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Clonality and Measurable Residual Disease (MRD)
Testing on the Illumina® NextSeqTM Sequencers
Lymphoid and Myeloid Malignancies
INTRODUCTION
Next-generation sequencing (NGS) technology is rapidlytransforming the paradigm of how gene rearrangement
information can be used in the study of hematologicalmalignancies. While traditional PCR-gel or PCR-capillary
based gene rearrangement assays have limitations withsamples containing low tumor burden or require expert
data interpretation, NGS-based methods effectively addressthese limitations due to its superior sensitivity and specificity.
The high resolution of NGS allows for more precise
differentiation between clonal and polyclonal T-cell
receptor (TCR) or immunoglobulin heavy chain (IGH) generearrangements and the identification of genomic
mutations associated with FLT3 and NPM1 gene variants.
Sequencing also provides accurate assessments of SomaticHypermutation, which an important prognostic factor for CLL.
NGS can uncover minute traces of malignant cells lingering
after treatment, providing a significant advantage overtraditional methods that might miss these low-burden
populations. This translates to a heightened ability to detectmeasurable residual disease (MRD), with potential clinicalapplications including identification of early relapse anddisease evolution as well as optimizing treatment strategies.

Benefits of using NGS for MRD detection include:
Higher Sensitivity and Specificity: NGS allows for thedetection of low-frequency mutations, enhancing diagnostic
accuracy and the ability to monitor disease progression
accurately.1
Improved Clonality Assessment: With higher resolution andmore objective interpretation, NGS overcomes limitationsof gel or capillary based methods, such as the inability todifferentiate clonal sequences from polyclonal background,leading to fewer false positives.1

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Quantitative Analysis: NGS enables precise assessments ofclonality, Somatic Hypermutation and monitoring of disease
burden over time, which is crucial for MRD assessment,
developing treatment strategies, and predicting relapse.1
Concordance with Established Methods: NGS-based assays
show high concordance rates with traditional gel or capillary
based methods demonstrating their clinical utility,
comparable accuracy and higher specificity, particularly
in diagnosing T-cell malignancies.1,2Enhanced Mutation Characterization: NGS facilitatesdetailed characterization of clonal populations, identificationof intraclonal diversity and gene mutations, which may aid
in prognostic assessments.
The National Comprehensive Cancer Network (NCCN)
guidelines recommend MRD testing for several lymphoid andmyeloid cancers, including multiple myeloma (MM), acutelymphoblastic leukemia (ALL), and chronic lymphocytic
leukemia (CLL), and acute myeloid leukemia (AML).3,4,5,6,7
European LeukemiaNet (ELN) guidelines recommend MRD
testing following induction and consolidation courses toassess remission status and determine kinetics of disease
response, and sequentially beyond consolidation to detect
impending morphologic relapse.8
Over the past decade, Invivoscribe has pioneered the useof NGS to identify gene rearrangements and track residual
disease over time. This innovation has led to the developmentof a suite of lymphoid and myeloid assays for use with
Illumina’s MiSeq™ and MiSeq™ NGS platforms, which
provide target-specific sequences to aid hematologic
disease research.
Today, the use of NGS technology for hematological diseaseresearch has become widely adopted, driving innovation
for increased read depth, superior sequencing accuracy,
streamlined data generation, and higher sample throughput
without sacrificing cost.

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These technological improvements have created a highdemand to adapt existing NGS assays for higher-throughput
platforms, such as Illumina’s NextSeq Systems. Here we
present a performance comparison between the MiSeq and
NextSeq 1000 platforms using identical libraries generated
by our assays.

As part of the LympoTrack MRD Solution, LymphoTrack Low
Positive Controls are used as an external quality control foreach run, while LymphoQuant® Internal Controls are spiked
into each sample to calculate clonal cell equivalents.
These RUO DNA controls are designed for use with
LymphoTrack Assays and LymphoTrack MRD Software
to track clonal sequences.

INVIVOSCRIBE NGS ASSAYS OVERVIEW

The LymphoTrack MRD Bundled Solutions are offered forT- or B-Cell assessment enabling researchers to accurately
detect and trend clonal evolution in longitudinal studies with
unprecedented simplicity. In summary, LymphoTrack MRD
Solutions offer improved sensitivity, objectivity, specificity, andquantitative capabilities compared to traditional methods,
making them a promising tool for future clinical applications
in lymphoproliferative diseases.

LymphoTrack® Assays for Lymphoproliferative
Disorders
Invivoscribe’s LymphoTrack® assays for MiSeq represent asignificant improvement over PCR-based clonality assays dueto their enhanced accuracy, specificity, and ability to provide
detailed clonal information empower researchers with theinformation necessary to drive innovation and understandingof lymphoproliferative disorders. Therefore, these assays havethree important and complementary uses:
1. Provides critical information on the existence of clonality.
2. Provides detailed sequence information on the degreeof Somatic Hypermutation (IGH FR1 and IGHV Leader
specific).
3. Identifies sequence information required to track clones
in subsequent samples.
Primers included in the master mixes are designed with
Illumina adapters, incorporating up to 24 different indices.
This design provides a streamlined approach consisting of asingle-step PCR and the ability to pool amplicons from several
different samples and targets into one sequencing run for
additional cost efficiencies. Assay interpretation and analysis
is completed by the associated RUO LymphoTrack Software,
which generates objective reports and data visualization.
Extending application utility, LymphoTrack Assays can also be
used to identify and track measurable residual disease (MRD)in lymphoproliferative diseases.
LymphoTrack MRD Solutions can be used to track levels of
MRD in subjects following therapy and throughout remission.

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MRD NGS Assays for Myeloid Diseases
In addition to the LymphoTrack assays, Invivoscribe’s NGS
products now includes two pivotal Research Use Only (RUO)
measurable residual disease assays for AML. Our innovative
FLT3 ITD MRD Assay (RUO), targets the FMS-like tyrosine
kinase 3 (FLT3) gene which encodes a receptor tyrosine kinasethat is normally expressed on many cell types, including
hematologic stem cells. Mutation of the FLT3 receptor,
by internal tandem duplication (ITD) of the juxtamembrane
domain, causes constitutive activation of the FLT3 receptor.
FLT3 ITD mutations are present in about 25% of patients with
AML and are characterized by an increased risk of relapse.
The NPM1 MRD Assay (RUO), targets the Nucleophosmin(NPM1) gene which encodes for a protein involved incellular activities that may relate to proliferative andgrowth-suppressive roles in the cell. As one of the mostcommonly mutated genes in AML, NPM1 gene mutations
occur in about one-third of the cases of primary AML.
Of the subjects with an NPM1 mutation at diagnosis,
roughly 50% relapse during the first 3 years, particularly
those with a concurrent FLT3-ITD mutation.9
Recent research suggests the presence of ITD and NPM1
mutations can be indicative of disease outcome. In a study
evaluating the persistence of residual FLT3-ITD and NPM1
mutations among subjects with AML in first remission prior
to allogeneic cell transplant, those with an allele fraction≥0.01% trended towards elevated risk of relapse andmortality compared to those without these mutations.10

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Each MRD assay kit includes 24 uniquely indexed mastermixes, allowing multiple samples and targets to be sequenced
on the same flow cell improving cost efficiency. Assayinterpretation and data analysis are completed with either the
FLT3 ITD MRD Software or NPM1 MRD Software in under one
hour, providing sequence annotations and objective results.

NGS INSTRUMENT OVERVIEW
Illumina MiSeqTM

The Illumina MiSeq is a benchtop sequencer designed
for targeted sequencing applications. It is known for its highaccuracy and relatively low throughput, making it suitable
for small to medium-sized laboratories.
Metric Description
Sequencing Reads and Length: Depth Capable of generating
up to 50 million paired-end reads per run, with a read lengthof up to 2x300 bp.

TAT: Faster than MiSeq, with runs completing in approximately
34 hours depending on the assay, kit, and workflow being
performed.
Cost-Effectiveness: Higher initial cost per run, but morecost-effective for high throughput needs due to the larger
number of samples that can be processed simultaneously.

MATERIALS AND METHODS
Positive DNA from cell lines or clinical samples were contrived
using negative DNA as a background to create panels for eachassay. The evaluated assays included V-J rearrangement(NGS clonality) assays for immunoglobulin heavy chain IGH(IGHV Leader, FR1, FR2 and FR3), immunoglobulin light chain(IGK), T-cell receptor gamma (TRG), T-cell receptor beta (TRB),
and MRD assays for FLT3 internal tandem duplications andNPM1 mutations. The workflow for Invivoscribe's NGS assays
is shown below in Figure 1.

Quality: >70% bases higher than Q30 for 2x300 bp.
TAT: Approximately 56 hours per run, depending on the assay,
kit, and workflow being performed.

PCR

PCR ProductPurification*1-2 Rounds

Cost-Effectiveness: Cost per run is relatively low, making it
economical for laboratories with moderate sample volumes.
*Clonality Assays = 1 round
MRD assays = 2 rounds

Illumina NextSeq 1000TM
The NextSeq 1000 is a recent addition to Illumina's NGS
portfolio, offering higher throughput and more flexibility
compared to the MiSeq. It is designed to support a broader
range of applications, from targeted sequencing towhole-exome sequencing.

PCR Product
Quantificationand Library
Pooling

Data Analysis

Sequencing

Metric Description
Sequencing Reads and Length: Depth Capable of generating
up to 3.6 billion paired-end reads per run, with a read lengthof up to 2x300 bp.
Quality: ≥85% bases higher than Q30 for 2x300 bp.

Figure 1: Workflow for Invivoscribe’s NGS Assays

Three pooled libraries were generated:
Library A: comprised of seven V-J Clonality Rearrangement
Assay Samples
Library B: comprised of FLT3 MRD Assay Samples
Library C: comprised of NPM1 MRD Assay Samples

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Library A was sequenced at 2x301 cycles on the MiSeq with aloading concentration of 18 pM using the MiSeq Reagent Kit
v3 and on the NextSeq 1000 with a loading concentration of
650 pM and 12.5% PhiX using NextSeq 1000/2000 P1 reagents,
respectively. FASTQ files from both platforms were analyzed
using the same in-house developed LymphoTrack Dx Software- MiSeq (v2.4.3). Top percent reads from each of the target
specific V-J rearrangements were compared between the
NGS platforms.
Libraries B (FLT3 ITD) and C (NPM1) were sequenced
separately each at 2x301 cycles on the MiSeq with a loading
concentration of 14 pM and 12.5% PhiX using the MiSeq
Reagent Kit v3 and sequenced together in a combined libraryon the NextSeq 1000 with a loading concentration of 650 pM
and 12.5% PhiX using NextSeq 1000/2000 P2 reagents.

Table 2: Clonality Assay Results Correlation between Instruments
Assay

Correlation(R2)

IGH Leader

0.997

IGH FR1

0.998

IGH FR2

0.999

IGH FR3

0.999

IGK

0.999

TRG

0.999

TRB

0.996

Key Run Metrics
MiSeq

NextSeq 1000

PF: 32 million

PF: 161 million

Q30: 77.02%

Q30: 86.86%

Sequencing time:~56 hours

Sequencing time:~34 hours

Plotting the results from each clonality assay to compare theinstruments is depicted in Figures 2 & 3.

FASTQ files from both platforms were analyzed using thesame in-house developed FLT3-ITD MRD Software (v1.2)
and NPM1 MRD Software (v1.1.1). Detected variant readfrequencies (VRF) from each of the target specific mutations
were compared between the NGS platforms.

Table 1: Illumina Materials
VendorDescription

Catalog #Illumina

MiSeq Reagent Kit v3

MS-102-3003

Illumina

NextSeq 1000/2000 P1 Reagents

20075294

Illumina

NextSeq 1000/2000 P2 Reagents

20075295A

B

C

Figure 2: IGHV, IGH FR1/2/3 & IGK Clonality Comparison

RESULTS

Clonality Assays
The full suite of Invivoscribe’s clonality NGS assays includes
4 targets within the IGH gene (Leader, FR1, FR2, and FR3),
as well as assays for the IGK, TRG, and TRB genes. Each ofthese were combined into a single amplification library which
was run on both the MiSeq and the NextSeq 1000. This librarygenerated 32 million pass filter (PF) reads with the MiSeq andgenerated 161 million PF reads with the NextSeq 1000, 5-foldmore PF reads as compared to MiSeq. A comparison of the
top percent reads generated from both instruments yielded
relative R2 of the correlations all greater than 0.99.
Each assay’s relative R2 values are listed in Table 2.

Figure 3: TRB, TRG Clonality Comparison

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MRD Assays
Invivoscribe’s myeloid MRD assay menu includes the FLT3
ITD MRD Assay and NPM1 MRD Assay. Separate libraries
were generated for each target (FLT3 ITD and NPM1) andsequenced individually on the MiSeq to ensure a suitable
read depth was achieved. The MiSeq generated 19 million
PF reads with the FLT3 ITD MRD library, and 14 million PF
reads with the NPM1 MRD library.
The same libraries were then combined and sequenced
on the NextSeq 1000, resulting in 11-fold more PF reads(413 million), as compared to the two MiSeq runs. The results
generated from both instruments were compared and therelative R2 of the variant read frequencies (VRF) correlationfor each were greater than 0.999. Each assay relative R2
values are listed in Table 3.
Table 3: MRD Assay Results Correlation between Instruments
Assay

Correlation(R2)

Key Run Metrics
MiSeq

NextSeq 1000

PF: 19 million

FLT3 ITD
MRD

NPM1 MRD

0.999

1.000

Q30: 89.00%
Sequencing time:~55 hours

PF: 413 million

PF: 14 million

Sequencing
time: ~44 hours

Q30: 89.94%

Figure 4: FLT3 ITD MRD & NPM1 MRD Comparison

The NextSeq 1000 can achieve even greater sensitivity for
MRD detection due to its deeper sequencing capability,
potentially obtaining sensivitity levels as low as 10-6.
This allows more precise detection of low-frequency clones,
which can be observed when comparing detection levels
to those achieved by the MiSeq, as exhibited in Table 4 for
FLT3 ITD MRD and Table 5 for NPM1 MRD.

Q30: 89.63%

Sequencing time:~56 hours

Plotting the results from each MRD assay to compare theinstruments is depicted in Figure 4.

Table 4: FLT3 ITD MRD Detection Results
ITD Insertion
LengthVRF
Target

MiSeq
(VRF)

NextSeq1000 (VRF)

5.00E-02

3.56E-02

3.51E-02

5.00E-03

3.23E-03

3.46E-03

5.00E-04

4.11E-04

3.96E-04

5.00E-05

3.28E-05

3.88E-05

1.00E-06

NOT DETECTEDNOT DETECTED

5.00E-02

1.39E-03

1.45E-03

5.00E-03

9.04E-05

1.11E-04

5.00E-04

1.93E-05

1.18E-05

5.00E-05

NOT DETECTED

1.34E-06

1.00E-06

NOT DETECTEDNOT DETECTEDFor Research Use Only (RUO). Not for use in Diganostic Procedures.

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MediumInsert:42 bp

LongInsert:172 bp

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Table 5: NPM1 MRD Detection Results
NPM1 Mutation
Type

VRF
Target

MiSeq
(VRF)

NextSeq1000 (VRF)

1.00E-05

1.30E-05

9.55E-06

1.00E-06

NOT DETECTEDNOT DETECTED

5.00E-05

1.18E-05

8.74E-06

1.00E-05

NOT DETECTED

2.27E-06

5.00E-05

5.03E-05

4.59E-05

1.00E-05

5.57E-06

1.03E-05

5.00E-05

3.86E-05

9.68E-05

1.00E-05

NOT DETECTED

1.62E-06

Type: A

Type: B

Type: D

Type: Other

CONCLUSION
Invivoscribe's NGS assays demonstrate high concordance
between the MiSeq and NextSeq 1000 platforms for bothclonality and MRD detection. Both platforms yielded highly
correlated results (R² > 0.99) for all clonality and MRD assays,
indicating reliable detection of gene rearrangements andmutations.
These results demonstrate that Invivoscribe’s RUO
LymphoTrack MiSeq and AML NGS MRD assays arecompatible with the NextSeq 1000.
Moreover, the increased read depth of the NextSeq 1000
platform enables pooling more libraries for sequencing
at a greater cost efficiency as well as enhanced sensitivity,
exhibited by the detection of 3 dilution levels missed by the
MiSeq. This provides evidence of enhanced sensitivity, which
is important when assessing residual disease.
However, the NextSeq 1000 offers a significant advantage
in throughput. During this investigation, the NextSeq 1000
generated a substantially greater number of reads comparedto the MiSeq (up to 5-fold for clonality and 11-fold for MRD).
This translates to:· Enhanced confidence in negative results: a deeper
sequencing run translates to a higher number of reads
analyzed. This can lead to greater confidence in negative
MRD results, potentially reducing the risk of false negatives
and unnecessary treatment interventions; AND· Improved efficiency: While the difference is modest, the
NextSeq 1000 demonstrated slightly faster run times for bothclonality and MRD assays. This can contribute to improved
workflow efficiency, allowing laboratories to process more
samples and potentially reduce turnaround times.
Invivoscribe's NGS assays offer reliable performance on both
platforms. However, for laboratories seeking to optimize
workflow efficiency, unlock the potential for increased
sensitivity in MRD detection, and gain deeper sequencing
insights, the NextSeq 1000 emerges as the preferred platform.
Its enhanced sequencing depth can empower clinicians withmore precise information to guide personalized treatment
decisions for their patients.

ABOUT INVIVOSCRIBE
Invivoscribe is a global, vertically integrated biotechnology
company dedicated to Improving Lives with Precision
Diagnostics®. For nearly thirty years, Invivoscribe has
improved the quality of healthcare worldwide by providing
high quality standardized research reagents, tests, andbioinformatics tools to advance the field of precision
medicine. Invivoscribe has a successful track recordof partnerships with global pharmaceutical companies
interested in developing and commercializing companion
diagnostics, and provides expertise in both regulatory andlaboratory services. Providing distributable reagents and kits,
as well as clinical trial services through its globally locatedclinical lab subsidiaries (LabPMM), Invivoscribe is an ideal
partner from proof of concept through commercialization.

· Potential for increased sensitivity: Improved detectionof low-frequency clones - particularly crucial for MRD
analysis where identifying even minute traces of residual
disease is critical;

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Invivoscribe’s RUO NGS Assays and Software for use with Illumina Sequencers

Catalog #Product Description

Quantity

71210009

LymphoTrack IGH FR1 Assay Kit A – MiSeq

8 indices – 5 reactions each71210039

LymphoTrack IGH FR1 Assay Panel – MiSeq

24 indices – 5 reactions each71210149

LymphoTrack IGH FR1 Assay Panel B – MiSeq

24 indices – 5 reactions each71210089

LymphoTrack IGH FR2 Assay Kit A – MiSeq

8 indices – 5 reactions each71210099

LymphoTrack IGH FR2 Assay Panel – MiSeq

24 indices – 5 reactions each71210109

LymphoTrack IGH FR3 Assay Kit A – MiSeq

8 indices – 5 reactions each71210119

LymphoTrack IGH FR3 Assay Panel – MiSeq

24 indices – 5 reactions each71210129

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

8 indices per FR region– 5 reactions each71210139

LymphoTrack IGH FR1/2/3 Assay Panel – MiSeq

24 indices per FR region – 5 reactions each71210059

LymphoTrack IGHV Leader Somatic Hypermutation Assay Kit A – MiSeq

8 indices – 5 reactions each71210069

LymphoTrack IGHV Leader Somatic Hypermutation Assay Panel – MiSeq

24 indices – 5 reactions each71220009

LymphoTrack IGK Assay Kit A – MiSeq

8 indices – 5 sequencing reactions each71220019

LymphoTrack IGK Assay Panel – MiSeq

24 indices – 5 sequencing reactions each72250009

LymphoTrack TRB Assay Kit A – MiSeq

8 indices – 5 reactions each72250019

LymphoTrack TRB Assay Panel – MiSeq

24 indices – 5 reactions each72270019

LymphoTrack TRG Assay Kit A – MiSeq

8 indices – 5 reactions each72270009

LymphoTrack TRG Assay Panel – MiSeq

24 indices – 5 reactions each14120019

FLT3 ITD MRD Assay

96 Reactions

14160019

NPM1 MRD Assay

96 Reactions

S100003

LymphoTrack Enterprise Software

1 software package

S100005

FLT3 ITD MRD Software

1 software package

S100004

NPM1 MRD Software

1 software package

To learn more about Invivoscribe and LabPMM, visit us online at invivoscribe.com or call us at +1.858.224.6600.
References | 1. Ho et al. J Mol Diag. 2021 May;23(7): 805–815. | 2. Arcila et al. J Mol Diag. 2019 March;21(2): 330–342. | 3. NCCN Clinical Practice Guidelines in Oncology: Multiple
Myeloma. Version 2.2020. | 4. NCCN Clinical Practice Guidelines in Oncology: Acute Lymphoblastic Leukemia. Version 2.2019. | 5. NCCN Clinical Practice Guidelines in Oncology: PediatricAcute Lymphoblastic Leukemia. Version 1.2020. | 6. NCCN Clinical Practice Guidelines in Oncology: Chronic Lymphocytic Leukemia. Version 1.2020. | 7. NCCN Clinical Practice Guidelines in
Oncology: Acute Myeloid Leukemia. Version 4.2023. | 8. Dohner et al. Blood. 2017 Jan 26; 129(4): 424–447. | 9. Kronke et al. Blood. 2013 Jul 4; 122 (1): 100–108. | 10. Dillon et al. JAMA. 2023
Mar 7; 329(9):745-755.

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