Instructions for Use
LymphoTrack® IGHV Leader Somatic Hypermutation Assay – MiSeqTM
Identify and track clonal B cell immunoglobulin heavy chain (IGH) gene rearrangements using next-generation sequencing with
the Illumina® MiSeq and to assess the extent of somatic hypermutation (SHM) in the variable (V) heavy chain gene sequence inchronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) samples.
This assay is for Research Use Only. Not for use in Diagnostic Procedures.
Schematic depiction of the IGH gene locus:

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

Catalog #71210059
71210069

Products
LymphoTrack IGHV Leader Somatic Hypermutation Assay Kit A – MiSeq
LymphoTrack IGHV Leader Somatic Hypermutation Assay Panel – MiSeq

Quantity
8 indices – 5 reactions each
24 indices – 5 reactions eachPage 2 of 3

Table of Contents
1.

ASSAY USE ............................................................................................................................................................................................. 32.

SUMMARY AND EXPLANATION OF THE TEST ..................................................................................................................................................... 3
Background................................................................................................................................................................................................3
Summary ...................................................................................................................................................................................................3

3.

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

4.

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

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

5.

INSTRUMENTS.......................................................................................................................................................................................... 8
Thermal cycler ...........................................................................................................................................................................................8
Magnetic Stand .........................................................................................................................................................................................8
Real-time PCR Instrument .........................................................................................................................................................................8
Illumina MiSeq Instrument ........................................................................................................................................................................86.

SPECIMEN COLLECTION AND PREPARATION ...................................................................................................................................................... 9
Precautions ................................................................................................................................................................................................9Interfering Substances ...............................................................................................................................................................................9
Specimen Requirements and Handling......................................................................................................................................................9
Sample Storage..........................................................................................................................................................................................97.

ASSAY PROCEDURE ................................................................................................................................................................................. 10
Materials Provided ..................................................................................................................................................................................10
Materials Required (not provided) ..........................................................................................................................................................10
Reagent Preparation................................................................................................................................................................................10
Amplification ...........................................................................................................................................................................................11
AMPure XP Purification ...........................................................................................................................................................................11
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 ..........................................................................................................................................................................15
MiSeq Sample Sheet Setup (Windows 7 or Windows 10) .......................................................................................................................15
MiSeq Run Start .......................................................................................................................................................................................16

8.

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

9.

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

10.

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

11.

SAMPLE DATA ....................................................................................................................................................................................... 18

12.

TROUBLESHOOTING GUIDE ........................................................................................................................................................................ 19

13.

TECHNICAL AND CUSTOMER SERVICE............................................................................................................................................................ 20

14.

REFERENCES .......................................................................................................................................................................................... 20

15.

SYMBOLS ............................................................................................................................................................................................. 20

16.

LEGAL NOTICE ....................................................................................................................................................................................... 21

17.

LYMPHOTRACK IGHV LEADER SOMATIC HYPERMUTATION ASSAY - MISEQ: SINGLE PAGE GUIDE ................................................................................ 22

18.

APPENDIX A: BUILDING A SEQUENCING LIBRARY WITH MULTIPLE NGS TARGETS .................................................................................................... 23

LymphoTrack IGHV Leader Somatic Hypermutation Assay – MiSeq
For Research Use Only. Not for use in Diagnostic Procedures

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1. Assay Use
This Research Use Only assay targets the clonal VH segments of the IGH gene to identify clonal IGH VH–JH rearrangements, theassociated VH–JH region DNA sequences, provides the distribution frequency of VH region and JH region segment utilization,and the degree of somatic hypermutation of rearranged genes using the Illumina MiSeq platform.

2. Summary and Explanation of the Test
Background
The immunoglobulin heavy chain (IGH) gene locus on chromosome 14 (14q32.3) includes 46-52 functional and 30 nonfunctional variable (VH) gene segments, 27 functional diversity (DH) gene segments, and 6 functional joining (JH) genesegments spread over 1,250 kilobases. The VH gene segments contain three conserved framework (FR) and twovariable complementarity-determining regions (CDRs).
Lymphoid cells are different from 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 theIGH molecules are assembled from multiple polymorphic gene segments that undergo rearrangements and selection,
generating VH–DH–JH combinations that are unique in both length and sequence. Since leukemias and lymphomas
originate from the malignant transformation of individual lymphoid cells, an individual’s leukemic or lymphoma cells
generally share one or more cell-specific or “clonal” antigen receptor gene rearrangements. Therefore, tests that
detect IGH clonal rearrangements can be useful in the study of B- and T-cell malignancies.
In addition, immunoglobulin heavy chain variable region (IGHV) gene hypermutation status provides important
prognostic information for patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL).
The presence of IGHV somatic hypermutation (SHM) is defined as greater or equal to 2% difference from the germline
VH gene sequence, whereas less than 2% difference is considered evidence of no somatic hypermutation. The status ofsomatic hypermutation for clone(s) has clinical relevance for B-CLL, as there is a clear distinction in the median survivalof patients with and without somatic hypermutation. Hypermutation of the IGHV region is strongly predictive of agood prognosis while lack of mutation predicts a poor prognosis.2
Initially, clonal rearrangements were identified using Restriction Fragment, Southern Blot Hybridization (RF-SBH)
techniques. However, these tests proved cumbersome, labor-intensive, required large amounts of DNA and were notsuitable for analysis of many of the less diverse antigen receptor loci.
During the last several decades, the use of RF-SBH assays has been supplanted by PCR-based clonality tests developedby Alexander Morley3, and are considered the current gold-standard method. These assays identify clonality on thebasis of over-representation of amplified VH–DH–JH (or incomplete DH–JH products) gene rearrangement following theirseparation using gel or capillary electrophoresis. Though sensitive and suitable for testing small amounts of DNA, theseassays cannot readily differentiate between clonal populations and multiple rearrangements that might lie beneath asingle-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 of particular importance, as once the unique clonespecific DNA sequence is identified, the sequence can be used in subsequent tests to track and follow the clonal cell
populations.
SummaryThis LymphoTrack IGHV Leader Somatic Hypermutation Assay – MiSeq represents a significant improvement overexisting clonality assays using fragment analysis, as it efficiently detects the majority of IGH gene rearrangements using
a single multiplex master mix and identifies the DNA sequence specific for each clonal gene rearrangement. Therefore,
this assay has three important and complementary uses: it provides critical information in the existence of clonality,
identifies sequence information required to track those clones in subsequent samples and provides detailed sequence
information on the degree of SHM.
The single multiplex master mix targets the Leader (VHL) and the joining (J) gene regions of IGH. 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 and targets (generated with other LymphoTrack Assays
LymphoTrack IGHV Leader Somatic Hypermutation Assay – MiSeq
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for the Illumina MiSeq instrument) onto one MiSeq flow cell, allowing up to 24 samples per target to be analyzed inparallel 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 and a somatic hypermutation positive control 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).4

3. Principles of the Procedure
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 from anormal or polyclonal population is amplified using DNA primers that flank the V–J region, amplicons that are unique inboth sequence and length are generated, reflecting the heterogeneous population. In some cases, where lymphocyte
DNA is absent, no amplicons will be generated. Samples containing IGH clonal populations yield one or two prominent
amplified products of the same length and sequence which are detected with significant frequency within a diminishedpolyclonal background.
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
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 theconcentration of PCR amplicons allows equal amplicon representation in the final pooled library that is loaded onto the
MiSeq for sequencing.
Next-Generation Sequencing (NGS)
Sanger sequencing methods represent the most popular in a range of ‘first-generation’ nucleic acid sequencing
technologies. Newer methods, which leverage tremendously parallel sequencing approaches, are often referred to as
NGS. These technologies can use various combination strategies of template preparation, sequencing, imaging, andbioinformatics for genome alignment and assembly.

LymphoTrack IGHV Leader Somatic Hypermutation Assay – MiSeq
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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 are amplified toform local clonal colonies (bridge amplification). Four types of reversible terminator bases (RT-bases) are added, andthe 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 thenext base.
Multiplexing Amplicons
This product 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. Eachof 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 the bioinformatics
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 same index)
to be pooled together to generate the library to be sequenced on a single flow cell. An example would be to sequence
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 gene targets it isimportant to use the appropriate sequencing chemistry. The number of sequencing cycles must be sufficient tosequence 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 haveshorter amplicon sizes, use the MiSeq v2 (300 or 500 cycle) sequencing kit and adjust the cycle settings in the sample
sheet. For further instructions, please refer to Appendix A (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 of reads per
sample, divide the total number of reads for the flow cell by the number of samples that will be multiplexed. Illumina
also manufacturers other flow cells that utilize the same sequencing chemistry, but these generate fewer reads. Whenusing these alternative flow cells, consider that fewer total reads either means less depth per sample or fewer
samples can be run on the flow cell to achieve the same depth per sample.
IGHV Somatic Hypermutation (SHM) Evaluation
When analyzing the somatic hypermutation status of samples, the bioinformatics software will provide the mutation
rate based upon the percent mismatch of the clonal amplicons as compared to germline reference genes, a predictionof whether the protein translation would be in or out of frame, a prediction of whether mutations or generearrangements result in a pre-mature stop codon, and the percentage of VH gene coverage for the region targeted by
the assay.
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.7 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 havedescribed 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.8,9The 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.
LymphoTrack IGHV Leader Somatic Hypermutation Assay – MiSeq
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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.

4. Reagents
Reagent Components
Table 1. Available Kits
Catalog#

Description# of Indexed Master Mixes

Total Reactions

71210059

LymphoTrack IGHV Leader Somatic Hypermutation Assay Kit A – MiSeq

8 indices – 5 sequencing runs each4071210069

LymphoTrack IGHV Leader Somatic Hypermutation Assay Panel – MiSeq

24 indices – 5 sequencing runs each

120

Table 2. Kit Components

Reagent

Master Mixesǂ

Positive Control
DNAs+

Negative
Control DNA

71210059# of Units

71210069# of Units1

IGH Leader MiSeq 01
IGH Leader MiSeq 02

1
1

1
1

IGH Leader MiSeq 03

1

1

IGH Leader MiSeq 04
IGH Leader MiSeq 05

1
1

1
1

IGH Leader MiSeq 06

1

1

IGH Leader MiSeq 07
IGH Leader MiSeq 08

1
1

1
1

IGH Leader MiSeq 09

01

IGH Leader MiSeq 10
IGH Leader MiSeq 11

0
0

1
1

0

1

0
0

1
1

IGH Leader MiSeq 15

01

IGH Leader MiSeq 16
IGH Leader MiSeq 18

0
0

1
1

IGH Leader MiSeq 19

01

IGH Leader MiSeq 20
IGH Leader MiSeq 21

0
0

1
1

IGH Leader MiSeq 2201

IGH Leader MiSeq 23
IGH Leader MiSeq 25

0
0

1
1

IGH Leader MiSeq 27

01

Reagent Components

IGH Leader MiSeq 12
IGH Leader MiSeq 13
IGH Leader MiSeq 14

Unit
Quantity

250 µLIGH SHM POS (+)(
40880008)

45 µL

1

3

(

IGH POS (+)
40880009)

45 µL

1

3

(

NGS NEG (-)
40920018)

45 µL

1

3

Storage
Temperature

Notes

N/A

IGH V4-59_08 / IGH J4_02 DNA in
tonsil DNA with ≥ 2% mutation ratecompared to the germline sequenceor

IGH V1-46_03 / IGH J4_02 DNA in
tonsil DNA
Tonsil DNA, highest sequencefrequency can vary between lots

Note: There are no preservatives used in the manufacturing of these kits.
Note1: Indices 17, 24 and 26 are not used in these kits.
Note+: While the IGH Positive Control DNA is positive for clonality in the IGH locus, it is not positive for the presence of somatic hypermutation. The IGH SHM
Positive Control DNA has been characterized as positive for somatic hypermutation in the IGH locus

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

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
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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 come with the purchase of these kits.
Do not mix or combine reagents from kits with different lot numbers.
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.
Dispose of unused reagents and waste in accordance with country, federal, state and local regulations.
Perform all laboratory procedures with standard personal protective equipment (gloves, laboratory coats andprotective eye wear). Follow good laboratory practices and universal precautions when working with specimens.
Do not pipette by mouth. Do not eat, drink, or smoke in laboratory work areas. Wash hands thoroughly after
handling specimens and assay reagents. Handle specimens in approved biological safety containment facilities andopen only in certified biological safety cabinets. Use molecular biology grade water for the preparation of
specimen DNA.
Due to the high analytical sensitivity of 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
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

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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 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 verify this assay.
Thermal cyclerUse or Function: Amplification of DNA samples
Suggested Instrument: Veriti™ Thermal Cycler or equivalent
Performance Characteristics and Specifications:o Minimum Thermal Range: 15ºC to 96ºC
o Minimum Ramping Speed: 0.8ºC/sec

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

Magnetic Stand

Use or Function: Purification of PCR products
Suggested Instrument:o Ambion® Magnetic Stand 96 (AM10027)
A32782) oro Agencourt SPRIPlate® 96 Ring Super Magnet Plate (o Thermo Fisher Scientific DynaMag™-96 Side Skirted Magnet (12027) or equivalentPerformance Characteristics and Specifications: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 PCR Instrument or equivalent
Performance Characteristics and Specifications:o Able to detect SYBR Green wavelength

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

Illumina MiSeq Instrument

Use or Function: Sequence normalized DNA library
Performance Characteristics and Specifications:o Compatible with MiSeq Reagent Kit v3



Follow manufacturer’s installation, operation, calibration and maintenance procedures
See sections 7.11 MiSeq Flow Cell Loading, 7.12
MiSeq Sample Sheet Setup, and 7.13 MiSeq Run Start for MiSeq parameters.

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6. Specimen Collection and Preparation
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.
Interfering Substances
The following substances are known to interfere with PCR:

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

Divalent cation chelators
Low retention pipette tips
EDTA (not significant at low concentrations)
HeparinSpecimen Requirements and Handling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.

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7. Assay Procedure
Materials Provided
See Table 2 for materials provided with each kit.
Materials Required (not provided)
Table 3. Materials Required (not provided)

Reagent/Material

Required or Recommended Reagents/Suppliers

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 and 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

SterilePCR Purification Kit

Beckman Coulter, Inc:
• Agencourt AMPure XP

A63880N/A

PCR Purification

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

AM10027
12027
or

N/A

Amplicon & Library
Quantification
MiSeq Run

MiSeq Software
Dilution Buffer A

KAPA Biosystems:• KAPA Library Quantification Kit – Illumina
Illumina:• MiSeq Reagent v3 kit• (600 cycles)••

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

A32782
KK4824

N/A

MS-102-3003

Standard flow cell

N/A

N/A

N/A

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

Reagent Preparation
To ensure DNA samples do not contain 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; product sizes may vary ±5 bp due to size standard
and instrument variability. This is especially important for DNA from 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.
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7.3.1.

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

7.3.2.


7.3.3.
7.3.4.

Include two controls in each run (one positive and one negative) as well as one NTC (the SHM control is
optional).
Use molecular biology grade water for the NTC 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 it in the PCR thermal cycler.
Table 4. Reaction Setup

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.
Note:

Amplify the samples using the PCR program from Table 5.
The LymphoTrack IGHV Leader SHM Assay – MiSeq PCR program has different cycling conditions than the other LymphoTrack Assays.
Table 5. 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

32x

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

AMPure XP Purification
Purification of the PCR products from samples, positive and negative controls, and no template controls was performed
during assay verification using the Agencourt AMPure XP PCR Purification system.
Preparation:
7.5.1.

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.

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Page 12 of 13

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.
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 atroom temperature.


7.5.8.
7.5.9.

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

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

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 -20°C or go to the next step.
The gel image in Figure 1 illustrates the effectiveness of a typical purification (showing amplicons before andafter purification).

Figure 1:
Example of apurification
result for
ampliconsfrom theLymphoTrack
IGHV Leader
Somatic
Hypermutation
Assay – MiSeq
Master Mixes.
The image was
generated byrunning
unpurified andpurifiedproducts on
the LabChip GX.

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Page 13 of 14

Quantification of Amplicons
The following steps were performed to quantify purified PCR amplicons generated from samples, positive, negative andno 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:

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.

Step B:
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 the thermal program in Table 7 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 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 the following
equation:
𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑜𝑜𝑜𝑜 𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 (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 IGHV Leader SHM Assay is 660 bp (A = 660).
(Note: Sequence length includes additional nucleotides that are necessary for sequencing.)

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

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Page 14 of 15

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 (withthe exception of NTC, which can be excluded).


7.7.2.

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:
Step B:

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.

Setup qPCR for Library Quantification
Refer to Table 6 for qPCR set up and Table 7 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 IGHV Leader SHM Assay is 660 bp (A = 660).
(Note: Sequence length includes additional nucleotides that are necessary for sequencing.)

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

Preparation of the Library for the MiSeq Sequencing Run
At the end of this section, the concentration of the library DNA will be between 12 - 20 pM for MiSeq reagent kit v3.
For multiplexing amplicons from different LymphoTrack Assays for the MiSeq into a single library, please refer toAppendix A: Building a Sequencing Library with Multiple NGS Targets(section 18).
7.10.1.

Determine the amount of library to be prepared based on the concentration of the pooled library from the qPCR
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

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 the bottomof 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:

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



If the diluted ssDNA library concentration is 40 pM (the initial concentration was 4 nM), dilute to the desired
MiSeq loading concentration using the examples shown in Table 10:
Table 10. Preparation of the Library for MiSeq Loading
7.10.8.
7.10.9.

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).
Verify 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.
MiSeq Sample Sheet Setup (Windows 7 or Windows 10)
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 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 up the Sample Sheet.

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.
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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. This set of samples/targets should begiven 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)

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 11. Sample Selection index names

LymphoTrack Assay – MiSeq PCR
Master Mix Index
id01
id02
id03
id04
id05
id06
id07
id08
id09
id10
id11
id12
id13
id14
id15
id16
id18
id19
id20
id21
id22
id23
id25
id27

Index
Sequence
ATCACGCGATGT
TTAGGCTGACCAACAGTGGCCAATCAGATC
ACTTGAGATCAGTAGCTT
GGCTACCTTGTAAGTCAAAGTTCCATGTCA
CCGTCCGTCCGCGTGAAAGTGGCCGTTTCGCGTACGGAGTGGACTGAT
ATTCCT

TruSeq DNA Single Indexes Set A B(LRM “Index Kit”)
AR001
AR002
AR003
AR004
AR005
AR006
AR007
AR008
AR009
AR010
AR011
AR012
AR013
AR014
AR015
AR016
AR018
AR019
AR020
AR021
AR022
AR023
AR025
AR027

MiSeq Run Start
Start the MiSeq run following the MiSeq Control Software Instructions. The approximate MiSeq run times are indicatedin Table 12.
Table 12. MiSeq Run Times

MiSeq Reagent Kit

Read Length

MCS Version

Total MiSeq Run Timev3

2x301 bp

2.6 or later~ 56 hours

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

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8. Data Analysis
The LymphoTrack IGHV Leader Somatic Hypermutation Assay – MiSeq was designed to produce sequencing data that can beanalyzed using the LymphoTrack Software – MiSeq package provided on the associated CD (75000009), which is included
with the assay purchase. This CD includes detailed instructions for installation and use of the software package.
The MiSeq sequencing run of samples prepared with the LymphoTrack IGHV Leader Somatic Hypermutation Assay provides
FASTQ files that can be easily processed into fully analyzed data using the LymphoTrack Software - MiSeq.

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%
IGH SHM Positive Control mutation rate ≥ 2.0%
MiSeq Run Validity* Q30 > 70% for v3 (2x301)*Note:

Q30 from all analytical validations has met the above criteria from Illumina MiSeq Q30 specification. However, Q30 score may varydepending 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 be invalid.

10. Limitations of ProcedureThis assay does not identify 100% of clonal cell populations.
PCR-based assays are subject to interference by degradation of DNA or inhibition of PCR amplification due to heparin orother agents that might be present in the analyzed sample.
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.

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Page 18 of 19

11. Sample Data

Figure 2: This table, generated via the LymphoTrack Reporter, 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 nucleotides (nts) different. Sequences were generated using the LymphoTrack IGHV Leader
Somatic Hypermutation Assay –MiSeq and analyzed using the LymphoTrack Software – MiSeq (75000009).

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Page 19 of 20

12. Troubleshooting Guide
Table 13. 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 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 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 < 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.

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13. Technical and Customer Service
Thank you for purchasing our LymphoTrack IGHV Leader Somatic Hypermutation Assay – MiSeq. We appreciate yourbusiness. We are happy to assist you with understanding this assay, and will provide ongoing technical assistance Monday
through Friday to keep the assay 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. Tonegawa, S., (1983). Somatic Generation of Antibody Diversity. Nature 302, 575–581.
2. Ghia, P. et al., (2007). ERIC recommendations on IGHV gene mutational status in chronic lymphocytic leukemia.
3.
4.
5.
6.
7.
8.
9.






Leukemia 21, 1–3.
Trainor, KJ. et al., (1990). Monoclonality in B-lymphoproliferative disorders detected at the DNA level. Blood 75, 2220–
2222.
Miller, JE. (2013). Principle of Immunoglobulin and T Cell Receptor Gene Rearrangement. In Cheng, L., Zhang, D., Eble, J.
N. (Eds), Molecular Genetic Pathology (2nd Ed., sections 30.2.7.13 and 30.2.7.18). New York, USA: Springer
Science & Business Media.
Davi, F., et al., (2008). Determination of IGHV gene mutational status in chronic lymphocytic leukemia: bioinformatics
advances meet clinical needs. Leukemia 22, 212-214.
Ghia, P., et al., (2005). Geographic patterns and pathogenetic implications of IGHV gene usage in chronic lymphocytic
leukemia: the lesson of the IGHV3-21 gene. Blood 105, 1678-1685.
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.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 the European
Community

Lot Number

Manufacturer

Storage Conditions

Consult Instructions for Use

LymphoTrack IGHV Leader Somatic Hypermutation Assay – MiSeq
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Page 21 of 22

16. Legal NoticeThis product is covered by one or more of the following patents and patent applications owned by or exclusively licensed to
Invivoscribe, Inc. (IVS). United States Patent Number 7,785,783, United States Patent Number 8,859,748 (together withdivisional application claims relating to the same original application), European Patent Number EP 1549764B1 (validated in
16 countries, and augmented by related European Patents Numbered EP2418287A3 and EP 2460889A3), Japanese Patent
Number JP04708029B2, Japanese Patent Application Number 2006-529437, Brazil Patent Application Number PI0410283.5,
Canadian Patent Number CA2525122, Indian Patent Number IN243620, Mexican Patent Number MX286493, Chinese Patent
Number CN1806051 and Korean Patent Number 101215194.
Use of this product may require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). Any necessary
license to practice amplification methods or to use reagents, amplification enzymes or equipment covered by third party
patents is the responsibility of the user and no such license is granted by Invivoscribe, Inc., expressly or by implication.
©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 Scientific andits subsidiaries.
KAPA™ is a trademark of Kapa Biosystems.
Microsoft®, Windows®, and Excel® are registered trademarks of Microsoft Corporation.

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17. LymphoTrack IGHV Leader Somatic Hypermutation Assay - MiSeq: Single
Page Guide
Using gloved hands, remove the Master Mixes from the freezer. Allow the tubes to thaw; then gently vortex to mix.
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 and no template controls.
Add 0.2 µL Taq DNA polymerase (@5 U/µL) to each Master Mix.
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.
Add 5 µL of molecular biology grade water to the well containing the respective Master Mix for no template control, andpipette up and down 5-10 times to mix.
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

32x

Remove the amplification plate from the thermal cycler.
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.
Quantify amplicons using the KAPA library quantification kit according to the kit instructions. Dilute amplicons 1:4,000
before proceeding to qPCR.
Pool equal amounts of amplicons from samples (do not include the no template control), dilute 1:1,000 and quantify thelibrary using the KAPA library quantification kit.
Denature and dilute the library to 12 - 20 pM for MiSeq reagent kit v3 (MCS v2.6 or later).
Load 600 µL of denatured and diluted library onto the MiSeq Reagent Cartridge.
Set up a MiSeq sample sheet Manager, and upload the sample sheet to the instrument (if necessary).
Start the MiSeq run.
Analyze and visualize the acquired data using the associated LymphoTrack Software - MiSeq package.

LymphoTrack IGHV Leader Somatic Hypermutation Assay – 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 from thesame 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 Table 14 to determine the cycle settings and Illumina MiSeq Reagent kits to be usedwith 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 14 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

Determine the concentration of each individual library (e.g., IGHV Leader, IGH FR1, IGH FR2, IGH FR3, IGK, TRB and TRG).
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 15. Calculation of Individual Library Inputs to Generate a Multi-Targeted Sequencing Library for the MiSeq Run

Library

Case A

Assay Name

Concentration(nM)

IGHV Leader

2.3

IGH FR1

Individual Library Volume (µL)
Case B
Case C
Case D

Case E

Case F

n=3

n=2

n=7

n=6

n=5

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

2.3

IGK

3.5

X

1.6

1.9

TRG

2.6

Y

2.2

2.6

TRB

2

Z

2.9

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

17.1

16.7

5.7
7.7

16.9

LymphoTrack IGHV Leader Somatic Hypermutation Assay – MiSeq
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 16 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 15, mix the applicable libraries first, then add 18 µL to thedenaturing reaction as shown in Table 16.
Table 16. 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 gently 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 17. Vortex briefly to mix and pulse centrifuge the sample.
Table 17. 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 IGHV Leader Somatic Hypermutation Assay – MiSeq
For Research Use Only. Not for use in Diagnostic Procedures

280337 Rev. M | March 2024