
▪

If the assay is not being used immediately, store at -85ºC to -65ºC.
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 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 four 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 specific instruments listed below are recommended based on the methods used to validate this assay.
5.1.

Thermal cycler▪
▪
▪

Use or Function: Amplification of DNA samples
Suggested Instrument: Veriti™ Dx Thermal Cycler or equivalent
Performance Characteristics and Specifications:
o
o

▪
▪
5.2.

Follow manufacturer’s installation, operation, calibration and maintenance procedures.
See section 7.4 Amplification for thermal cycler program.

Magnetic Stand▪
▪

Use or Function: Purification of PCR products
Suggested Instrument:
o
o
o

▪

▪

Precipitate paramagnetic beads

See section 7.5 AMPure XP Purification for PCR product purification methods.

Real-time PCR Instrument▪
▪
▪

Use or Function: Quantify purified PCR products
Suggested Instrument: Applied Biosystems® 7500 Fast Dx Real-Time PCR Instrument or equivalent
Performance Characteristics and Specifications:
o

▪
▪
5.4.

Ambion® Magnetic Stand 96* (AM10027)
Agencourt SPRIPlate® 96 Ring Super Magnet Plate* (A32782)
Thermo Fisher Scientific DynaMag™-96 Side Skirted Magnet* (12027)

Performance Characteristics and Specifications:o5.3.

Minimum Thermal Range: 15ºC to 96ºC
Minimum Ramping Speed: 0.8ºC/sec

Can detect SYBR Green wavelength

Follow manufacturer’s installation, operation, calibration and maintenance procedures.
See section 7.6 Quantification of Amplicons for real-time PCR program.

Illumina MiSeq Instrument*▪
▪

Use or Function: Sequence normalized DNA library
Performance Characteristics and Specifications:
o
o

▪
▪

Compatible with MiSeq Reagent Kit v2*
Compatible with MiSeq Reagent Kit v3* when multiplexing

Follow manufacturer’s installation, operation, calibration and maintenance procedures
See section 7.11 MiSeq Flow Cell Loading, 7.12 MiSeq Sample Sheet Setup and 7.13 MiSeq Run Start for MiSeq
parameters.
*Warning: These are not CE-marked products

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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 L 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 be free of PCR inhibitors.
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 for materials provided.

7.2.

Materials Required (not provided)

Table 3. Materials Required (not provided)

Required or Recommended
Reagents/Suppliers

Reagent/Material

DNA Polymerase

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

Calibrated Pipettes

N/A

N/A

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

PCR Plates or Tubes

N/A

N/A

DNase / RNase / PCR inhibitor-freeFilter Barrier Pipette Tips

N/A

N/A

Sterile, RNase/DNase/Pyrogen-free

Microcentrifuge Tubes

N/A

N/A

SterileA63880

N/A

AM10027
12027
or

N/A

PCR Purification Kit

Beckman Coulter, Inc:•
Agencourt AMPure XP

PCR Purification

Thermo Fisher Scientific:•
Ambion Magnetic Stand 96•
DynaMag-96 Side Skirted Magnet
orBeckman Coulter:•
Agencourt SPRIPlate 96 Ring Super
Magnet Plateor Equivalent

Amplicon & Library
Quantification

MiSeq Run

MiSeq Software

KAPA Biosystems:•
KAPA Library Quantification Kit –Illumina

KK4824

N/A

MS-102-2002
orMS-102-2003
orMS-102-3003

Standard flow cell

MiSeq Control Software v2.6 or later
Local Run Manager v2.0 or later

N/A

N/A

N/A

N/A

Prepare a 10 mM Tris-HCl, pH 8.0+ 0.05% Tween 20 solution

Illumina:•
MiSeq Reagent v2 kit (300 cycles)or•v2 kit (500 cycles)or•v3 kit (600 cycles) when multiplexing••

Dilution Buffer A

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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 andgenerates a series of amplicons of 100, 200, 300, 400 and 600 bp; sizing may vary +/- 5 bp due to size standard and/or
instrument differences. Verifying the DNA integrity is especially important for challenging specimens e.g., FFPE
tissue.
Always use the 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 process.
7.3.1.
7.3.2.

Using gloved hands, remove the Master Mixes from the freezer. Allow the tubes to thaw; then gently vortex
to mix followed by a very brief centrifugation.
In a containment hood or dead air box, pipette 45 L from each Master Mix tube into a clean PCR plate (onewell for 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 L of Taq DNA polymerase (@5 U/L) to each well containing aliquoted Master Mixes.
Add 5 L of sample DNA (at a minimum concentration of 10 ng/L), 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 and place it in the PCR thermal cycler.
Table 4. Reaction Setup

7.4.

Reagent

VolumeMaster Mix

45.0 μL

Taq DNA polymerase

0.2 μL

Sample or Control DNA

5.0 μL

Total Volume

50.2 μL

Amplification
7.4.1.

Amplify the samples using the PCR program from Table 5.

If multiplexing multiple targets, please refer to Appendix A (section 20) for other LymphoTrack Dx Assay - MiSeq thermal cycling conditions.
Table 5. PCR Program

Step

Temperature

Time

Cycle

1
2

95 °C

7 minutes

1

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

Note: Set the heated lid to 105°C and reaction volume to 50 μL

7.4.2.

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.

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7.5.

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

Preparation:
7.5.1.

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.

Please refer to Appendix A (section 20) for AMPure XP reagent volumes used in other LymphoTrack Dx Assay - MiSeq PCR products.

7.5.2.

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 × 50 µL (n is number of samples to be purified).

Prepare a fresh stock (0.5 mL for each sample to be purified) of 80% ethanol using sterile water.

Binding of Amplicons to Magnetic Particles:
7.5.4.

Add 50 μL of the aliquoted, room temperature Agencourt AMPure XP reagent to each sample to be purified.
▪
▪
▪

7.5.5.

Place the mixed samples on a DynaMag-96 Side Skirted or Ambion Magnetic Stand-96 and incubate at room
temperature for 5 minutes to allow 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 10 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 95 μL, aspirate the cleared 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 80% ethanol to each sample. Incubate for 30 seconds
at room 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.

Elution:
7.5.10.

Remove the plate from the magnetic stand. Add 25 μL of 10 mM Tris-HCl, pH 8.0 buffer.
▪
▪

7.5.11.
7.5.12.
7.5.13.

Mix by pipetting until homogeneous.
Make sure all magnetic particles are in solution.

Incubate at room temperature for 2 minutes.
Place the plate on the magnetic stand for 5 minutes or until the supernatant has cleared.
Transfer 22 μL of the eluate to a fresh plate. 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 thenext step.
The gel image in Figure 1 illustrates the effectiveness of a typical purification (showing amplicons before andafter purification).

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Figure 1: Example ofa purification result
for amplicons fromthe TRG MiSeq
Master Mix. The
image was generated
by running
unpurified andpurified products on
the LabChip GX.

7.6.

Quantification of Amplicons
The following steps were performed during assay validation to quantify purified PCR amplicons generated fromsamples, as well as positive, negative and no template controls using the KAPA library quantification kit (KAPA
Biosystems).
7.6.1.

Dilution of amplicons
Dilution Buffer A below refers to: 10 mM Tris-HCl, pH 8.0 + 0.05% Tween 20

1: 4,000 Final:
Step A:
2 μL of purified amplicon eluate + 198 µL of Dilution Buffer A.
Mix well by pipetting up and down 10 times.
Step B:
5 μL from Step A + 195 μL of Dilution Buffer A.
Mix well by pipetting up and down 10 times.
7.6.2.

Set up a qPCR run for amplicon quantification following Table 6 for each reaction (please refer to the KAPA
library quantification kit instructions for more details):
Table 6. qPCR Setup

Reagent

7.6.3.

Volume

PCR-grade water

3.6 μL

KAPA SYBR FAST qPCR Master Mix containing Primer Premix

12.0 μL

ROX

0.4 μL

Diluted amplicons or Standard (1-6)

4.0 μL

Total Volume20.0 μL

Follow Table 7 for the thermal program for qPCR.
Table 7. qPCR Program

Step

Temperature

Time

Cycle

1

95°C

5 minutes

1

95°C

30 seconds

60°C

45 seconds (plate read)2

7.6.4.

35x

Use the data from the qPCR run to check for contamination by calculating the ΔCt values between the controls(positive and negative) and the NTC, using the following equation:
ΔCt = Ct (NTC) – Ct (Control)
If the ΔCt ≥ 4.0 for both controls, proceed to the next step. If the ΔCt < 4.0 for either control, see section 14
Troubleshooting Guide for further instructions.

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If multiplexing multiple targets, please refer to Appendix A (section 20) for the qualifying ΔCt value for other LymphoTrack Dx Assay - MiSeq
PCR products.

7.6.5.

Use the data from the qPCR run to determine the concentration of amplicon for each sample using thefollowing equation:
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑢𝑛𝑑𝑖𝑙𝑢𝑡𝑒𝑑 𝑎𝑚𝑝𝑙𝑖𝑐𝑜𝑛 (𝑛𝑀) =452 × 𝐴𝑣𝑔 𝐶𝑜𝑛𝑐. (𝑝𝑀)𝐶𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑 𝑏𝑦 𝑞𝑃𝐶𝑅
×4
A

452 represents the average fragment length (bp) of the KAPA Illumina DNA Standard.
A = The average fragment length of amplicons generated using the TRG Assay = 300bp. (A = 300).
Sequence length includes additional nucleotides that are necessary for sequencing.
Note:

7.7.

If multiplexing multiple targets, please refer to Appendix A (section 20) for the average fragment length of amplicons generated forother LymphoTrack Dx Assay - MiSeq

Pooling and Quantification of Library
The quantity of library DNA loaded onto the MiSeq flow cell is critical for generating optimal cluster density andobtaining high quality data in a sequencing run. Quantification of the library by qPCR is strongly recommended.
7.7.1.

Based on the amplicon concentration calculated from the qPCR results, add an equal amount of amplicons(with the exception of NTC, which can be excluded).
▪
▪

7.7.2.
7.8.

e.g., dilute each amplicon to 4 nM in a total volume of 10 μL using Dilution Buffer A as diluent. Combine 10
μL of each 4 nM amplicon.
For samples that have < 4 nM concentration, add the maximum amount of sample possible (10 μL) and do
not add any Dilution Buffer A to that sample.

Gently vortex to mix followed by a brief centrifugation.

Dilution of the Pooled Library
1:1,000 Final:
Step A:
2 μL of pooled library + 198 μL of Dilution Buffer A.
Mix well by pipetting up and down 10 times.
Step B:
20 μL from Step A + 180 μL of Dilution Buffer A.
Mix well by pipetting up and down 10 times.

7.9.

Setup qPCR for Library Quantification
Refer to Table 6 for qPCR setup and Table 7 for the thermal program.
7.9.1.

Determine the concentration of the pooled library from the qPCR results.
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑢𝑛𝑑𝑖𝑙𝑢𝑡𝑒𝑑 𝑎𝑚𝑝𝑙𝑖𝑐𝑜𝑛 (𝑛𝑀) =452 × 𝐴𝑣𝑔 𝐶𝑜𝑛𝑐. (𝑝𝑀)𝐶𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑 𝑏𝑦 𝑞𝑃𝐶𝑅
A

452 represents the average fragment length (bp) of the KAPA Illumina DNA Standard.
A = The average fragment length of amplicons generated using the TRG Assay = 300bp. (A = 300).
Sequence length includes additional nucleotides that are necessary for sequencing.
Note:

7.10.

If multiplexing multiple targets, please refer to Appendix A (section 20) for the average fragment length of amplicons generated forother LymphoTrack Dx Assay - MiSeq

Preparation of the Library for the MiSeq Sequencing Run
At the end of this section, the concentration of the library DNA will be 12 pM for MiSeq reagent kit v2 and 20 pM for
MiSeq reagent kit v3 when multiplexing. For multiplexing amplicons from different LymphoTrack Dx Assays for the
MiSeq into a single library, please refer to section 20: Appendix A: Building a Sequencing Library with Multiple NGS
Targets.

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7.10.1.

Determine the amount of library to be prepared based on the concentration of the pooled library from theqPCR results and dilute if necessary:▪
▪

7.10.2.

If the library is more than 4 nM, dilute the library to 4 nM in a final volume of 10 μL using Dilution Buffer A.
If the library is less than 4 nM, use 10 μL of the library directly for the next step.

Use the following instructions to denature the library DNA.
▪
▪

Prepare a fresh solution of 0.2 N NaOH. A fresh solution is essential for completely denaturing sample DNA
and for optimal cluster generation on the MiSeq.
Add 10 μL of 0.2 N NaOH to the diluted library (10 μL) prepared in the previous step.
Table 8. Library Denaturation

Reagent

7.10.3.

7.10.4.

VolumeDiluted library

10 μL

0.2 N NaOH

10 μL

Total Volume

20 μL

Vortex briefly to mix the solution and then briefly centrifuge to ensure all the solution has settled to thebottom of the tube. Incubate for 5 minutes at room temperature to denature the dsDNA library into single
stranded DNA (ssDNA).
Add 980 μL of pre-chilled HT1 buffer (provided in MiSeq Reagent kits) to the tube containing the denatured
library DNA:
Table 9. HT1 Buffer Addition

Reagent

7.10.5.
7.10.6.
7.10.7.

VolumeDenatured library

20 μL

HT1 Buffer

980 μL

Total Volume

1000 μL

Vortex briefly to mix, then pulse centrifuge the diluted and denatured library DNA solution.
Place the diluted and denatured library on ice until the next step.
Remove the diluted ssDNA library from ice and use the following instructions to dilute the library further in
preparation for loading onto the MiSeq.

If multiplexing multiple targets, please refer to Appendix A (section 20) for loading concentration and MiSeq reagent kit.
For MiSeq Control Software (v2.6 or later):
The concentration of the library DNA is 12 pM for MiSeq reagent kit v2.
The concentration of the library DNA is 12 to 20 pM for MiSeq reagent kit v3 when multiplexing.

▪

If the diluted ssDNA library concentration is 40 pM (i.e., the initial concentration was 4 nM), dilute to thedesired MiSeq loading concentration using the following examples:
Table 10. Preparation of the Library for MiSeq Loading▪
▪
7.10.8.
7.10.9.

Final Concentrations

12 pM

Denatured library

300 μLHT1 Buffer

700 μL

Final NaOH Concentration (mM)

0.60 mM

If the diluted ssDNA library concentration is less than 40 pM (the initial concentration was less than 4 nM),
dilute the denatured DNA appropriately to the desired MiSeq loading concentration (e.g. 12 pM).
Be sure that the final NaOH concentration is not more than 1.0 mM.

Invert the final library 5 times to mix and pulse centrifuge.
Place the final prepared library on ice until it is loaded onto the MiSeq Reagent Cartridge.

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7.11.

MiSeq Flow Cell Loading
Load 600 μL of the Final Prepared Library onto a MiSeq Reagent Cartridge.

7.12.

MiSeq Sample Sheet Setup
Please refer to the latest Illumina documentation for Sample Sheet creation. Upload the sample sheet to the MiSeq
instrument. If using Illumina-associated software (such as Local Run Manager [LRM]), select TruSeq Nano DNA for the
Library Prep Kit and TruSeq DNA Single Indexes Set A B for the Index Kit.

Characters in the sample name:•Create a unique name and identifier for each sample. If duplicate samples are run, a similar name can be used (i.e., Sample1a andSample1b).
•
Failure to provide unique names to samples sequenced on the same flow cell will result in only one sample being analyzed by the
LymphoTrack Dx Software – MiSeq during the analysis process.
•Use only alphanumeric and hyphen characters (A-Z, a-z, 0-9, , -) when preparing the Sample Sheet.

Sample name when multiplexing:
List each index in the Sample Sheet only one time; any necessary tracking information for samples sequenced with multiple targets using
the same index must be included within one Sample ID field (which is incorporated into the FASTQ file name).
Keep track of all samples and targets in a MiSeq run that are sequenced using the same index. This set of samples/targets should be givena unique identifier to include in the Sample ID field in the Sample Sheet. Please keep in mind that the Sample ID has a strict 40-character
limit when choosing a naming convention.

The Sample Name field in the Sample Sheet is incorporated by default into the FASTQ file name instead of the
Sample ID when information is entered into this field. Please leave this field blank or copy the information that wasentered for the Sample ID. If alternative information is entered into the Sample Name field, please make sure that it
includes a unique identifier and conforms to the recommendations above for tracking samples.
Important!

Adapter sequences are not recognized by the LymphoTrack Dx Software – MiSeq.



If using Illumina-associated software (such as Local Run Manager (LRM)), adapter trimming must be
selected when creating the sample sheet.

Table 11. Indices used with LymphoTrack Dx Assay master mixes

LymphoTrack Dx Assay –MiSeq PCR Master Mix Index

Index
Sequence

TruSeq DNA Single Indexes Set A
B (LRM “Index Kit”)

id01
id02

ATCACGCGATGT

AR001
AR002

id03

TTAGGC

AR003

id04
id05

TGACCAACAGTG

AR004
AR005

id06

GCCAAT

AR006

id07
id08

CAGATC
ACTTGA

AR007
AR008

id09

GATCAG

AR009

id10
id11

TAGCTT
GGCTAC

AR010
AR011

id12

CTTGTA

AR012

id13
id14

AGTCAAAGTTCC

AR013
AR014

id15

ATGTCA

AR015

id16
id18

CCGTCCGTCCGC

AR016
AR018

id19

GTGAAA

AR019

id20

GTGGCC

AR020

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Table 11. Indices used with LymphoTrack Dx Assay master mixes

LymphoTrack Dx Assay –MiSeq PCR Master Mix Index

7.13.

Index
Sequence

TruSeq DNA Single Indexes Set A
B (LRM “Index Kit”)

id21

GTTTCG

AR021

id22
id23

CGTACG

AR022

id25

GAGTGGACTGAT

AR023
AR025

id27

ATTCCT

AR027

MiSeq Run Start
Start the MiSeq run following the MiSeq Control Software Instructions. The approximate MiSeq run times areindicated in Table 12.

Table 12. MiSeq Run Times

MiSeq Reagent Kit

Read Length

MCS Version

Total MiSeq Run Time

2x151 bpv2.6 or later~ 24 hours

2x251 bpv2.6 or later~ 39 hours

2x301 bpv2.6 or later~ 56 hours

v2
v3

Note: Using a kit with fewer cycles will not be sufficient to generate read lengths required for this assay.

8. Data Analysis
The LymphoTrack Dx TRG Assay – MiSeq was designed to produce sequencing data that can be analyzed using theLymphoTrack Dx Software – MiSeq package provided on the associated CD (95000009), which is included with the assaypurchase. This CD includes detailed instructions for installation and use of the software package.

9. Expected Values
The calculations generated by the software are rounded to the nearest tenth to determine assay result.

▪
▪
▪

TRG Positive Control top % reads ≥ 2.5%
NGS Negative Control top % reads < 1.0%
MiSeq Run Validityooo*Note:%Q30 > 80% for v2 (2x151)%Q30 > 75% for v2 (2x251)%Q30 > 70% for v3 (2x301)
Q30 from all analytical validations has met the above criteria from Illumina MiSeq Q30 specification. However, the Q30
score may vary depending on sample quality. If Q30 falls below Illumina Q30 specification, check the index Q30 value
from the LymphoTrack Dx Report after data analysis by the LymphoTrack Dx Software - MiSeq. If an index Q30 score on
the LymphoTrack Dx Report does not meet the Illumina specification, consider that index to be invalid.
MiSeq v2 chemistry has been validated for this single-target assay. MiSeq v3 chemistry has been validated for assay
multiplexing.

10. Limitations of Procedure▪
▪
▪

This assay does not identify 100% of clonal cell populations.
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.
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.

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Always interpret the results of molecular clonality tests in the context of clinical, histological, and immunophenotypicdata.

11. Interpretation and Reporting
Use the Merged Read Summary report to identify the top merged read sequences and their frequencies prior to clonality
determination using the criteria listed below. Refer to section 8: Data Analysis for more information on the Merged Read
Summary report. There are some clonal processes that may result in the detection of two or more clones. Examples of thisinclude a dominant population with a small sub-clonal population or when multiple lymphoproliferative disorders arepresent. It is especially important that these cases are interpreted within their clinical context.
* Be cautious in making interpretation if “none” is present for V, D and/or J gene in suspected clonal reads. “None” is assigned when thealignment does not meet the minimum quality threshold due to poor alignment.
Table 13. Interpretation Criteria

Criterion 1

The total number of reads foreach sample is ≥ 20,000.

The total number of reads foreach sample is ≥ 10,000 and <20,000.

Criterion 2

Criterion 3

Result

The % reads for a suspected clonal merged
sequence is > 2X the % reads for the 3rd mostfrequent merged sequence.1

EVIDENCE OF CLONALITY
DETECTED

The % reads for a suspected clonal merged
sequence is ≤ 2X the % reads for the 3rd mostfrequent merged sequence.1

No evidence of clonality
detected

The % reads for a suspected clonal merged
sequence is > 2X the % reads for the 3rd mostfrequent merged sequence.1

EVIDENCE OF CLONALITY
DETECTED

The % reads for a suspected clonal merged
sequence is ≤ 2X the % reads for the 3rd mostfrequent merged sequence.1

No evidence of clonality
detected

N/A

Not evaluable

The top merged sequence has≥ 2.5% of the total reads.

The top merged sequence has≥ 5.0% of the total reads.

The total number of reads foreach sample is < 10,000.

N/A

1Software calculations are rounded to the nearest tenth for comparison.

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Figure 2: Interpretation of data based on Criteria from Table 13.

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12. Sample Data

Figure 3:

This table, generated via the LymphoTrack Dx Software - MiSeq, shows the top 10 reads from the read summary merged with the top
500 reads; a read will merge with another if they are only 1 or 2 base pair (bp) different. Sequences were generated using theLymphoTrack Dx TRG Assay – MiSeq and analyzed using the LymphoTrack Dx Software – MiSeq (95000009).

13. Performance Characteristics
The LymphoTrack Dx TRG Assay – MiSeq results were compared to the laboratory diagnostic report and the concordance (oroverall percent agreement), positive percent agreement (PPA) and negative percent agreement (NPA) were: 89% (51/57
cases), 81% and 97%, respectively.
Table 14. Comparison between LymphoTrack Dx TRG Assay – MiSeq and the Laboratory Diagnostic Report

Laboratory Diagnostic Report
Clonal

Non-Clonal

Clonal22

1

Non-Clonal

529

LymphoTrack Dx TRG Assay- MiSeq

The analytical performance of the LymphoTrack Dx TRG Assay – MiSeq was evaluated by testing spiked clonal cell line DNA
into tonsil DNA at different dilutions. The Limit of Detection (LoD) was observed at 5% DNA dilution. The highest % reads
from tonsil DNA was < 1%. The linear regression R2 was > 0.98 for a range of 0 to 10% DNA dilution. The coefficient ofvariation (CV%) across 8 runs from 2 operators, 2 reagent lots and 2 instruments was less than 15% when testing 5% and
10% DNA dilutions.

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14. Troubleshooting Guide
Table 15. Troubleshooting Guide

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

Test the sample using the Specimen Control Size
Ladder available from Invivoscribe
Sample and reagent preparation

Sample DNA integrity is low(

20960021 for ABI detection or
20960020 for gel detection)

Quantification of amplicons using the
KAPA library quantification kit

ΔCt < 4.0
ΔCt = Ct (NTC) – Ct (Control)

Check the standard curve in qPCR.
Check for contamination and repeat the KAPA
qPCR for all samples and controls.
If ΔCt < 4.0 again, redo the PCR and qPCR for all
samples and controls.

Library creation by ampliconquantification and pooling

Amplicon concentrationis less than 1 nM

Check the standard curve in qPCR and repeat PCR
if less than 1 nM

Sample sheet not foundImproperly formatted sample sheet
Fluidics check failed
MiSeq run set up

Disk space low

Refer to Illumina troubleshooting
Or call Illumina Tech Support+1-800-809-4566

Empty waste bottle
Network disconnected
RFID Failure
MiSeq run

%Q30 < 80% for v2 (2x151)*%Q30 < 75% for v2 (2x251)*%Q30 < 70% for v3 (2x301)*

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

CD installation

LymphoTrack Dx Software does not install
properly

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

Data analysis

LymphoTrack Dx Software stops running

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 amplification after PCR

Repeat the assay* Q30 from all analytical validations has met the above criteria from Illumina MiSeq Q30 specification. However, the Q30 score may vary depending on
sample quality. If Q30 falls below Illumina Q30 specification, check the index Q30 value from the LymphoTrack Dx Report after data analysis by the
LymphoTrack Dx Software - MiSeq. If an index Q30 score from the LymphoTrack Dx Report does not meet the Illumina specification, consider that index
to be invalid.

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15. Technical and Customer Service
Thank you for purchasing our LymphoTrack Dx TRG Assay – MiSeq. We appreciate your business. We are happy to assist
you with understanding this assay 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
Technical Service: support@invivoscribe.com | Customer Service: sales@invivoscribe.com | Website: www.invivoscribe.com

16. References
1.
2.
3.

4.

▪
▪
▪
▪
▪
▪

Tonegawa, S. (1983). Somatic Generation of Antibody Diversity. Nature 302, 575-581
Trainor KJ. et al., (1990). Monoclonality in B-lymphoproliferative disorders detected at the DNA level. Blood 75, 22202222
Miller, JE. (2013). Principle of Immunoglobulin and T Cell Receptor Gene Rearrangement. In Cheng, L., Zhang, D., Eble,
JN. (Eds), Molecular Genetic Pathology (2nd Ed., sections 30.2.7.13 and 30.2.7.18). New York, USA: Springer
Science & Business Media.
JJM van Dongen et al., (2003). Design and standardization of PCR primers and protocols for detection of clonal
immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of the
BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 17, 2257–2317.
LymphoTrack Dx Software – MiSeq Package Instructions for Use (95000009)
https://www.beckmancoulter.com
http://www.illumina.com
http://www.invitrogen.com
http://www.kapabiosystems.com
http://www.thermofisher.com

17. Symbols
The following symbols are used in labeling for Invivoscribe NGS diagnostic products.
Catalog Number

Expiration Date

Reagent Volume

Authorized Representative
European Community

Lot Number

Consult Instructions for Use

Storage Conditions

For In Vitro Diagnostic Use

Unique Device Identifier

Manufacturer

UK Conformity Assessed

UK Responsible Person

Swiss Authorized Representative

European Conformity

in

the

Software Application

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18. 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 EP 2418287A3 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). Anynecessary license to practice amplification methods or to use reagents, amplification enzymes or equipment covered bythird party patents is the responsibility of the user and no such license is granted by Invivoscribe, Inc., expressly or by
implication.
©2024 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.
Illumina® and MiSeqTM are registered trademarks of Illumina, Inc.
Beckman Coulter ®, Agencourt®, AMPure® and SPRIPlate® are registered trademarks of Beckman Coulter, Inc.
Roche® is a registered trademark and EagleTaq™ is a trademark of Roche.
Veriti®, Sybr®, Ambion®, Applied BioSystems® and Life Technologies® are registered trademarks of Thermo Fisher Scientificand its subsidiaries.
KAPA™ is a trademark of Kapa Biosystems.
Microsoft®, Windows® and Excel® are registered trademarks of Microsoft Corporation.

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19. LymphoTrack Dx TRG Assay - MiSeq: Single Page Guide
19.1.

Using gloved hands, remove the Master Mixes from the freezer. Allow the tubes to thaw; then gently vortex to mix.

19.2.

In a containment hood or dead air box, pipette 45 L of Master Mix into individual wells of a PCR plate. One well foreach Master Mix and one Master Mix per sample, positive, negative or no template controls.

19.3.

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

19.4.

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

19.5.

Add 5 L of molecular biology grade water to the well containing the respective Master Mix for no template control
and pipette up and down 5-10 times to mix.

19.6.

Amplify target DNA using the following 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

19.7.

Remove the amplification plate from the thermal cycler.

19.8.

Purify the PCR products using the Agencourt AMPure XP PCR Purification system. Add 50 μL of particles to each 50 µL
reaction; elute DNA in 25 µL eluate.

19.9.

Quantify amplicons using the KAPA library quantification kit according to the kit instructions. Dilute amplicons 1:4,000
before proceeding to qPCR.

19.10.

Pool equal amounts of amplicons from samples (do not include the no template control), dilute 1:1,000 and quantify
the library using the KAPA library quantification kit.

19.11.

Denature and dilute the library to 12 pM for MiSeq reagent kit v2 and 12 - 20 pM for MiSeq reagent kit v3 when
multiplexing (MCS v2.6 or later).

19.12.

Load 600 μL of denatured and diluted library to the MiSeq Reagent Cartridge.

19.13.

Set up a MiSeq sample sheet, and upload the sample sheet to the instrument (if necessary).

19.14.

Start the MiSeq run.

19.15.

Analyze and visualize the acquired data using the associated LymphoTrack Dx Software – MiSeq package.

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20. Appendix A: Building a Sequencing Library with Multiple NGS Targets
When running multiple targets using different LymphoTrack Dx Assay - MiSeq in parallel, it is important to note theprocedural differences between each assay. For example, the IGHV Leader assay utilizes 32 PCR cycles and should be placedon a separate thermal cycling run than other LymphoTrack Dx Assays which utilize only 29 PCR cycles. Table 16, below,
summarizes these procedural differences. For full instructions, please see the Instructions For Use for the respective
LymphoTrack Dx Assay – MiSeq.
Table 16 LymphoTrack Dx Assay procedural differences

LymphoTrack Dx Assay - MiSeq

Procedure
Step

Description

7.4.1

IGHV
Leader

IGH FR1

IGH FR2

IGH FR3

IGK

TRG

TRB

Number of PCR Cycles

32

29

29

29

29

29

29

7.5.1

Volume of AMPure XP Reagent(Ratio)50 µL(1:1 ratio)

50 µL(1:1 ratio)

50 µL(1:1 ratio)

50 µL(1:1 ratio)

50 µL(1:1 ratio)

50 µL(1:1 ratio)

35 µL(0.7:1 ratio)

7.6.4

Contamination Check
ΔCt Value =Ct (NTC) – Ct (Control)

ΔCt ≥ 4.0

ΔCt ≥ 4.0

ΔCt ≥ 4.0

ΔCt ≥ 4.0

ΔCt ≥ 4.0

ΔCt ≥ 4.0

ΔCt ≥ 3.0

7.6.5
and
7.9.1

A
(Average Fragment Length)660 bp450 bp390 bp260 bp410 bp300 bp400 bpLoading Concentration

12 - 20 pM

12 pM

12 pM

12 pM

8 pM

12 pM

12 pM

MiSeq Reagent Kit for SingleTarget Sequencing*v3 (600)

v2 (500)

v2 (500)v2 (300)
v2 (500)

v2 (500)v2 (300)
v2 (500)

v2 (500)

Sample Sheet Settings for:
Cycles Read 1*
Cycles Read 2*

301

251

251

151

251

151

251

7.10.7

7.12*Note:

MiSeq v2 chemistry has been validated for these single-target assays. MiSeq v3 chemistry has been validated for IGHV Leader and assay multiplexing.

Two or more sequencing libraries generated from the same LymphoTrack gene target master mixes (e.g., two TRG
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. Please refer to the following
table to determine the cycle settings and Illumina MiSeq Reagent kits to be used with different combinations of targets. It is
recommended to use MiSeq reagent kit v3 when sequencing all seven targets together in order to achieve enough reads per
sample.
Table 17 Cycle Settings and Reagent Kits for a Multi-Targeted MiSeq Run

Multiplexing Targets

Sample Sheet Settings

MiSeq Reagent Kit

Loading
Concentration

Catalog #Only IGH FR3 and TRG together

151 cycles Read 1
151 cycles Read 2

v2 kit (300 cycle)orv2 kit (500 cycle)

12 pM (v2)or
20 pM (v3)MS-102-2002
orMS-102-2003

Any combination of these targets
together:
IGH FR1, IGH FR2, IGH FR3,
IGK, TRB and TRG

251 cycles Read 1
251 cycles Read 2

v2 kit (500 cycle)
up to 4 targetsorv3 kit (600 cycle)

12 pM (v2)or
20 pM (v3)MS-102-2003
orMS-102-3003

When combining any of the assays
with IGHV Leader

301 cycles Read 1
301 cycles Read 2

v3 kit (600 cycle)

20 pM (v3)MS-102-3003

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20.1.

Determine the concentration of each individual library (e.g., IGHV Leader, IGH FR1, IGH FR2, IGH FR3, IGK, TRB and
TRG).

20.2.

Determine the amount of each library to be denatured.
In the table below, Case A, B, C, D, E and F are different examples of assay multiplexing (e.g., Case A is a multiplex of
IGHV Leader, IGH FR1, IGH FR2, IGH FR3, IGK, TRB and TRG). T, U, V, W, X, Y and Z are volumes in µL.
n=number of targets being loaded to a MiSeq cartridgeT=

40 fmole/[n x IGHV Leader library concentration (nM)]U=

40 fmole/[n x IGH FR1 library concentration (nM)]V=

40 fmole/[n x IGH FR2 library concentration (nM)]W=

40 fmole/[n x IGH FR3 library concentration (nM)]X=

40 fmole/[n x IGK library concentration (nM)]Y=

40 fmole/[n x TRG library concentration (nM)]Z=

40 fmole/[n x TRB library concentration (nM)]

Note: The 40 fmole value corresponds to the 20 µL of 2 nM at the end of step 20.3.
Table 18. Calculation of Individual Library Inputs to Generate a Multi-Targeted Sequencing Library for the MiSeq Run

Library
Assay Name

Individual Library Volume (µL)

Concentration

Case A

Case B

Case C

Case D

Case E

Case F

(nM)

n=7

n=6

n=5

n=4

n=3

n=2

IGHV Leader

2.3

T

2.5

2.9

3.5

4.3

IGH FR1

1.5

U

3.8

4.4

5.3

6.7

8.9

IGH FR2

4

V

1.4

1.7

2

2.5

3.3

IGH FR3

2.1

W

2.7

3.2

3.8

4.8

6.4

IGK

3.5

X

1.6

1.9

2.3

TRG

2.6

Y

2.2

2.6

TRB

2

20.3.

Z

2.9

T+U+V+W+X+Y+Z

17.1

16.7

5.7
7.7

16.9

18.3

18.6

13.4

Denature the combined libraries to 2 nM.
▪

Add reagents according to Table 19 based on the amount determined in the previous step.

If T+U+V+W+X+Y+Z >18, such as in case D and E from Table 18, mix the applicable libraries first, then add 18 µL to thedenaturing reaction as shown in the following table.
Table 19. Library Denaturation

Reagent

Volume (µL)

IGHV Leader LibraryT

IGH FR1 LibraryU

IGH FR2 LibraryV

IGH FR3 LibraryW

IGK LibraryX

TRG LibraryY

TRB LibraryZ

1N NaOH

2

10 mM Tris-HCl pH 8.0, 0.05% Tween 20
Total

LymphoTrack Dx TRG Assay – MiSeq

18 – (T+U+V+W+X+Y+Z)
20

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Vortex briefly to mix the solution, and then briefly centrifuge to ensure all the solution has settled to the bottom of the
tube. Incubate for 5 minutes at room temperature to denature the combined library DNA into single strands.
20.4.

Dilute the denatured library to 40 pM.
Add 980 μL of pre-chilled HT1 buffer (provided in MiSeq Reagent kit) to the tube containing the 20 μL denatured libraryDNA. Vortex briefly to mix and pulse centrifuge the sample.

20.5.

Prepare the denatured library for loading onto the MiSeq.
Dilute the library to 12 pM for MiSeq reagent kit v2 and 20 pM for MiSeq reagent kit v3 when multiplexing (MCS v2.6
or later) by following Table 20. Vortex briefly to mix and pulse centrifuge the sample.
Table 20. Preparation of the Combo Library for MiSeq Loading

VolumeReagent
12 pM

20 pM

40 pM library

300 µL500 µL

Chilled HT1 Buffer

700 µL500 µL

Total

1000 µL

1000 µL

20.6.

Load 600 µL of the combined denatured library from the previous step onto a MiSeq Reagent Cartridge.

20.7.

Set up a MiSeq sample sheet, and upload the sample sheet to the instrument (if necessary).

20.8.

Start the MiSeq run.

20.9.

Analyze and visualize the acquired data using the associated LymphoTrack Dx Software – MiSeq package.

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