Instructions for Use

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq®

TM

Identify and track B-cell immunoglobulin heavy chain (IGH) gene rearrangements using next-generation sequencing
with the Illumina® MiSeq.
This assay is for Research Use Only. Not for use in Diagnostic Procedures.
Schematic depiction of the IGH gene locus and the framework (FR) regions targeted:

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

Catalog #71210009
71210039
71210149
71210089
71210099
71210109
712101197121012971210139

Products

LymphoTrack IGH FR1 Assay Kit A – MiSeq
LymphoTrack IGH FR1 Assay Panel – MiSeq
LymphoTrack IGH FR1 Assay Panel B – MiSeq
LymphoTrack IGH FR2 Assay Kit A – MiSeq
LymphoTrack IGH FR2 Assay Panel – MiSeq
LymphoTrack IGH FR3 Assay Kit A – MiSeq
LymphoTrack IGH FR3 Assay Panel – MiSeq
LymphoTrack IGH FR1/2/3 Assay Kit A – MiSeq
LymphoTrack IGH FR1/2/3 Assay Panel – MiSeq

Quantity

8 indices – 5 reactions each
24 indices – 5 reactions each
24 indices – 5 reactions each
8 indices – 5 reactions each
24 indices – 5 reactions each
8 indices – 5 reactions each
24 indices – 5 reactions each
8 indices per FR region– 5 reactions each
24 indices per FR region – 5 reactions eachPage 2 of 3

Table of Contents
1.

ASSAY 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.
3.6.
3.7.

4.

Thermal cycler ........................................................................................................................................................................................ 14
Magnetic Stand ....................................................................................................................................................................................... 14
Real-time PCR Instrument....................................................................................................................................................................... 14
Illumina MiSeq Instrument ..................................................................................................................................................................... 14

SPECIMEN COLLECTION AND PREPARATION .................................................................................................................................................... 15
6.1.
6.2.
6.3.
6.4.

7.

Reagent Components ............................................................................................................................................................................... 7
Warnings and Precautions ...................................................................................................................................................................... 13
Storage and Handling.............................................................................................................................................................................. 13

INSTRUMENTS ........................................................................................................................................................................................ 14
5.1.
5.2.
5.3.
5.4.

6.

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

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

5.

Background ............................................................................................................................................................................................... 3
Summary................................................................................................................................................................................................... 4

Precautions ............................................................................................................................................................................................. 15
Interfering Substances ............................................................................................................................................................................ 15
Specimen Requirements and Handling ................................................................................................................................................... 15
Sample Storage ....................................................................................................................................................................................... 15

ASSAY PROCEDURE .................................................................................................................................................................................. 16
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.................................................................................................................................................................................. 16
Materials Required (not provided) ......................................................................................................................................................... 16
Reagent Preparation ............................................................................................................................................................................... 17
Amplification........................................................................................................................................................................................... 17
AMPure XP Purification........................................................................................................................................................................... 18
Quantification of Amplicons ................................................................................................................................................................... 19
Pooling and Quantification of Library ..................................................................................................................................................... 20
Dilution of the Pooled Library ................................................................................................................................................................. 21
Setup qPCR for Library Quantification .................................................................................................................................................... 21
Preparation of the Library for the MiSeq Sequencing Run ..................................................................................................................... 21
MiSeq Flow Cell Loading ......................................................................................................................................................................... 22
MiSeq Sample Sheet Setup .................................................................................................................................................................... 22
MiSeq Run Start ...................................................................................................................................................................................... 24

8.

DATA ANALYSIS ...................................................................................................................................................................................... 24

9.

EXPECTED VALUES ................................................................................................................................................................................... 25

10.

LIMITATIONS OF PROCEDURE ...................................................................................................................................................................... 25

11.

SAMPLE DATA ........................................................................................................................................................................................ 25

12.

TROUBLESHOOTING GUIDE ........................................................................................................................................................................ 27

13.

TECHNICAL AND CUSTOMER SERVICE ............................................................................................................................................................ 28

14.

REFERENCES .......................................................................................................................................................................................... 28

15.

SYMBOLS .............................................................................................................................................................................................. 28

16.

LEGAL NOTICE ........................................................................................................................................................................................ 29

17.

LYMPHOTRACK IGH FR1/FR2/FR3 ASSAY - MISEQ: SINGLE PAGE GUIDE ........................................................................................................... 30

18.

APPENDIX A: BUILDING A SEQUENCING LIBRARY WITH MULTIPLE NGS TARGETS ..................................................................................................... 31

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 3 of 4

1. Assay Use
The LymphoTrack IGH FR1 Assay – MiSeq 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 frequency
distribution of VH region and JH region segment utilization, and the degree of somatic hypermutation (SHM) of rearranged
genes using the Illumina MiSeq platform.
The LymphoTrack IGH FR2 Assay – MiSeq 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 thefrequency distribution of VH region and JH region segment utilization using the Illumina MiSeq platform.
The LymphoTrack IGH FR3 Assay – MiSeq 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 thefrequency distribution of VH region and JH region segment utilization using the Illumina MiSeq platform.
Data from all three framework regions is needed to determine evidence of clonality for a sample.

1.
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
non-functional variable (VH) gene segments, 27 functional diversity (DH) gene segments, and 6 functional joining (JH)
gene segments 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 the other somatic cells in the body; during development, the antigen receptor
genes in lymphoid cells undergo somatic gene rearrangement.1 For example, during B-cell development, genes
encoding the IGH 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 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 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 thegermline VH gene sequence, whereas less than 2% difference is considered evidence of no somatic hypermutation.
The status of somatic hypermutation for clone(s) has clinical relevance for B-CLL, as there is a clear distinction in the
median survival of patients with and without somatic hypermutation. 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
developed by Alexander Morley3, and are considered the current gold-standard method. These assays identify
clonality on the basis of over-representation of amplified VH–DH–JH (or incomplete DH–JH products) generearrangement following their separation using gel or capillary electrophoresis. Though sensitive and suitable fortesting 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 clonal populations in subsequent analyses. This second limitation can be ofparticular importance, as once the unique clone-specific DNA sequence is identified, the sequence can be used insubsequent tests to track and follow the clonal cell populations.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 4 of 5

2.2.

Summary
The LymphoTrack IGH (FR1, FR2 and FR3) Assays for MiSeq (sold separately and as a set) represent a significant
improvement over existing clonality assays using fragment analysis as they efficiently detect IGH generearrangements, and at the same time, identify the DNA sequence specific for each clonal gene rearrangement.
Therefore, these assays have three important and complementary uses: they provide critical information on theexistence of clonality, identify sequence information required to track those clones in subsequent samples andprovide 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 sitesof the involved VH gene segments can impede DNA amplification.4
Primers included in the master mixes are designed with Illumina adapters and up to 48 different indices. This method
allows for a one-step PCR, and pooling of amplicons from several different samples and targets (generated with other
LymphoTrack Assays for the Illumina MiSeq instrument) onto one MiSeq flow cell, allowing for up to 48 samples pertarget to be analyzed in parallel in a single run.
The associated RUO LymphoTrack Software for MiSeq 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 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 the B and T lymphocytes arise as a result of thesegenetic 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 IGH 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 the Agencourt® AMPure®
XP system. This method utilizes solid-phase reversible immobilization (SPRI) paramagnetic bead technology for highthroughput 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 amore purified PCR product for downstream analysis and amplicon quantification.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 5 of 6

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
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 as each RT-base is added, and then cleaved to allow
incorporation of the next base.

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.
Each of these 48 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 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: Building a Sequencing Library with
Multiple NGS Targets (section 18).
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 20-25 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. Illumina also manufactures other flow cells that utilize the same sequencing chemistry, but generate
fewer reads. When using these alternative flow cells one must consider that fewer total reads either means lessdepth per sample or fewer samples can be run on the flow cell to achieve the same depth per sample.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 6 of 7

3.6.

IGHV Somatic Hypermutation (SHM) Evaluation
For evaluation of the somatic hypermutation rate of the IGHV region, the LymphoTrack IGH FR1 Assay Master Mixes
can be used; however, this only targets a portion of the IGHV region as the sequence upstream of the primer bindingsite 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 germlinereference genes, a prediction of whether the protein translation would be in or out of frame, a prediction of whether
mutations or gene rearrangements result in a pre-mature stop codon, and the percentage of VH gene coverage forthe region targeted by the assay.

3.7.

Minimal Residual Disease Evaluation
The correlation of MRD status with overall survival rate was initially demonstrated for subjects with chronic
lymphocytic leukemia using multi-parameter flow analysis.6 However, flow-based methods have proven difficult tostandardize outside individual centers. Therefore, flow cytometry is not a suitable technology for international
standardization, as it is difficult to compare results from different centers. Fortunately, a number of investigators
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.7,8
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 (280364) and the MRD Technical Bulletin (M-0031) 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 and confidence.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 7 of 8

4. Reagents
4.1.

Reagent Components

Table 1. Available Kits

Catalog#

Description# of Indexed Master Mixes

Total Reactions

71210009

LymphoTrack IGH FR1 Assay Kit A – MiSeq

8 indices – 5 sequencing runs each4071210039

LymphoTrack IGH FR1 Assay Panel – MiSeq

24 indices – 5 sequencing runs each12071210149

LymphoTrack IGH FR1 Assay Panel B – MiSeq

24 indices – 5 sequencing runs each12071210089

LymphoTrack IGH FR2 Assay Kit A – MiSeq

8 indices – 5 sequencing runs each4071210099

LymphoTrack IGH FR2 Assay Panel – MiSeq

24 indices – 5 sequencing runs each12071210109

LymphoTrack IGH FR3 Assay Kit A – MiSeq

8 indices – 5 sequencing runs each4071210119

LymphoTrack IGH FR3 Assay Panel – MiSeq

24 indices – 5 sequencing runs each12071210129

LymphoTrack IGH FR1/2/3 Assay Kit A – MiSeq(8+8+8) indices – 5 sequencing runs each

40+40+40

71210139

LymphoTrack IGH FR1/2/3 Assay Panel – MiSeq(24+24+24) indices – 5 sequencing runs each

120+120+120

Table 2. LymphoTrack IGH FR1 Assay Kit A – MiSeq Components (Reagents

Master Mixesǂ

Positive Control DNA

Reagent Components

71210009)

Unit
Quantity# of Units

IGH FR1 MiSeq 01

1

IGH FR1 MiSeq 02

1

IGH FR1 MiSeq 03

1

IGH FR1 MiSeq 04
IGH FR1 MiSeq 05

250 µL

1

1

IGH FR1 MiSeq 07

1

IGH FR1 MiSeq 08

1

IGH POS (+)
40880009)

45 µL

Notes

N/A

1

IGH FR1 MiSeq 06(

Storage
Temperature

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

1or

Negative Control DNA(

NGS NEG (-)
40920018)

45 µL

1

Tonsil DNA, highest sequencefrequency can vary between lotsNoteǂ: There are no preservatives used in the manufacturing of these kits.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 8 of 9

Table 3. LymphoTrack IGH FR1 Assay Panel – MiSeq Components (Reagents

Master Mixesǂ

Positive Control DNA
Negative Control DNA

Reagent Components

71210039)

Unit
Quantity# of Units1

IGH FR1 MiSeq 01

1

IGH FR1 MiSeq 02

1

IGH FR1 MiSeq 03

1

IGH FR1 MiSeq 04

1

IGH FR1 MiSeq 05

1

IGH FR1 MiSeq 06

1

IGH FR1 MiSeq 07

1

IGH FR1 MiSeq 08

1

IGH FR1 MiSeq 09

1

IGH FR1 MiSeq 10

1

IGH FR1 MiSeq 11

1

IGH FR1 MiSeq 12
IGH FR1 MiSeq 13

250 µL

1

1

IGH FR1 MiSeq 15

1

IGH FR1 MiSeq 16

1

IGH FR1 MiSeq 18

1

IGH FR1 MiSeq 19

1

IGH FR1 MiSeq 20

1

IGH FR1 MiSeq 21

1

IGH FR1 MiSeq 22

1

IGH FR1 MiSeq 23

1

IGH FR1 MiSeq 25

1

IGH FR1 MiSeq 27

1

IGH POS (+)
40880009)

45 µL

NGS NEG (-)(
40920018)

45 µL

Notes

N/A

1

IGH FR1 MiSeq 14(

Storage
Temperature3
or3

IGH V1-46_03 / IGH J4_02 DNA
diluted in tonsil DNA
Tonsil DNA, highest sequencefrequency can vary between lotsNoteǂ: There are no preservatives used in the manufacturing of these kits.
Note1: Indices 17, 24, and 26 are not used in this kit.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 9 of 10
Table 4. LymphoTrack IGH FR1 Assay Panel B - MiSeq Components (Reagents

Master Mixesǂ

Positive Control DNA
Negative Control DNA

Reagent Components

Unit
Quantity

71210149)# of Units

IGH FR1 MiSeq 17

1

IGH FR1 MiSeq 24

1

IGH FR1 MiSeq 26

1

IGH FR1 MiSeq 28

1

IGH FR1 MiSeq 29

1

IGH FR1 MiSeq 30

1

IGH FR1 MiSeq 31

1

IGH FR1 MiSeq 32

1

IGH FR1 MiSeq 33

1

IGH FR1 MiSeq 34

1

IGH FR1 MiSeq 35

1

IGH FR1 MiSeq 36
IGH FR1 MiSeq 37

250 µL

1
1

IGH FR1 MiSeq 39

1

IGH FR1 MiSeq 40

1

IGH FR1 MiSeq 41

1

IGH FR1 MiSeq 42

1

IGH FR1 MiSeq 43

1

IGH FR1 MiSeq 44

1

IGH FR1 MiSeq 45

1

IGH FR1 MiSeq 46

1

IGH FR1 MiSeq 47

1

IGH FR1 MiSeq 48

1

IGH POS (+)
40880009)

45 µL

3

NGS NEG (-)(
40920018)

45 µL

3

Notes

N/A

1

IGH FR1 MiSeq 38(

Storage Temperatureor

IGH V1-46_03 / IGH J4_02 DNA
diluted in tonsil DNA
Tonsil DNA, highest sequencefrequency can vary between lotsNoteǂ: There are no preservatives used in the manufacturing of these kits.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 10 of 11
Table 5. LymphoTrack IGH FR2 Assay Kit Components

Reagents

Master Mixesǂ

71210089 #of Units

71210099 #of Units

IGH FR2 MiSeq 01

1

1

IGH FR2 MiSeq 02

1

1

IGH FR2 MiSeq 03

1

1

IGH FR2 MiSeq 04

1

1

IGH FR2 MiSeq 05

1

1

IGH FR2 MiSeq 06

1

1

IGH FR2 MiSeq 07

1

1

IGH FR2 MiSeq 08

1

1

IGH FR2 MiSeq 09

01

IGH FR2 MiSeq 1001

IGH FR2 MiSeq 11

0

1

0

1

0

1

IGH FR2 MiSeq 14

01

IGH FR2 MiSeq 15

01

IGH FR2 MiSeq 1601

IGH FR2 MiSeq 18

01

IGH FR2 MiSeq 19

01

IGH FR2 MiSeq 20

01

IGH FR2 MiSeq 21

01

IGH FR2 MiSeq 2201

IGH FR2 MiSeq 23

01

IGH FR2 MiSeq 25

01

IGH FR2 MiSeq 27

0

1

1

3

Reagent Components

IGH FR2 MiSeq 12
IGH FR2 MiSeq 13

Positive Control DNA

IGH POS (+)(
40880009)

Negative Control DNA

NGS NEG (-)(
40920018)

Unit
Quantity

250 µL

45 µL

Storage
Temperature

N/A

or
45 µL

1

3

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: Indices 17, 24, and 26 are not used in these kits.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 11 of 12

Table 6. LymphoTrack IGH FR3 Assay Kit Components

Reagents

Master Mixesǂ

71210109 #of Units

71210119 #of Units

IGH FR3 MiSeq 01

1

1

IGH FR3 MiSeq 02

1

1

IGH FR3 MiSeq 03

1

1

IGH FR3 MiSeq 04

1

1

IGH FR3 MiSeq 05

1

1

IGH FR3 MiSeq 06

1

1

IGH FR3 MiSeq 07

1

1

IGH FR3 MiSeq 08

1

1

IGH FR3 MiSeq 09

01

IGH FR3 MiSeq 1001

IGH FR3 MiSeq 11

0

1

0

1

0

1

IGH FR3 MiSeq 14

01

IGH FR3 MiSeq 15

01

IGH FR3 MiSeq 1601

IGH FR3 MiSeq 18

01

IGH FR3 MiSeq 19

01

IGH FR3 MiSeq 20

01

IGH FR3 MiSeq 21

01

IGH FR3 MiSeq 2201

IGH FR3 MiSeq 23

01

IGH FR3 MiSeq 25

01

IGH FR3 MiSeq 27

0

1

1

3

Reagent Components

IGH FR3 MiSeq 12
IGH FR3 MiSeq 13

Positive Control DNA

IGH POS (+)(
40880009)

Negative Control DNA

NGS NEG (-)(
40920018)

Unit
Quantity

250 µL

45 µL

Storage
Temperature

N/A

or
45 µL

1

3

Notes

IGH V1-46_03 / IGH
J4_02 DNA diluted in
tonsil DNA
Tonsil DNA, highest
sequence frequency
can vary between lotsNoteǂ: There are no preservatives used in the manufacturing of these kits.
Note1: Indices 17, 24, and 26 are not used in these kits.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 12 of 13

Table 7. LymphoTrack IGH FR1/2/3 Assay Kit Components

71210129# of Units per FR

71210139# of Units per FRǂ

IGH FR3 MiSeq 01

1

1

IGH FR2 MiSeq 02

IGH FR3 MiSeq 02

1

1

IGH FR1 MiSeq 03

IGH FR2 MiSeq 03

IGH FR3 MiSeq 03

1

1

IGH FR1 MiSeq 04

IGH FR2 MiSeq 04

IGH FR3 MiSeq 04

1

1

IGH FR1 MiSeq 05

IGH FR2 MiSeq 05

IGH FR3 MiSeq 05

1

1

IGH FR1 MiSeq 06

IGH FR2 MiSeq 06

IGH FR3 MiSeq 06

1

1

IGH FR1 MiSeq 07

IGH FR2 MiSeq 07

IGH FR3 MiSeq 07

1

1

IGH FR1 MiSeq 08

IGH FR2 MiSeq 08

IGH FR3 MiSeq 08

1

1

IGH FR1 MiSeq 09

IGH FR2 MiSeq 09

IGH FR3 MiSeq 09

01

IGH FR1 MiSeq 10

IGH FR2 MiSeq 10

IGH FR3 MiSeq 1001

IGH FR1 MiSeq 11

IGH FR2 MiSeq 11

IGH FR3 MiSeq 11

01

IGH FR1 MiSeq 12

IGH FR2 MiSeq 12

IGH FR3 MiSeq 1201

IGH FR1 MiSeq 13

IGH FR2 MiSeq 13

IGH FR3 MiSeq 13

01

IGH FR1 MiSeq 14

IGH FR2 MiSeq 14

IGH FR3 MiSeq 14

01

IGH FR1 MiSeq 15

IGH FR2 MiSeq 15

IGH FR3 MiSeq 15

01

IGH FR1 MiSeq 16

IGH FR2 MiSeq 16

IGH FR3 MiSeq 1601

IGH FR1 MiSeq 18

IGH FR2 MiSeq 18

IGH FR3 MiSeq 18

01

IGH FR1 MiSeq 19

IGH FR2 MiSeq 19

IGH FR3 MiSeq 19

01

IGH FR1 MiSeq 20

IGH FR2 MiSeq 20

IGH FR3 MiSeq 20

01

IGH FR1 MiSeq 21

IGH FR2 MiSeq 21

IGH FR3 MiSeq 21

01

IGH FR1 MiSeq 22

IGH FR2 MiSeq 22

IGH FR3 MiSeq 2201

IGH FR1 MiSeq 23

IGH FR2 MiSeq 23

IGH FR3 MiSeq 23

01

IGH FR1 MiSeq 25

IGH FR2 MiSeq 25

IGH FR3 MiSeq 25

01

IGH FR1 MiSeq 27

IGH FR2 MiSeq 27

IGH FR3 MiSeq 27

0

1

FR1 Master Mixes1

FR2 Master Mixes1

FR3 Master Mixes1

IGH FR1 MiSeq 01

IGH FR2 MiSeq 01

IGH FR1 MiSeq 02

(
(

Unit
Quantity

250 µL

Control DNA*

Notes

Unit
Quantity

71210129# of Units

71210139# of Units

IGH POS (+)
40880009)

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

45 µL

2

6

NGS NEG (-)
40920018)

Tonsil DNA, highest sequencefrequency can vary between lots

45 µL

2

6

Note1: The storage temperature for master mixes is -65ºC to -85ºC.
Note*: The storage temperature for controls is 2ºC to 8ºC or -65ºC to -85ºC.
Note: There are no preservatives used in the manufacturing of these kits.
Noteǂ: Indices 17, 24, and 26 are not used in these kits.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

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Page 13 of 14

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.

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 kitsbeyond 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 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 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 plasticware whenever possible to avoid contamination.

Storage and HandlingIf the assay is not being used immediately, store at -65ºC to -85ºC.
The optimum storage temperature for DNA controls is 2ºC to 8ºC, but DNA can also be stored at -65ºC to -85ºC.
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 ofcycles 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.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

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Page 14 of 15

5. Instruments
The specific instruments listed in below are recommended based on the methods used to verify the assay.
5.1.

Thermal cyclerUse or Function: Amplification of DNA samples
Suggested Instrument: Veriti™ 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 Real-Time Instrument or equivalent
Performance Characteristics and Specifications:
o



5.4.

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

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

Follow manufacturer’s installation, operation, calibration, and maintenance procedures.
See section 7.10 MiSeq Flow Cell Loading, 7.12 MiSeq Sample Sheet Setup, and 7.13 MiSeq Run Start for MiSeq
parameters.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

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Page 15 of 16

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 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.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

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7. Assay Procedure
7.1.

Materials Provided
See Tables 2 – 7 for materials provided in each kit.

7.2.

Materials Required (not provided)

Table 8. Materials Required (not provided)

Required or Recommended
Reagents/Suppliers

Reagent/Material

DNA Polymerase

Roche:• EagleTaq™ DNA PolymeraseorInvivoscribe, Inc.
• 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

A32782

KK4824

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

MS-102-2003
orMS-102-3003

When only sequencing IGH FR3:
• MiSeq Reagent v2 kit (300 cycles)or• MiSeq Reagent v2 kit (500 cycles)

MS-102-2002
orMS-102-2003••

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

N/A

N/A

N/A

Dilution Buffer A

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

N/AStandard flow cell

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

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Page 17 of 18

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 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 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 (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 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 and place in the PCR thermal cycler.
Table 9. Reaction Setup

Reagent

7.4.

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 10.

Please note the LymphoTrack IGHV Leader SHM Assay – MiSeq PCR program has different cycling conditions than the other LymphoTrack
Assays.
Table 10. PCR Program

7.4.2.

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.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

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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 verification 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.
AMPure XP reagent volume added to 50 µL of PCR product by assay:
TRB: 35 µL
IGHV Leader, IGH FR1, IGH FR2, IGH FR3, IGK and TRG: 50 µL

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 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.
Incubate 10 minutes at room temperature.
The color of the mixture should appear homogenous after mixing.

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 the nextstep.
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 MiSeq Master Mixes respectively.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

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Page 19 of 20

Figure 1: Example ofa purification result
for amplicons fromthe IGH FR1 MiSeq
Master Mixes. The
image was generated
by running unpurified
and purified productson the LabChip GX.

Figure 2: Example of apurification result for
amplicons from the IGH
FR2 MiSeq Master
Mixes. The image was
generated by running
unpurified and purified
products on the
LabChip GX.

Figure 3: Example ofa purification result
for amplicons fromthe IGH FR3 MiSeq
Master Mixes. The
image was generated
by running unpurified
and purified productson the LabChip GX.

7.6.

Quantification of Amplicons
The following steps were performed to quantify purified PCR amplicons generated from samples, positive, negative,
and no template controls using the KAPA library quantification kit (KAPA Biosystems) during assay verification.
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:
Step B:

2 μL of purified amplicon eluate + 198 µL of Dilution Buffer A.
Mix well by pipetting up and down 10 times.
5 μL from Step A + 195 μL of Dilution Buffer A.
Mix well by pipetting up and down 10 times.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

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7.6.2.

Set up a qPCR run for amplicon quantification following Table 11 for each reaction (please refer to the KAPA
library quantification kit instructions for more details):
Table 11. 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 12 for the thermal program for qPCR.
Table 12. 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 ∆Ct < 4.0 for either control, see section 12
Troubleshooting Guide for further instructions.
Note: ΔCt for the TRB assay is set different than the other LymphoTrack assays:
TRB ΔCt ≥ 3.0
IGHV Leader, IGH FR1, IGH FR2, IGH FR3, IGK and TRG ΔCt ≥ 4.0

7.6.5.

Use the data from the qPCR run to determine the concentration of amplicon for each sample using thefollowing equation:
𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑜𝑜𝑜𝑜 𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢 𝑎𝑎𝑎𝑎𝑎𝑎𝑙𝑙𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 (𝑛𝑛𝑛𝑛) =452 × 𝐴𝐴𝐴𝐴𝐴𝐴 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶. (𝑝𝑝𝑝𝑝)𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑏𝑏𝑏𝑏 𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞
×4
A

A= The average fragment length of amplicons generated by the assay:

The average fragment length of amplicons generated using the IGH FR1 Assay is 450 bp (A = 450).
The average fragment length of amplicons generated using the IGH FR2 Assay is 390 bp (A = 390).
The average fragment length of amplicons generated using the IGH FR3 Assay is 260 bp (A = 260).
(Note: Sequence length includes additional nucleotides that are necessary for sequencing).

The average fragment length of KAPA Illumina DNA Standard is 452 bp.

7.7.

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.
It is recommended that a separate pool is created for each LymphoTrack Assay and corresponding target (IGH FR1,
FR2 or FR3). After final quantification of a pooled library for each target according to section 7.6, LymphoTrack
Assays can be sequenced individually continuing with the instructions provided in section 7.8, or can be multiplexed
together according to the instructions provided in Appendix A (section 18).

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

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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 for 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.
20 μL from Step A + 180 μL of Dilution Buffer A.
Mix well by pipetting up and down 10 times.

Step B:
7.9.

Setup qPCR for Library Quantification
Refer to Table 11 for qPCR setup and Table 12 for the thermal cycler program.
7.9.1.

Determine the concentration of the pooled library from the qPCR results.
𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑜𝑜𝑜𝑜 𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 (𝑛𝑛𝑛𝑛) =452 × 𝐴𝐴𝐴𝐴𝐴𝐴 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶. (𝑝𝑝𝑝𝑝)𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑏𝑏𝑏𝑏 𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞
A

A= The average fragment length of amplicons generated by the assay:

The average fragment length of amplicons generated using the IGH FR1 Assay is 450 bp (A = 450).
The average fragment length of amplicons generated using the IGH FR2 Assay is 390 bp (A = 390).
The average fragment length of amplicons generated using the IGH FR3 Assay is 260 bp (A = 260).
(Note: Sequence length includes additional nucleotides that are necessary for sequencing).

The average fragment length of KAPA Illumina DNA Standard is 452 bp.

7.10.

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 12 - 20
pM for MiSeq reagent kit v3. For multiplexing amplicons from different LymphoTrack Assays for the MiSeq into asingle library, please refer to Appendix A: Building a Sequencing Library with Multiple NGS Targets.

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 13. Library Denaturation

Reagent

VolumeDiluted library

10 μL

0.2 N NaOH

10 μL

Total Volume

20 μL

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7.10.3.
7.10.4.

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 singlestranded DNA (ssDNA).
Add 980 μL of pre-chilled HT1 buffer (provided in MiSeq Reagent kits) to the tube containing the denatured
library DNA:
Table 14. 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:

For MiSeq Control Software (MCS v2.6 or later):
The concentration of the library DNA must be 12 pM for MiSeq reagent kit v2.
The concentration of the library DNA must be between 12 to 20 pM for MiSeq reagent kit v3.



If the diluted ssDNA library concentration is 40 pM (the initial concentration was 4 nM), dilute to thedesired MiSeq loading concentration using the examples shown in Table 15.
Table 15. Preparation of the Library for MiSeq Loading
7.10.8.
7.10.9.
7.11.

Final Concentrations

12 pM

15 pM

20 pM

Denatured library

300 μL375 μL500 μLHT1 Buffer

700 μL625 μL500 μL

Final NaOH Concentration (mM)

0.60 mM

0.75 mM

1.0 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 (12 to 20 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.

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]) with the LymphoTrack Kit A or
Panel versions, select TruSeq Nano DNA for the Library Prep Kit and TruSeq DNA Single Indexes Set A B for the Index
Kit. If using Panel B, use ScriptSeqV2 or ScriptSeq Complete for the Index Kit.

Characters in the sample name:•••Create a unique name and identifier when naming each sample. If duplicate samples are run, a similar name can be used (i.e.,
Sample1a and Sample1b).
Failure to provide unique names to samples sequenced on the same flow cell will result in only one sample being analyzed by the
LymphoTrack Software – MiSeq during the analysis process.
Use only alphanumeric and hyphen characters (A-Z, a-z, 0-9, -,) when preparing the Sample Sheet.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 23 of 24

Sample name when multiplexing:
Each index can only be listed in the Sample Sheet once; therefore, any necessary tracking information for samples sequenced withmultiple 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. Give this set of samples/targets a
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.
Examples of Sample ID naming 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_Leader (multiple samples sequenced with multiple assays using the same index)
 Pool12_A012 (Pool12 refers to all samples/targets sequenced with index A012 and tracked elsewhere)

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 for tracking samples.
Important!

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

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

Table 16. Sample Selection index names

LymphoTrack Assay – MiSeq
PCR Master Mix Index

Index
Sequence

LymphoTrack Assay
Kit or Panel

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

ScriptSeqV2 / ScriptSeq Complete(LRM “Index Kit”)

id01

ATCACG

Kit A / Panel

AR001

A001

id02

CGATGT

Kit A / Panel

AR002

A002

id03

TTAGGC

Kit A / Panel

AR003

A003

id04

TGACCA

Kit A / Panel

AR004

A004

id05

ACAGTG

Kit A / Panel

AR005

A005

id06

GCCAAT

Kit A / Panel

AR006

A006

id07

CAGATC

Kit A / Panel

AR007

A007

id08

ACTTGA

Kit A / Panel

AR008

A008

id09

GATCAG

Panel

AR009

A009

id10

TAGCTT

Panel

AR010

A010

id11

GGCTAC

Panel

AR011

A011

id12

CTTGTA

Panel

AR012

A012

id13

AGTCAA

Panel

AR013

A013

id14

AGTTCC

Panel

AR014

A014

id15

ATGTCA

Panel

AR015

A015

id16

CCGTCC

Panel

AR016

A016

id18

GTCCGC

Panel

AR018

A018

id19

GTGAAA

Panel

AR019

A019

id20

GTGGCC

Panel

AR020

A020

id21

GTTTCG

Panel

AR021

A021

id22

CGTACG

Panel

AR022

A022

id23

GAGTGG

Panel

AR023

A023

id25

ACTGAT

Panel

AR025

A025

id27

ATTCCT

Panel

AR027

A027

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 24 of 25
Table 16. Sample Selection index names

LymphoTrack Assay – MiSeq
PCR Master Mix Index

Index
Sequence

LymphoTrack Assay
Kit or Panel

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

ScriptSeqV2 / ScriptSeq Complete(LRM “Index Kit”)

id17

GTAGAG

Panel B

N/A

A017

id24

GGTAGC

Panel B

N/A

A024

id26

ATGAGC

Panel B

N/A

A026

id28

CAAAAG

Panel B

N/A

A028

id29

CAACTA

Panel B

N/A

A029

id30

CACCGG

Panel B

N/A

A030

id31

CACGAT

Panel B

N/A

A031

id32

CACTCA

Panel B

N/A

A032

id33

CAGGCG

Panel B

N/A

A033

id34

CATGGC

Panel B

N/A

A034

id35

CATTTT

Panel B

N/A

A035

id36

CCAACA

Panel B

N/A

A036

id37

CGGAAT

Panel B

N/A

A037

id38

CTAGCT

Panel B

N/A

A038

id39

CTATAC

Panel B

N/A

A039

id40

CTCAGA

Panel B

N/A

A040

id41

GACGAC

Panel B

N/A

A041

id42

TAATCG

Panel B

N/A

A042

id43

TACAGC

Panel B

N/A

A043

id44

TATAAT

Panel B

N/A

A044

id45

TCATTC

Panel B

N/A

A045

id46

TCCCGA

Panel B

N/A

A046

id47

TCGAAG

Panel B

N/A

A047

id48

TCGGCA

Panel B

N/A

A048

7.13.

MiSeq Run Start
Start the MiSeq run following the MiSeq Control Software Instructions. The approximate MiSeq run times areindicated in Table 17.

Table 17. MiSeq Run Times for LymphoTrack

MiSeq Reagent Kit
v2
v3

Read Length

MCS Version

Total MiSeq Run Time

2x151 bp (FR3 only)

2.6 or later~24 hours

2x251 bp

2.6 or later~ 39 hours

2x301 bp

2.6 or later~ 56 hours

8. Data Analysis
The LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq were designed to produce sequencing data that can be analyzed using
the RUO LymphoTrack Software – MiSeq package provided on the associated CD (75000009), which is included withthe assay purchase. This CD includes detailed instructions for installation and use of the software package.
Samples prepared with LymphoTrack IGH (FR1, FR2 and/or FR3) Assays can be easily processed into fully analyzed data using
the LymphoTrack Software - MiSeq.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 25 of 26

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

IGH Positive Control top % reads ≥ 2.5%
NGS Negative Control top % reads < 1.0%
IGH SHM Positive Control (40880008, can be purchased separately) top % reads ≥ 2.5%
MiSeq Run Validity*oooQ30 > 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, Q30 score may vary depending on sample quality. If Q30 falls below Illumina Q30 specification, check the index Q30 value fromthe LymphoTrack Report after data analysis by the LymphoTrack Software - MiSeq. If an index Q30 score on the LymphoTrack Report does
not meet the Illumina specification, consider that index to be invalid.)

10. Limitations of Procedure







These assays do 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 LymphoTrack IGH FR1 Assay – MiSeq only targets a portion of the IGHV region, as the sequence upstream of theprimer binding site will not be assessed. The Research Use Only LymphoTrack IGHV Leader Somatic Hypermutation
Assay for the Illumina MiSeq (71210059 or
71210069) can be purchased separately if complete IGHV
sequencing is required.
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.

11. Sample Data

Figure 4: This table,
generated via the
LymphoTrack Software MiSeq, shows the top 10
reads from the readsummary merged with the
top 500 reads; a read will
merge with another if they
are only 1 or 2 nucleotides(nts) different. Sequenceswere generated using theLymphoTrack IGH FR1Assay –MiSeq andanalyzed using theLymphoTrack Software –MiSeq (
75000009).

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 26 of 27

Figure 5: This table,
generated via the
LymphoTrack Software MiSeq, shows the top 10
reads from the readsummary merged withthe top 500 reads; a read
will merge with anotherif they are only 1 or 2
nucleotides (nts)different. Sequenceswere generated using
the LymphoTrack IGH
FR2 Assay –MiSeq andanalyzed using theLymphoTrack Software –MiSeq (
75000009).

Figure 6: This table,
generated via the
LymphoTrack Software MiSeq, shows the top 10
reads from the readsummary merged withthe top 500 reads; a read
will merge with another ifthey are only 1 or 2
nucleotides (nts)different. Sequenceswere generated using theLymphoTrack IGH FR3Assay –MiSeq andanalyzed using theLymphoTrack Software –MiSeq (
75000009).

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 27 of 28

12. Troubleshooting Guide
Table 18. 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

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)

Quantification of amplicons using the
KAPA library quantification kit

ΔCt < 4.0

Check the standard curve in qPCR
Check for contamination and repeat the KAPA
qPCR for all samples and controls.
If ∆Ct < 4.0 again, redo 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 and reagent preparation

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

Disk space low
Empty waste bottle

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

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 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

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, Q30 score may vary depending on
sample quality. If Q30 falls below Illumina Q30 specification, check the index Q30 value from the LymphoTrack Report after data analysis by the
LymphoTrack Software - MiSeq. If an index Q30 score on the LymphoTrack Report does not meet the Illumina specification, consider that index to beinvalid

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 28 of 29

13. Technical and Customer Service
Thank you for purchasing our LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq. We appreciate your business. We are
happy to assist you with understanding these assays, and will provide ongoing technical assistance Monday through Friday
to keep the assays performing efficiently in your laboratory.
Contact Information
Invivoscribe, Inc
10222 Barnes Canyon Road | Building 1 | San Diego | California 92121-2711 | USA
Phone: +1 858 224-6600 | Fax: +1 858 224-6601 | Business Hours: 7:00AM - 5:00 PM PST/PDT
Technical Service: support@invivoscribe.com | Customer Service: sales@invivoscribe.com | Website: www.invivoscribe.com

14. References
1.
2.
3.
4.
5.
6.
7.
8.







Tonegawa, S. (1983). Somatic Generation of Antibody Diversity. Nature 302, 575–581.
Ghia, P. et al. (2007). ERIC recommendations on IGHV gene mutational status 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.
Evans PA. et al. (2007). Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple
immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 21,
207–14.
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.
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 – MiSeq Package Instructions for Use (75000009)
https://www.beckmancoulter.com
http://www.illumina.com
http://www.invitrogen.com
http://www.kapabiosystems.com
http://www.thermofisher.com

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

Expiration Date

Reagent Volume

Authorized Representative in theEuropean Community

Lot Number

Manufacturer

Storage Conditions

Consult Instructions for Use

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024

Page 29 of 30

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). 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.
Verti®, 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.

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

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Page 30 of 31

17. LymphoTrack IGH FR1/FR2/FR3 Assay - MiSeq: Single Page Guide
17.1.

Using gloved hands, remove the Master Mixes from the freezer. Allow the 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. One well foreach Master Mix and one Master Mix per sample, positive, negative, or no template controls.

17.3.

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

17.4.

Add 5 µL of 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 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.

17.6.

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

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 50 μL of particles to each 50 µL
reaction; elute DNA in 25 µL eluate.

17.9.

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

17.10.

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.

17.11.

Denature and dilute the library to 12 pM for MiSeq reagent kit v2 and 12 - 20 pM for MiSeq reagent kit v3 (MCS 2.6 orlater).

17.12.

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

17.13.

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

17.14.

Start the MiSeq run.

17.15.

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

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

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18. Appendix A: Building a Sequencing Library with Multiple NGS Targets
This appendix contains instructions for multiplexing any combination of LymphoTrack IGHV Leader, IGH FR1, IGH FR2, IGH
FR3, IGK, TRB and TRG Assays together into a single sequencing library. Two or more sequencing libraries generated fromthe same LymphoTrack gene target master mixes (e.g., two TRG sequencing libraries, either from the same or different kitlots) 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 Table 19 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 7
targets together in order to achieve enough reads per sample.
Table 19. Cycle Settings and Reagent Kits for a Multi-Targeted MiSeq Run

Multiplexing Targets

Sample Sheet Settings

MiSeq Reagent Kit

Catalog #Only IGH FR3 and TRG together

151 cycles Read 1
151 cycles Read 2

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

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)

MS-102-2003
orMS-102-3003

When combining any of the assays together with:
IGHV Leader

301 cycles Read 1
301 cycles Read 2

v3 kit (600 cycle)

MS-102-3003

18.1.

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

18.2.

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 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 18.3.
Table 20. Calculation of Individual Library Inputs to Generate a Multi-Targeted Sequencing Library for the MiSeq Run

Individual Library Volume (µL)

Library

Case A
n=7

Case B
n=6

Case C
n=5

Case D
n=4

T

2.5

2.9

3.5

4.3

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

Z

2.9

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

17.1

Assay Name

Concentration (nM)

IGHV Leader

2.3

IGH FR1

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

16.7

Case E
n=3

Case F
n=2

5.7
7.7

16.9

For Research Use Only. Not for use in Diagnostic Procedures

18.3

18.6

13.4

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18.3.

Denature the combined libraries to 2 nM.


Add reagents according to Table 21 based on the amount determined from the previous step.

If T+U+V+W+X+Y+Z >18, such as in case D and E from Table 20, mix the applicable libraries first, then add 18 µL to
the denaturing reaction as shown in Table 21.
Table 21. 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

18 – (T+U+V+W+X+Y+Z)

Total

20

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.
18.4.

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.

18.5.

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 (MCS v2.6 or later) by following
Table 22. Vortex briefly to mix and pulse centrifuge the sample.
Table 22. Preparation of the Combo Library for MiSeq Loading

Reagent

18.6.

Volume
12 pM

20 pM

40 pM library

300 µL500 µL

Chilled HT1 Buffer

700 µL500 µL

Total

1000 µL

1000 µL

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

LymphoTrack IGH (FR1/FR2/FR3) Assays – MiSeq

For Research Use Only. Not for use in Diagnostic Procedures

280377 Rev. J | March 2024