Instructions for Use

IGH Somatic Hypermutation Assay v2.0
To sequence and identify mutational status of clonal IGH gene rearrangements.
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Manufactured in U.S.A.

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

Catalog #51010030
51010031
51010040
51010041

Products
IGH Somatic Hypermutation Assay v2.0 for Gel Detection
IGH Somatic Hypermutation Assay v2.0 for ABI Fluorescence Detection
IGH Somatic Hypermutation Assay MegaKit v2.0 for Gel Detection
IGH Somatic Hypermutation Assay MegaKit v2.0 for ABI Fluorescence Detection

Quantity
33 Reactions
33 Reactions
330 Reactions
330 Reactions

Page 2 of 24

Table of Contents
1.
2.

3.

4.

5.

6.

7.

8.

9.
10.

11.

12.
13.
14.
15.

ASSAY USE ............................................................................................................................................................................................. 3
SUMMARY AND EXPLANATION OF THE TEST ..................................................................................................................................................... 3
2.1.
Background ....................................................................................................................................................................................... 3
2.2.
Summary ........................................................................................................................................................................................... 3
PRINCIPLES OF THE PROCEDURE .................................................................................................................................................................... 3
3.1.
Polymerase Chain Reaction (PCR) ..................................................................................................................................................... 3
3.2.
Gel Electrophoresis Detection ........................................................................................................................................................... 4
3.3.
Differential Fluorescence Detection .................................................................................................................................................. 4
3.4.
IGH Somatic Hypermutation (SHM) Analysis ..................................................................................................................................... 5
REAGENTS .............................................................................................................................................................................................. 6
4.1.
Reagent Components ........................................................................................................................................................................ 6
4.2.
Warnings and Precaution .................................................................................................................................................................. 7
4.3.
Storage and Handling ........................................................................................................................................................................ 7
INSTRUMENTS ......................................................................................................................................................................................... 8
5.1.
Thermal cycler ................................................................................................................................................................................... 8
5.2.
Agarose or Polyacrylamide Gel Electrophoresis Unit ........................................................................................................................ 8
5.3.
UV Illuminator Unit ........................................................................................................................................................................... 8
5.4.
ABI 310, ABI 3100 or ABI 3130 (For ABI Fluorescence Detection Assays) ......................................................................................... 8
SPECIMEN COLLECTION AND PREPARATION ..................................................................................................................................................... 9
6.1.
Precautions ........................................................................................................................................................................................ 9
6.2.
Interfering Substances ...................................................................................................................................................................... 9
6.3.
Specimen Requirements and Handling ............................................................................................................................................. 9
6.4.
gDNA Sample Preparation ................................................................................................................................................................. 9
6.5.
gDNA Sample Storage ....................................................................................................................................................................... 9
6.6.
RNA and cDNA Sample Preparation .................................................................................................................................................. 9
6.7.
cDNA Synthesis Protocol ................................................................................................................................................................. 10
6.8.
RNA and cDNA Sample Storage ....................................................................................................................................................... 10
ASSAY PROCEDURE ................................................................................................................................................................................. 11
7.1.
Materials Provided .......................................................................................................................................................................... 11
7.2.
Materials Required (not provided) .................................................................................................................................................. 11
7.3.
Reagent Preparation ....................................................................................................................................................................... 13
7.4.
Amplification ................................................................................................................................................................................... 14
7.5.
Detection ......................................................................................................................................................................................... 14
7.6.
Quality Control ................................................................................................................................................................................ 15
7.7.
Recommended Positive Controls .................................................................................................................................................... 16
7.8.
Sequencing of PCR Product ............................................................................................................................................................. 16
DATA INTERPRETATION ............................................................................................................................................................................ 17
8.1.
Analysis ............................................................................................................................................................................................ 17
8.2.
Sample Interpretation ..................................................................................................................................................................... 18
LIMITATIONS OF PROCEDURE ...................................................................................................................................................................... 18
EXPECTED RESULTS .................................................................................................................................................................................. 18
10.1.
Expected Size of Amplified Products ............................................................................................................................................... 18
10.2.
Sample Data .................................................................................................................................................................................... 19
HYPERMUTATION ANALYSIS AND REPORTING ................................................................................................................................................. 20
11.1.
Analysis ............................................................................................................................................................................................ 20
11.2.
IVS-0013 Clonal Control Data .......................................................................................................................................................... 21
11.3.
Sample Data .................................................................................................................................................................................... 21
TECHNICAL AND CUSTOMER SERVICE ........................................................................................................................................................... 23
REFERENCES .......................................................................................................................................................................................... 23
SYMBOLS ............................................................................................................................................................................................. 24
LEGAL NOTICE ....................................................................................................................................................................................... 24
15.1.
Warranty and Liability ..................................................................................................................................................................... 24
15.2.
Patents and Trademarks .................................................................................................................................................................. 24

IGH Somatic Hypermutation Assay v2.0

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 3 of 24

1. Assay Use
The Research Use Only IGH Somatic Hypermutation Assay v2.0 is used to identify clonal rearrangements of theimmunoglobulin heavy (IGH) chain gene and determine the somatic mutation status of the variable (V) gene sequence andis useful for the study of:

Identifying clonal rearrangements of the IGH chain gene
Assessing the extent of somatic hypermutation in the variable region of the immunoglobulin heavy chain gene
Evaluating new research and methods in malignancy studies

2. Summary and Explanation of the Test
BackgroundRearrangements of the antigen receptor genes occur during ontogeny in B and T lymphocytes. These generearrangements are unique in length and sequence for each cell. Therefore, polymerase chain reaction (PCR) assays
can be used to identify lymphocyte populations derived from a single cell by detecting the unique V-J generearrangements present within these antigen receptor loci.1 This PCR-based assay employs multiple consensus DNA
primers that target conserved genetic regions within the immunoglobulin heavy chain (IGH) gene. This test is used todetect and sequence the majority of clonal IGH rearrangements from either genomic DNA (gDNA) or complementary
DNA (cDNA). Clonal products can be detected using a variety of methods, including gel and capillary electrophoresis.
The primers that target the leader (VHL) and framework 1 (FR1) regions have been designed to include a universal
sequencing tag at the 5’end. This design allows for bi-directional sequencing of clonal PCR products with just one
sequencing-tag specific forward primer and one JH reverse primer. The presence of IGH somatic hypermutation(SHM) is defined as greater or equal to 2% difference from the germline variable (V) gene sequence, whereas lessthan 2% difference is considered evidence of no somatic hypermutation.

SummaryThis test amplifies either gDNA or cDNA that lies between the upstream leader (VHL) or framework 1 (FR1) regions
and the downstream joining (J) region of the IGH gene. The test utilizes two (2) different master mixes:
Hypermutation Mix 1 v2.0 and Hypermutation Mix 2 v2.0. The Hypermutation Mix 1 v2.0 targets sequences between
the leader and joining regions. Therefore the amplicon product(s) span the entire variable (V) region, which contains
the FR1, CDR1 (complementarity-determining region 1), FR2, CDR2, FR3 and CDR3 regions. The Hypermutation Mix 2
v2.0 targets sequences between the framework 1 (FR1) and joining (J) regions. The resulting amplicons include a
portion of the FR1 region to the downstream J region. Accordingly products do not include the complete FR1
sequence.

3. Principles of the Procedure
Polymerase Chain Reaction (PCR)
PCR assays are routinely used for the identification of clonal lymphocyte populations. Each B-cell has a single
productive IGH gene rearrangement (consisting of the combination of a variable (V) region, a diversity (D) region anda joining (J) region) that is unique in both length and sequence. Therefore, when gDNA or cDNA from a normal or
polyclonal population is amplified using primers that flank the V-J region, a bell-shaped curve (Gaussian distribution)of amplicon products within an expected size range is produced. On a gel, this distribution of products is seen as asmear. This Gaussian distribution reflects the heterogeneous population of V-D-J rearrangements. In cases wherelymphocytes are not present, no product is seen. For gDNA or cDNA from samples containing a clonal population, theyield is one or two prominent amplified products (amplicons) within a diminished polyclonal background. Two
products are produced in cases where the initial rearrangement was non-productive and was followed byrearrangement of the other homologous chromosome.
Since the antigen receptor genes are polymorphic (consisting of a heterogeneous population of related DNA
sequences), it is difficult to employ a single set of primer sequences to target all of the conserved flanking regions
around the V-D-J rearrangement. N-region diversity and somatic mutation further diversify the genetic sequences in
IGH Somatic Hypermutation Assay v2.0

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 4 of 24

these regions. Therefore multiplex master mixes, which target several regions such as the leader (L) or framework(FR) regions, are required to identify the majority of clonal rearrangements. As indicated, clonal rearrangements are
identified as prominent, single-sized products within the background of different-sized amplicon products that form a
Gaussian distribution around a statistically favored, average-sized rearrangement. As expected, primers that amplify
from the L or FR regions, produce a correspondingly different size-range of V-D-J products.

Figure 1.

Depicted is a simple representation of the organization of a rearranged immunoglobulin heavy chain gene on chromosome 14. Black
arrows represent the relative positions of primers that target the conserved Leader (L) and Framework 1 (FR1) regions, and thedownstream consensus JH gene segments.

Gel Electrophoresis Detection
Gel electrophoresis, such as agarose gel electrophoresis or non-denaturing polyacrylamide gel electrophoresisis(PAGE), is commonly used to resolve the different amplicon products based on their size, charge and conformation.
Since DNA is negatively charged, when an electrical potential (voltage) is applied across the gel containing PCR
products, the electrical field causes the amplicons to migrate through the gel. Smaller DNA fragments are able toeasily migrate through the gel matrix, whereas larger DNA fragments migrate more slowly. This causes a separationof the amplicon products based on size. Ethidium bromide or other DNA intercalating dye can then be used to stain
and detect these products in the gel.
A heteroduplex analysis can also be performed and run on a polyacrylamide gel to separate clonal and non-clonal PCR
products. A heteroduplex analysis involves denaturing the PCR products at a high temperature, then quickly reannealing the DNA strands by suddenly reducing the temperature. This causes a large portion of DNA strands toincorrectly bind to other non-homologous strands creating loops in the DNA. These loops cause a significant
reduction in the ability of the DNA to migrate through a polyacrylamide gel. However, if the majority of the PCR
products are clonal, when a heteroduplex analysis is performed, most of these PCR products will correctly re-anneal
with a homologous strand. These PCR products will run normally through the polyacrylamide gel. Therefore in aclonal sample with a polyclonal background, a heteroduplex analysis will cause most of the polyclonal product to run
much slower through the polyacrylamide gel, thereby increasing their separation and the ability to identify the clonalband(s).

Differential Fluorescence Detection
Differential fluorescence detection is commonly used to resolve the different-sized amplicon products using acapillary electrophoresis instrument. Primers can be conjugated with several different fluorescent dyes
(fluorophores) so that they can produce different emission spectra upon excitation by a laser in the capillaryelectrophoresis instrument. In this manner, different fluorescent dyes can correspond to different targeted regions.
This detection system results in unsurpassed sensitivity, single nucleotide resolution, differential product detection
and relative quantification. In addition, the use of agarose and polyacrylamide gels, as well as the use of carcinogens
such as ethidium bromide, can virtually be eliminated. Further, differential detection allows accurate, reproducible
and objective interpretation of primer-specific products and automatic archiving of data. Inter-assay and intra-assay
reproducibility in size determination using capillary electrophoresis is approximately 1 to 2 nucleotides.

IGH Somatic Hypermutation Assay v2.0

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 5 of 24

IGH Somatic Hypermutation (SHM) Analysis
Clonal PCR products are identified and are gel extracted and sequenced. Analysis for somatic hypermutation (SHM)
includes sequencing of the full variable region (FR1-FR3) or a partial variable region (CDR1-FR3) to determine
mutational status. Mutational status is determined by comparing the sequence of the IGHV region of the sample tothe most homologous germline V sequence. Sequences that differ by more than 2% from their corresponding
germline sequences are considered highly mutated whereas sequences that differ by less than 2% are consideredunmutated.4 Listed below are several websites available to aid in IGH Somatic Hypermutation Analyses:
V BASE – The MRC Centre for Protein Engineering’s Database of human antibody genesoo

http://www.vbase2.org
Analysis tools: DNAPLOTNCBI – National Center for Biotechnology Informationoo

http://www.ncbi.nlm.nih.gov/igblast/
Analysis tools: IgBLAST (Basic Local Alignment Search Tool)

IGH Somatic Hypermutation Assay v2.0

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 6 of 24

4. Reagents
Reagent Components
Table 1. Available Kits

Catalog #Description

Total Reactions

51010030

IGH Somatic Hypermutation Assay v2.0 - Gel Detection

33 Reactions

51010031

IGH Somatic Hypermutation Assay v2.0 - ABI Fluorescence Detection

33 Reactions

51010040

IGH Somatic Hypermutation Assay MegaKit v2.0 - Gel Detection

330 Reactions

51010041

IGH Somatic Hypermutation Assay MegaKit v2.0 - ABI Fluorescence Detection

330 Reactions

Table 2. Reagent Components

Reagent

Positive Control DNA
and RNA

Catalog #()
40891090
40880730

Negative (Normal)
Control DNA

40920010

Sequencing Primers

31010380

Sequencing Primers

30000000

Reagent Components(active ingredients)
IVS-0013 Clonal Control RNA
400 µg/mL of RNA in water.

IVS-0013 Clonal Control DNA

200 µg/mL of DNA in 1/10th TE solution.

IVS-0000 Polyclonal Control DNA

200 µg/mL of DNA in 1/10th TE solution.

IGH JH Primer – Unlabeled

100 µM of DNA primer in water.

Primer - Hypermutation – Unlabeled
100 µM of DNA primer in water.

Unit
Quantity

Assay Kit #of Units

Assay
MegaKit# of Units

100 µL1

5100 µL

1

5

Storage Temp.

or
100 µL

1

5

10 µL

1

5

10 µL

1

5

1500 µL

1

101500 µL

1

101500 µL

1

101500 µL

1

101500 µL

1

101500 µL

1

10orDifferential Fluorescence Detection
Hypermutation Mix 1 v2.0 – 6FAM
21010171
Master Mixes

Hypermutation Mix 2 v2.0 – 6FAM
21010181

Template
Amplification Control
Master Mix

Multiple oligonucleotides targeting the leader
region of the immunoglobulin heavy chain gene in abuffered salt solution.

20960021

Multiple oligonucleotides targeting the framework 1
region of the immunoglobulin heavy chain gene in abuffered salt solution.

Specimen Control Size Ladder – 6FAM

Multiple oligonucleotides targeting housekeeping
genes.

Gel Detection
Hypermutation Mix 1 v2.0 - Unlabeled21010170
Master Mixes

Hypermutation Mix 2 v2.0 - Unlabeled21010180

Template
Amplification Control
Master Mix
Note:

Multiple oligonucleotides targeting the leader
region of the immunoglobulin heavy chain gene in abuffered salt solution.

20960020

Multiple oligonucleotides targeting the framework 1
region of the immunoglobulin heavy chain gene in abuffered salt solution.

Specimen Control Size Ladder - Unlabeled

Multiple oligonucleotides targeting housekeeping
genes.

There are no preservatives used during the manufacturing process of this kit.

IGH Somatic Hypermutation Assay v2.0

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 7 of 24

Warnings and Precaution
















This product is For Research Use Only
Use the assay kit as a system. Do not substitute other manufacturer’s reagents. Dilution, reducing amplification
reaction volumes or other deviation in this protocol may affect the performance of this test and/or nullify any limited
sublicense that comes with the purchase of this testing kit.
Materials are stable until the labeled expiration date when stored and handled as directed. Do not use kits beyond
their expiration date.
Adherence to the protocol will assure optimal performance and reproducibility. Ensure use of correct thermal cycler
program, as other programs may provide inaccurate/faulty data, such as false positive and false negative results.
Do not mix or combine reagents from kits with different lot numbers.
Wear appropriate personal protective equipment and follow good laboratory practices and universal precautions
when working with specimens. Handle all 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 analytical sensitivity of this test, use extreme care to avoid the contamination of reagents or amplification
mixtures with samples, controls or amplified materials. Closely monitor all reagents for signs of contamination (e.g.,
negative controls giving positive signals). Discard reagents suspected of contamination.
To minimize contamination, wear clean gloves when handling samples and reagents and routinely clean work areasand pipettes prior to doing PCR.
Autoclaving does not eliminate DNA contamination. Follow uni-directional work flow in the PCR laboratory between
separate work areas; begin with Master Mix preparation, move to specimen preparation, then to the amplification andfinally to detection. Do not bring amplified DNA into the areas designated for master mix or specimen preparation.
Dedicate all pipettes, pipette tips and any equipment used in a particular area to that area of the laboratory.
Use sterile, disposable plastic ware whenever possible to avoid RNase, DNase or cross-contamination.

Storage and Handling






For any duration other than immediate use, store assay kits at -85°C to -65°C.
The optimum storage temperature for DNA controls is 2°C to 8°C, but for long term storage DNA controls can be stored
at -85°C to -65°C.
All reagents and controls must be thawed and vortexed or mixed thoroughly prior to use to ensure that they areresuspended completely. Excessive vortexing may shear DNA and cause labeled primers to lose their fluorophores.
Materials are stable until the labeled expiration date when stored and handle as directed. Do not use kits beyond their
expiration date.
Due to high salt concentrations, PCR master mixes are sensitive to freeze/thaw cycles. Aliquot master mixes into sterileo-ring screw-cap tubes if necessary.

IGH Somatic Hypermutation Assay v2.0

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 8 of 24

5. Instruments
Thermal cycler

Use or function: Amplification of DNA samples
Performance Characteristics and Specification:
o
o




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

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

Agarose or Polyacrylamide Gel Electrophoresis Unit

Use or function: DNA fragment separation
Performance Characteristics and Specification:oCapable of maintaining 35-135 V for extended times

Follow manufacturer’s installation, operation, calibration and maintenance procedures.
See section 7.5 Detection for more details.

UV Illuminator Unit

Use or function: DNA detection
Performance Characteristics and Specification:oCapable of emitting light at a wavelength of ~302 nm

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

ABI 310, ABI 3100 or ABI 3130 (For ABI Fluorescence Detection Assays)

Use or function: Fragment detection and analysis
Performance characteristics and Specification:o

The following capillary electrophoresis instruments will meet the performance needs for this assay:











ABI 310 Genetic Analyzer (1-capillary)
ABI 3100 Avant Genetic Analyzer (4-capillaries)
ABI 3100 Genetic Analyzer (16-capillaries)
ABI 3130 Genetic Analyzer (4-capillaries)
ABI 3130xL Genetic Analyzer (16-capillaries)

Follow manufacturer’s installation, operation, calibration and maintenance procedures.
Calibrate the ABI instrument with the Dye Set(s) for 6-FAM, HEX, NED, and ROX.
Use the default settings for your polymer and capillary type.
See section 7.5 Detection for more details.

IGH Somatic Hypermutation Assay v2.0

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 9 of 24

6. Specimen Collection and Preparation
Precautions
Biological specimens from humans may contain potentially infectious materials. Handle all specimens in accordancewith the OSHA Standard on Bloodborne Pathogens or Biosafety Level 2.

Interfering Substances
The following substances are known to interfere with PCR:Divalent cation chelators.
Low retention pipette tips
EDTA (not significant at low concentrations)
HeparinSpecimen Requirements and HandlingThis assay tests extracted and purified gDNA or cDNA derived from RNA. Common sources of gDNA and RNA include:

5 cc of peripheral blood, bone marrow biopsy or bone marrow aspirate anti-coagulated with heparin or EDTA (stored at
2ºC to 8ºC and shipped at ambient temperature)
Formalin-fixed paraffin embedded tissue or slides (stored and shipped at ambient temperature)

gDNA Sample Preparation
This assay can be used with either gDNA or cDNA.
6.4.1.
6.4.2.

Extract the gDNA from specimens as soon as possible.
Resuspend DNA to a final concentration of 100 µg to 400 µg per mL in 1/10th TE (1 mM Tris-HCl, pH 8.0; 0.1
mM EDTA) or in molecular biology grade or USP water.
This is a robust assay system; a wide range of DNA concentrations will generate a valid result.
Therefore, quantifying and adjusting DNA concentrations is generally not necessary.
Testing sample DNA with the Specimen Control Size Ladder master mix will ensure that DNA of sufficientquality and quantity was present to yield a valid result.

gDNA Sample Storage
Store gDNA at 2ᐤC to 8ᐤC or at -85ᐤC to -65ᐤC for long term storage.

RNA and cDNA Sample Preparation
This assay can be used with either gDNA or cDNA.
6.6.1.
6.6.2.

Extract total RNA or mRNA from specimens as soon as possible.
Resuspend RNA to a final concentration of 100 µg to 400 µg per mL in RNase-free molecular biology grade or
USP water.


6.6.3.

This is a robust assay system; a wide range of RNA concentrations will generate a valid result.
Therefore, quantifying and adjusting RNA concentrations is generally not necessary.

Synthesize cDNA from RNA, using the positive control RNA as a cDNA synthesis control (and use the cDNA fromthe positive control RNA as a positive PCR control when testing cDNA samples).

IGH Somatic Hypermutation Assay v2.0

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 10 of 24

cDNA Synthesis Protocol
6.7.1.

In a single microcentrifuge tube, mix the components following the table below:
Table 3. cDNA synthesis reaction set-up

6.7.2.
6.7.3.
6.7.4.
6.7.5.
6.7.6.

Component

Volume per sample

RNA

24.0 µL

5X SuperScript first-strand buffer

10.0 µL

25 mM dNTPs

2.0 µLwater

3.0 µL

100 mM DTT

5.0 µLrandom primers (3 µg/µL)

2.0 µL

Total volume

46.0 µL

Heat to 96ºC to 100ºC for 2 minutes and quick chill on ice.
Collect sample with a brief spin.
Add 2 µL of RNasin (5 units/µL) and mix.
Add 2 µL of SuperScript II and mix. (Final reaction volume of 50 µL.)
Place tubes at 42ºC for 90 minutes.

Note:
Use a heating block with a heated lid. If the heating block does not heat the caps, use a mineral overlay.
Otherwise condensation will change the effective concentration in the solution.
6.7.7.

Heat sample to 96ºC to 100ºC for 2 minutes and quick chill on ice. Collect sample at the bottom of the tube
with a brief spin.


6.7.8.

Use cDNA as is, it is not necessary to dilute further.

Test the sample cDNA with the Specimen Control Size Ladder master mix to ensure that cDNA of sufficientquality and quantity was present to yield a valid result.
The Specimen Control Size Ladder targets single gene exons (primers do not span intron/exon borders), and
if RNA or cDNA is contaminated with gDNA, the Specimen Control Size Ladder may amplify the gDNA andproduce results that are not indicative of the quality and quantity of the cDNA present.
Remove gDNA contamination from RNA samples with DNase; however, properly inactivate or remove the
DNase from the sample prior to cDNA synthesis.

RNA and cDNA Sample StorageStore RNA at -85ºC to -65ºC until use.
Store cDNA at 2ºC to 8ºC or at -85ºC to -65ºC for long term storage.

IGH Somatic Hypermutation Assay v2.0

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 11 of 24

7. Assay Procedure
Materials Provided
Table 4. Materials Provided

Gel Detection Kits
Catalog #ABI Fluorescence Detection Kits

Description

Catalog #Description

21010170

Hypermutation Mix 1 v2.0 - Unlabeled21010171

Hypermutation Mix 1 v2.0 – 6FAM

21010180

Hypermutation Mix 2 v2.0 – Unlabeled21010181

Hypermutation Mix 2 v2.0 – 6FAM

20960020

Specimen Control Size Ladder – Unlabeled20960021

Specimen Control Size Ladder – 6FAM

40880730

IVS-0013 Clonal Control DNA

40880730

IVS-0013 Clonal Control DNA

40891090

IVS-0013 Clonal Control RNA

40891090

IVS-0013 Clonal Control RNA

40920010

IVS-0000 Polyclonal Control DNA

40920010

IVS-0000 Polyclonal Control DNA

31010380

IGH JH Primer – Unlabeled

31010380

IGH JH Primer – Unlabeled

30000000

Primer – Hypermutation - Unlabeled

30000000

Primer – Hypermutation - Unlabeled

Materials Required (not provided)
Table 5. Materials Required (not provided)

Reagent/Material

Recommended Reagents/Materials and SuppliersPart Number

Notes
Includes 100 mM DTT and 5X
SuperScript first-strand buffer

Reverse Transcriptase

Thermo Fisher Scientific®:• SuperScript II (SSII) RT

18064-071

25mM dNTP mix

Thermo Fisher Scientific:• Nucleotide Mix, 25mM

R1121

Random Primers

Thermo Fisher Scientific:• Random Primers

Ribonuclease Inhibitor

DNA Polymerase

48190-011

Promega:• RNasin® Ribonuclease Inhibitor
Roche:• EagleTaq DNA Polymerase
Invivoscribe:• FalconTaq DNA Polymeraseor equivalent

N2115
05206944190

N/A
N/A
N/A

N/A

60970130

Molecular Biology Gradeor USP Water

N/A

N/A

DNase / RNase free

Calibrated Pipettes

N/A

N/A

Capable of accurately
measuring volumes between
1 μL and 1000 μL.

N/A

N/A
N/A

Thermal cycler

Bio-Rad:• MJ Research PTC-100 or PTC-200, PTC-220, PTC-240
Perkin-Elmer• PE 9600 or PE 9700

Vortex Mixer

N/A

N/A

PCR plates or tubes

N/A

N/A

SterileFilter barrier pipette tips

N/A

N/A

Sterile, RNase / DNase /Pyrogen-free

Microcentrifuge tubes

N/A

N/A

SterileGel Electrophoresis Unit

N/A

N/A

For agarose or
polyacrylamide gels

Agarose

Thermo Fisher Scientific:• MetaPhor Agarose• NuSieve 3:1 Agarose

BMA50181
BMA50090

IGH Somatic Hypermutation Assay v2.0

N/A

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 12 of 24
Table 5. Materials Required (not provided)

Reagent/Material

Recommended Reagents/Materials and SuppliersPart Number

Thermo Fisher Scientific:• UltraPure® 10 mg/mL Ethidium Bromide

15595-011

6% Polyacrylamide Gels

Thermo Fisher Scientific:• Novex® TBE Gels (6%, 12 well)EC62652Box

TBE Running Buffer

Thermo Fisher Scientific:• Novex TBE Running Buffer (5X)

LC6675

Gel Loading Buffer

Thermo Fisher Scientific:• 10X BlueJuice Gel Loading Buffer• Novex Hi-Density TBE Buffer (5X)

10816-015
LC6678100 bp DNA Ladder

Thermo Fisher Scientific:• TrackIt® 100 bp DNA Ladder

10488-058

ABI Capillary
Electrophoresis
Instrument

Thermo Fisher Scientific:• ABI 310, 3100 or 3130 series

Hi-Di Formamide

Thermo Fisher Scientific:• Hi-Di® Formamide

ROX Size Standards

Invivoscribe:• Hi-Di Formamide w/ROX size standards for ABI 310• Hi-Di Formamide w/ROX size standards for ABI 3100
Thermo Fisher Scientific:• GeneScan - 400HD [ROX]

Ethidium Bromide

Spectral Calibration Dye
Set D

Polymer

BufferGel Extraction Kit

TA Cloning Kit
Plasmid Isolation Kit

Thermo Fisher Scientific:• For ABI 3100 and 3130 instruments:o DS-30 Matrix Standard Kit (Dye Set D)• For ABI 310 instruments:
o NED Matrix Standardo And Fluorescent Amidite Matrix Standards [6FAM, TET,HEX, TAMRA, ROX]
Thermo Fisher Scientific:• POP-4 Polymer:o POP-4 for 310 Genetic Analyzerso POP-4 for 3100/3100-Avant Genetic Analyzerso POP-4 for 3130/3130xL Genetic Analyzers• POP-7 Polymer:o POP-7 for 3130/3130xL Genetic Analyzers

N/A

4311320

Notes
N/A
N/A
Dilute 1:5 prior to use.
N/A
N/A
N/A
N/A

60980051
60980061
4029854345827
402996

Dye set used to spectrally
calibrate ABI instrument for
use with 6-FAM, HEX, NED
and ROX

40154640283843163554352755

N/A

4352759

Thermo Fisher Scientific:• 10X Genetic Analyzer Buffer with EDTA

402824

Qiagen:• MinElute Gel Extraction Kit• QIAEX II Gel Extraction Kit

28604
20021

N/A

K4500-01

N/A

Thermo Fisher Scientific:• TOPO TA Cloning® Kit (with pCR®2.1-TOPO® vector) with
OneShot TOP10 Chemically Competent E. coli
Qiagen:• QIAprep Spin MiniPrep Kit

27104

IGH Somatic Hypermutation Assay v2.0

Dilute 1:10 in sterile water
before use

N/A

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 13 of 24

Reagent Preparation

7.3.1.
7.3.2.

Test all unknown samples with the Specimen Control Size Ladder master mix to ensure that no inhibitors of amplification
are present and there is gDNA or cDNA of sufficient quality and quantity to generate a valid result.
Test positive, negative and no template controls with each master mix.
Using gloved hands, remove the master mixes from the freezer. Allow the tubes to thaw completely; thengently vortex to mix.
In containment hood or dead air box, remove an appropriate aliquot from each master mix to individual clean,sterile microcentrifuge tubes.
Aliquot volumes are 45 µL for each reaction.
Add an additional reaction volume for every 15 reactions to correct for pipetting errors. Thus, for eachmaster mix (except for the Specimen Control Size Ladder), the number of reactions (n) is:
n =

n =




m =

7.3.3.



7.3.5.
7.3.6.

Total# of samples
+1
+1
+1

(run each sample in singlicate)
positive control DNA (IVS-0000 Polyclonal Control DNA)no template control (water)
to correct for pipetting errors# of samples + 3

Total

Therefore the total aliquot volume for the Specimen Control Size Ladder master mix is m × 45 µL.
The total Taq DNA polymerase added to each master mix is n × 0.25 µL and m × 0.25 µL for the Specimen
Control Size Ladder master mix.
Gently vortex to mix.

For each reaction, aliquot 45 µL of the appropriate master mix + DNA polymerase solution into individual wells
in a PCR plate or tube.
Add 5 µL of appropriate template (sample gDNA or cDNA, positive control DNA or cDNA, negative control DNA
or water) to the individual wells containing the respective master mix solutions. Pipette up and down several
times to mix.
Cap or cover the PCR plate.


7.3.7.

# of samples + 5

Add 1.25 units (or 0.25 µL @5 U/µL) of Taq DNA polymerase per reaction to each master mix.


7.3.4.

(run each sample in singlicate)
positive control DNA (required if testing gDNA samples)
cDNA from positive control RNA (required if testing cDNA samples)negative control DNA (IVS-0000 Polyclonal Control DNA)no template control (water)
to correct for pipetting errors

Therefore, the total aliquot volume for each master mix is n × 45 µL.
For the Specimen Control Size Ladder master mix, the number of reactions (m) can be calculated:m =

# of samples
+1
+1
+1
+1
+1

Samples are now ready to be amplified on a thermal cycler.

If amplification cannot be performed immediately following reagent preparation, the PCR plate or tubes canbe stored at 2ºC to 8ºC for up to 24 hours.
Quick Guide
For each master mix and n reactions, mix:
Master Mix
n × 45 μL
Taq DNA polymerasen × 0.25 μL
Vortex gently to mix.
Aliquot 45 μL of master mix + DNA polymerase solution into each reaction well.
Add 5 μL of appropriate Template to each well.
Total reaction volume =50 μL

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280287 Rev. F | October 2023
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Page 14 of 24

Amplification
7.4.1.

Amplify the samples using the following PCR program:Use the calculated option for temperature measurement with the BioRad MJ Research PTC thermal cyclers.
Table 6. Thermal cycling conditions

Step

Temperature

Duration

Cycles1

95ᐤC

7 minutes

1

2

95ᐤC

45 seconds

3

60ᐤC

45 seconds

4

72ᐤC

90 seconds

5

72ᐤC

10 minutes

1

15ᐤC

∞

1

6

7.4.2.
7.4.3.

35

Remove the amplification plate or tubes from the thermal cycler.
Proceed with one of the following detection methods in section 7.5 Detection.


Although amplified DNA is stable at room temperature for extended periods of time, store PCR products at
2ºC to 8ºC until detection.
Detection must be within 30 days of amplification.

Detection
Agarose Gel Detection
7.5.1.
7.5.2.
7.5.3.
7.5.4.
7.5.5.

This section is for detection of samples amplified with Unlabeled Master Mixes.
Prepare a 2% MetaPhor or NuSieve 3:1 agarose/1X TBE gel with large combs.
Place gel in electrophoresis unit and cover with 1X TBE buffer.
Mix 20 µL of each PCR product with 4 µL of 6X gel loading buffer.
Load 20 µL of this mixture into separate wells of the gel and 4 µL of the 100 bp DNA Ladder flanking thesamples.
Run at 100V for 1.5 to 2 hours.


7.5.6.
7.5.7.
7.5.8.
7.5.9.

Voltage and electrophoresis time depend on the PCR amplicon size, gel length and % of agarose in the gel.
Voltage and run time can be adapted accordingly.

Stain gel with ethidium bromide or other dye.
Place gel over UV illuminator and photograph data.
Interpret data. (See section 8: Data Interpretation)
Proceed with sequencing. (See section 7.8 Sequencing of PCR Product)

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Page 15 of 24

Polyacrylamide Gel Detection
7.5.10.
7.5.11.
7.5.12.
7.5.13.

This section is for detection of samples amplified with Unlabeled Master Mixes.
Assemble the electrophoresis unit using a non-denaturing, 6% polyacrylamide TBE gel and 1X TBE runningbuffer.
Mix 20 µL of each sample with 5 µL of ice-cold non-denaturing 5X bromophenol blue loading buffer.
Load all 20 µL of the mixture into individual wells of the gel.
Run gel at 110 V for 2-3 hours or 40-50V overnight.


7.5.14.
7.5.15.
7.5.16.
7.5.17.
7.5.18.

Voltage and electrophoresis time depend on the PCR amplicon size and polyacrylamide gel thickness.
Voltage and run time can be adapted accordingly.

Stain gels in 0.5 µg/mL ethidium bromide (in water or 0.5X TBE Buffer) for 5-10 minutes.
De-stain gels in water for 5-10 minutes. Repeat with new water.
Place gel over UV illuminator and photograph data.
Interpret data. (See section 8: Data Interpretation)
Proceed with sequencing. (See section 7.8: Sequencing of PCR Product)

Heteroduplex Analysis
This section is for detection of samples amplified with Unlabeled Master Mixes.

7.5.19.
7.5.20.
7.5.21.

Denature 20 µL of PCR products at 94ºC for 5 minutes.
Quick chill PCR products at 4ºC (on an ice water bath) for 60 minutes.
Proceed with polyacrylamide gel detection, in step 7.5.10.

ABI Fluorescence Detection
Note:
When using ABI instruments for fluorescence detection, there can often be a preceding peak, which isan artifact due to the detection method the ABI platforms use. Preceding peaks are sometimes skewed and havebases that slope on the right side towards the real peak. This is especially evident in the Specimen Control Size
Ladder master mix where the 96 nt peak has a preceding peak that occurs at 84 nt.
7.5.22.
7.5.23.
7.5.24.
7.5.25.
7.5.26.
7.5.27.
7.5.28.

In a new microcentrifuge tube, mix an appropriate amount (for a total of 10 µL per reaction) of Hi-Di
Formamide with ROX Size Standards. Vortex well.
In a new 96-well PCR plate, add 10 µL of Hi-Di Formamide with ROX size standards to individual wells for eachreaction.
Transfer 1 µL of each reaction to the wells containing Hi-Di Formamide with ROX size standards. Add only one
sample per well. Pipette up and down to mix.
Cap or cover the PCR plate or tubes.
Heat denature the samples at 95ºC for 2 minutes then snap chill on ice for 5 minutes.
Prepare a sample sheet and injection list for the samples.
Run the samples on an ABI capillary electrophoresis instrument according to the user manual.


7.5.29.
7.5.30.

Data are automatically displayed as size and color specific peaks.

Review profile and controls, report results. (see section 8: Data Interpretation)
Proceed with sequencing. (see section 7.8: Sequencing of PCR Product)

Quality Control
Positive and negative (or normal) controls are furnished with the kit and can be run in singlicate each time the assay
is performed to ensure proper performance of the assay. In addition, include a no template control (e.g. water) totest for contamination of the master mix or cross-contamination of reactions due to improper sterile technique. A
buffer control may also be added to ensure that no contamination of the buffer used to resuspend the samples has
occurred. The values for the positive controls are provided in Table 7. Additional controls and sensitivity controls(dilutions of positive controls into negative control) are available from Invivoscribe.

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280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 16 of 24

Recommended Positive Controls
The amplicon sizes listed were determined using an ABI instrument. Amplicon sizes seen on each specific capillaryelectrophoresis instrument may differ 1 to 4 nucleotides (nt) from those listed depending on the platform ofdetection and the version of the analysis software used. Once identified, the amplicon size as determined on eachspecific platform will be consistent from run to run. This reproducibility is extremely useful when monitoring disease
recurrence.
Note:“Color” indicates the color of products generated with the master mix when using the default color assignment on
ABI fluorescence detection systems.

Table 7. Recommended Positive Controls

DNA
Catalog #DNA Product Size (nt)RNA
Catalog #RNA Product
Size (nt)

Valid Size Range
IVS-0013 Clonal Control--40880730

500 - 570
536--40891090

415 – 485
461

Blue

Valid Size Range
IVS-0013 Clonal Control--40880730

310 - 380358--40891090

310 - 380
358

Blue

Valid Size Range
IVS-0000 Polyclonal Control--40920010

96 - 602
96, 197, 297, 397, 602

Master Mix

TargetColor

Hypermutation
Mix 1 v2.0

Leaderto JH

BlueHypermutation
Mix 2 v2.0

FR1 to JH

Specimen Control
Size LadderMultiple
Genes

aNote:

Control RNA and DNA

n/asame as DNA

Because smaller PCR fragments are preferentially amplified, it is not unusual for the 602 nt fragment generated by the Specimen Control Size Ladderto have a diminished signal or to be missing entirely. For ABI fluorescence detection, the 602 nt peak may not appear during normal run times. In
addition, the size of this peak may differ by over 30 nt when fragment size is extrapolated using the GeneScan - 400HD [ROX] size standards.

Sequencing of PCR Product7.8.1.

Direct Sequencing
Direct sequencing works best for samples with little to no background amplification and only one clonal
product.

7.8.1.1.

In two separate containers, prepare the appropriate concentration of each sequencing primer with the
PCR product:

7.8.1.2.
7.8.1.3.
7.8.2.

IGH JH Primer – Unlabeled andPrimer – Hypermutation – Unlabeled

Sequence the sample.
Proceed with section 11: Hypermutation Analysis and Reporting.

Gel Extraction
Use gel extraction with weak clonal bands in a polyclonal background or if there is more than one clonal band.

7.8.2.1.
Note:

After gel data has been analyzed and clonal bands identified, place gel over UV illuminator.

UV light is harmful and damaging to eyes and skin. Please use caution and wear a UV shield, lab coat and gloves
to cover eyes and exposed skin.
7.8.2.2.
7.8.2.3.
7.8.2.4.

Use a clean razor blade, scalpel or band pick to remove positive band from gel, avoid removing excess
gel and be careful not to cut into other bands as they will contaminate the sample.
Use a gel extraction kit to extract and elute the DNA in an appropriate buffer at a high concentration.
Proceed with either Direct Sequencing, above, or Cloning and Sequencing below.

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Page 17 of 24

7.8.3.

Cloning and Sequencing
Clone the band extracted PCR product using Invitrogen’s TOPO TA Cloning Kit. Please note that cloning is only
reliable when performed with unlabeled amplicons.

7.8.3.1.
7.8.3.2.
7.8.3.3.
7.8.3.4.
7.8.3.5.
7.8.3.6.
7.8.3.7.

Use 4 µL of the band extracted PCR product for the ligation reaction with the pCR2.1-TOPO vector.
Follow the manufacturer’s protocol for transforming and plating bacteria.
Pick at least 8 white colonies per band and grow in 2-4 mL of LB + ampicillin media in a shaking
incubator at 37°C overnight.
Spin each bacterial culture down at 6000 rpm in a centrifuge and pour off media.
Proceed with a plasmid DNA extraction using the QIAgen QIAprep Spin Miniprep Kit.
Sequence each plasmid sample with M13 forward and/or M13 reverse sequencing primers.
Proceed with section 11 Hypermutation Analysis and Reporting.

8. Data Interpretation
This assay is for research use only; not intended for diagnostic purposes. PCR based testing does not identify 100% of clonalcell populations; therefore, repeat testing by NGS may be advisable to rule out clonality. For accurate and meaningfulinterpretation ignore peaks that occur outside of the valid size range for each master mix.

Analysis8.1.1.
8.1.2.
8.1.3.
8.1.4.

Samples that fail to amplify following repeat testing can be reported as “A result cannot be reported on thisspecimen because there was DNA of insufficient quantity or quality for analysis”.
If samples run in duplicate yield differing results, re-test and/or re-evaluate samples for sample switching.
Examine all assay controls prior to interpretation of sample results.
The following table describes the analysis of each control and the decisions based upon the results.

Table 8. Control Analysis Summary

Type of Control

Expected Result

Aberrant Result

No Template Control

No amplification present, continue with analysis

Amplification present. Repeat the assay.

Polyclonal Control

Product size is consistent with expected size listed
in Table 9. No clonal rearrangements are present.
Continue with analysis.

Clonal rearrangements are present. Repeat theassay.

Positive Control

Product size is consistent with expected size listed(This can also be an extraction control if
positive control material is taken through in Table 9. Continue with analysis.
extraction processes)

Specimen Control Size Ladder (Thisamplification control is essential forsamples of unknown quantity andquality.)

Repeat the assay.

If all of the 96, 197, 297, 397, and 602 nt peaks areobserved, continue with analysis. Because smaller
If no bands are seen, repeat the assay. If only 1, 2,
PCR fragments are preferentially amplified, it is notor 3 bands are seen, re-evaluate sample for DNA
unusual for the 608 nt product to have adegradation unless specimen tests positive.
diminished signal or to be missing entirely.
Continue with analysis.

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Page 18 of 24

Sample Interpretation
Given that the controls produce expected results, interpret the samples as follows:
8.2.1.

One or two prominent positive peaksa within the valid size range indicates the sample is “Positive” for the“detection of clonal immunoglobulin heavy chain gene rearrangement(s), consistent with the presence of aclonal cell population.”
An absence of positive peaksa within the valid size range indicates the sample is “Negative” for the “detectionof clonal immunoglobulin heavy chain gene rearrangement(s).”8.2.2.
a

Note:

Criteria for defining a positive peak are as follows:
ABI Fluorescence Detection:
Products generated from samples that fall within the valid size range
and are at least three times the amplitude of the third largest peak in the polyclonal
background are consistent with a positive peak.
Gel Detection: Products generated from samples that fall within the valid size range and produce a
discrete band(s) distinct from any background smear are consistent with a positive
peak.





8.2.3.

Extract the positive samples from the gel and sequence (if ABI Fluorescence detection was initially used,
amplify with the unlabeled master mixes, available for purchase from Invivoscribe for gel detection and run ona gel.; see section 7.8: Sequencing of PCR Product).

9. Limitations of Procedure
The assay is subject to interference by degradation of DNA or inhibition of PCR due to heparin or other agents.

10. Expected Results
Expected Size of Amplified Products
The amplicon sizes listed were determined using an ABI platform. Amplicon sizes observed on each specific capillaryelectrophoresis instrument may differ 1 to 4 nucleotides (nt) from those listed depending on the platform ofdetection and the version of the analysis software used. Once identified, the amplicon size as determined on eachspecific platform will be consistent from run to run.
Note:

“Color” indicates the color of products generated with the master mix when using the default color assignment on
ABI fluorescence detection systems.

Table 9. Expected Size of Amplified Products

Master Mix

Hypermutation Mix 1v2.0

Hypermutation Mix 2
v2.0
Specimen Control
Size Ladder
Note:

TargetLeader to JH

FR1 to JH

Multiple
Genes

Color

Control DNA or RNA

Catalog #Blue

Valid Size Range (DNA)
IVS-0000 Polyclonal Control DNA
IVS-0013 Clonal Control DNA
Valid Size Range (RNA)
IVS-0013 Clonal Control RNA--40920010
40880730

Blue

Valid Size Range (DNA)
IVS-0000 Polyclonal Control DNA
IVS-0013 Clonal Control DNA
Valid Size Range (RNA)
IVS-0013 Clonal Control RNA--40920010
40880730

Blue

40891090

40891090

Valid Size Range (DNA & RNA)Any Human DNA or cDNA---

Product Size (nt)
500 - 570
500 - 570
536
415 - 485
461
310 - 380
310 - 380358
310 - 380358
96 – 602
96, 197, 297, 397, 602

Because smaller PCR fragments are preferentially amplified, it is not unusual for the 602 nt fragment for the Specimen Control Size Ladder to havea diminished signal or to be missing entirely. For ABI fluorescence detection the 602 nt peak may not appear during normal run times. In addition,
the size of this peak may differ by over 30 nt when the fragment size is extrapolated using the GeneScan - 400HD [ROX] size standards.

IGH Somatic Hypermutation Assay v2.0

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 19 of 24

Sample Data
Gel Detection: The data shown in Figure 2 and Figure 3 were generated using the indicated master mixes and run on a gel.

Figure 2.

Amplified products were generated using
Hypermutation Mix 1 v2.0

Figure 3.

Amplified products wereHypermutation Mix 2 v2.0.

generated

using

ABI Fluorescence Detection: The data shown in Figure 4 - Figure 6 were generated using the master mixes indicated and run on an ABI instrument.

Figure 4.

Figure 5.

Amplified products were generated using
Hypermutation Mix 1 v2.0 – 6FAM.

Amplified products were generated using
Hypermutation Mix 2 v2.0 – 6FAM.

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Page 20 of 24

Figure 6.

Products were generated using the Specimen
Control Size Ladder master mix.

11. Hypermutation Analysis and Reporting
Analysis11.1.1.

Align multiple sequence data obtained from a given sample and check for concordance. The majority of thesequences will be identical.


11.1.2.

For sequences obtained by cloning, ignore vector sequences.
For sequences obtained by direct sequencing, adjust sequences so that they are in the V to J orientation
instead of J to V.

Check sequences to ensure they represent real V-D-J product(s). This can be done with the aid of one or moreof the websites listed below:
V BASE – The MRC Centre for Protein Engineering’s Database of human antibody genesooNCBI – National Center for Biotechnology Informationoo
11.1.3.

Please note different databases may produce varying results and may have different amino acid numberingdefinitions for FR and CDR regions.

If the sequence was obtained using the Hypermutation Mix 1 v2.0, analysis of the full FR1-FR3 region canbe completed.
If the sequence was obtained using the Hypermutation Mix 2 v2.0, analysis of only the CDR1-FR3 region canbe completed.

Find the germline V region sequence that best corresponds to the sample sequence.
Align the germline V region sequence to the V region sequence of the sample.
Determine the number of mismatched bases and the total number of bases that are being compared.


11.1.7.
11.1.8.

http://www.ncbi.nlm.nih.gov/igblast/
Analysis tools: IgBLAST (Basic Local Alignment Search Tool)

After identifying a valid sequence, IGH somatic hypermutation analysis can be performed using one of thelisted websites; alternatively, this can be performed manually.


11.1.4.
11.1.5.
11.1.6.

http://www.vbase2.org
Analysis tools: DNAPLOT% 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 =𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛 𝑜𝑜𝑜𝑜 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚ℎ𝑒𝑒𝑒𝑒 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏

𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛 𝑜𝑜𝑜𝑜 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎% ℎ𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 = 100% − % 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑

Complete these steps for both the forward and reverse sequences.
If the forward and reverse sequences are discordant for mutational status, repeat the experiment.

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Page 21 of 24

IVS-0013 Clonal Control Data
Sequence data obtained from the positive IVS-0013 Clonal Control DNA or RNA will correspond to an unmutated, inframe VH1-46 to JH4 rearrangement.

Sample Data
The following sequence data is an example of an alignment using V BASE’s DNAPLOT analysis tool and the sequence
data of a sample. The sample was sequenced using the Hypermutation Mix 2, therefore only data from CDR1 to FR3(amino acids 31 to 95) was used. DNAPLOT gives five germline sequences with the highest degree of homology. In
this case, the sequence has the highest degree of homology with the germline sequence from DP-75 whichcorresponds to VH1-02.

EMBL

Locus

Z14071
X92208
Z12310
Z14213
X07448

1-02
1-02
1-02
1-02

𝑇𝑇ℎ𝑒𝑒 % 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 𝑓𝑓𝑓𝑓𝑓𝑓 𝑡𝑡ℎ𝑖𝑖𝑖𝑖 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 =

21

206

= 10.2%Name

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

DP-75/VI-2...+
1-1+
DP-8+
VHGL1.2
V35/VI-2b+

CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG GCC TCA GTG AAG GTC TCC
CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CTT GGG GCC TCA GTG AAG GTC TCC
CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG GCC TCA GTG AAG GTC TCC
CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG GCC TCA GTG AAG GTT TCC
CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG GCC TCA GTG AAG GTC TCC
H1

EMBL

Locus

Name

Z14071
X92208
Z12310
Z14213
X07448

1-02
1-02
1-02
1-02

DP-75/VI-2...+
1-1+
DP-8+
VHGL1.2
V35/VI-2b+

CDR1
22 23 24 25 26 27 28 29 30 31 31a 31b 32 33 34 35 36 37 38 39 40... ... ... ... ... ... ... ... ... GAC ... ... TAC TAT TTG CAC TGG GTG CGA CAG GCC
TGC AAG GCT TCT GGA TAC ACC TTC ACC -G- ... ... --- --- A-- --- --- --- --- --- --TGC AAG GCT TCT GGA TAC ACC TTC ACC -G- ... ... --- --- A ------------------------------------------- X- ---TGC AAG GCT TCT GGA TAC ACC TTC ACC -G- ... ... --- --- A-- --- --- --- --- --- --TGC AAG GCA TCT GGA TAC ACC TTC ACC AG- ... ... --- --- A-- --- --- --- --- --- --TGC AAG GCT TCT GGA TAC ACC TTC ACC -G- ... ... --- --- A-- --- --- --- --- --- --H2

EMBL

Locus

Name

Z14071
X92208
Z12310
Z14213
X07448

1-02
1-02
1-02
1-02

DP-75/VI-2...+
1-1+
DP-8+
VHGL1.2
V35/VI-2b+

EMBL

Locus

Name

Z14071
X92208
Z12310
Z14213
X07448

1-02
1-02
1-02
1-02

DP-75/VI-2...+
1-1+
DP-8+
VHGL1.2
V35/VI-2b+

EMBL

Locus

Name

Z14071
X92208
Z12310
Z14213
X07448

1-02
1-02
1-02
1-02

DP-75/VI-2...+
1-1+
DP-8+
VHGL1.2
V35/VI-2b+

CDR2
41 42 43 44 45 46 47 48 49 50 51 52 52a 52b 52c 53 54 55 56 57 58
CCT GGA CAA GGG TTT GAG TGG ATG GGA TGG ATC AAC CTT ... ... AAG AAT GGT GCC ATC AGG--- --- --- --- C-- --- --- --- --- --- --- --- -C- ... ... --C -G- --- -G- -CA -AC--- --- --- --- C-- --- --- --- --- --- --- --- -C- ... ... --C -G- --- -G- -CA -AC--- --- --- --- C-- --- --- --- --- --- --- --- -C- ... ... --C -G- --- -G- -CA -AC--- --- --- --- C-- --- --- --- --- --- --- --- -C- ... ... --C -G- --- -G- -CA -AC--- --- --- --- C-- --- --- --- --- C-- --- --- -C- ... ... --C -G- --- -G- -CA -AC

59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79
TAT GCA CAG AGG TTT GAG GAC AGG GTC ACC ATG ACC AGT GAC ACG TCC ATT AGC ACA GCC TAC--- --- --- -A- --- C-- -G- --- --- --- --- --- --G --- --- --- --C --- --- --- ----- --- --- -A- --- C-- -G- --- --- --- --- --- --G --- --- --- --C --- --- --- ----- --- --- -A- --- C-- -G- T-- --- --- --- --- --G --- --- --- --C --- --- --- ----- --- --- -A- --- C-- -G- --- --- --- --- --- --G --- --- --- --C --- --- --- ----- --- --- -A- --- C-- -G- --- --- --- -GT --- --G --- --- --- --C --- --- --- --80 81 82 82a 82b 82c 83 84 85 86 87 88 89 90 91 92 93 94 95
ATG GAG CTG GGC AGG CTG AAA TCA GAC GAC ACG GCT GTA TAT TAC TGT GCG AGA GAG--- --- --- A-- --- --- -G- --T --- --- --- --C --G --- --- --- --- --- ----- --- --- A-- --- --- -G- --T --- --- --- --C --G --- --- --- --- --- ----- --- --- A-- --- --- -G- --T --- --- --- --C --G --- --- --- --- --- --G-- --- --- A-- --- --- -G- --T --- --- --- --C --G --- --- --- --- --- ----- --- --- A-- --- --- -G- --T --- --- --- -TC --G --- --- --- --- --- ---IGH Somatic Hypermutation Assay v2.0

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 22 of 24

The following is the same sequence analyzed with the NCBI IgBlast tool. Again only data from CDR1 to FR3 (aminoacids 31 to 95) was used, and the sequence was also found to have the highest degree of homology with VH1-02(additional sequences were listed, but only the top three matches are shown below). IgBlast provides the percentage
of identity (or homology) given by (ID%). This sample is 90% homologous to the germline VH1-02.
% 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 = 100% − 90% = 10%ID%tmpseq_0

3

90
86
84

VH1-2
VH1-8
VH1-46

91
104
93

ID%

tmpseq_0

80

90
86
84

VH1-2
VH1-8
VH1-46

170
170
170

ID%

tmpseq_0

159

90
86
84

VH1-2
VH1-8
VH1-46

249
249
249

<-----CDR1---->--CTACTATTTGCAC
GG.......A.....
-------------..
--.......A.....

<-------------------FWR2----------------->TGGGTGCGACAGGCCCCTGGACAAGGGTTTGAGTGGATGGGA...........................C..............
...............A...........C..............
...........................C..............

<---------------------TGGATCA-ACCTTAAGAATGGTG 79.......-...C...C.G......169.....G.-...C...C.G....A 169
ATA....-...C..GTGG....A 169--CDR2-----------------------> <----------------------------------------------FWR
CCATCAGG-TATGCACAGAGGTTTGAGGAC AGGGTCACCATGACCAGTGACACGTCCATTAGCACAGCCTACATGGAGCT 158
G..CA.AC-..........A....C...G.
A..-....C..........A...CC...G.
G..CA..C-..C.......A...CC...G.

.................G...........C. . 248..A..............GA....C.....A. . 248..A..............G..........CG.......T.

248

3-------------------------------------------->GGGCAGGCTGAAATCAGACGACACGGCTGTATATTACTGTGCGAGA204.A.........G...T...........C..G.
.A....C....G...T..G........C..G.
.A....C....G...T..G........C..G.

294
294
294

The following is the same sequence analyzed with IMGT’s V-QUEST tool. Again only data from CDR1 to FR3 was used,however, IMGT has a different delineation for FR and CDR regions. Therefore the CDR1 - FR3 region used for thisanalysis corresponded to amino acids 27 through 104. Again the sequence was found to have the highest degree ofhomology with VH1-02 (additional sequences are listed).
% 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 =input
X62106 IGHV1-2*02
X92208 IGHV1-2*03
Z12310 IGHV1-2*04
X07448 IGHV1-2*01
L06612 IGHV1-46*03

25

231

= 10.8%<--------------------- F
R
1
I
M
G
T --------------1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...
CAG GTG CAG CTG GTG CAG TCT GGG GCT ... GAG GTG AAG AAG CCT GGG GCC TCACAG GTG CAG CTG GTG CAG TCT GGG GCT ... GAG GTG AAG AAG CTT GGG GCC TCACAG GTG CAG CTG GTG CAG TCT GGG GCT ... GAG GTG AAG AAG CCT GGG GCC TCACAG GTG CAG CTG GTG CAG TCT GGG GCT ... GAG GTG AAG AAG CCT GGG GCC TCACAG GTG CAG CTG GTG CAG TCT GGG GCT ... GAG GTG AAG AAG CCT GGG GCC TCA------------------------------>

input
X62106 IGHV1-2*02
X92208 IGHV1-2*03
Z12310 IGHV1-2*04
X07448 IGHV1-2*01
L06612 IGHV1-46*03____________________CDR1 – IMGT________
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36... ... ... ... ... ... ... ... AGA AAC ACA TTC AGC GAC TAC TAT ... ...
GTG AAG GTC TCC TGC AAG GCT TCT G-- T-- --C --- -C- -G- --- --- ... ...
GTG AAG GTC TCC TGC AAG GCT TCT G-- T-- --C --- -C- -G- --- --- ... ...
GTG AAG GTC TCC TGC AAG GCT TCT G-- T-- --C --- -C- -G- --- --- ... ...
GTG AAG GTC TCC TGC AAG GCT TCT G-- T-- --C --- -C- -G- --- --- ... ...
GTG AAG GTT TCC TGC AAG GCA TCT G-- T-- --C --- -C- AG- --- --- ... ...

input
X62106 IGHV1-2*02
X92208 IGHV1-2*03
Z12310 IGHV1-2*04
X07448 IGHV1-2*01
L06612 IGHV1-46*03<------------------- F
R
2
I
M
G
T
-------_________
37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54... ... TTG CAC TGG GTG CGA CAG GCC CCT GGA CAA GGG TTT GAG TGG ATG GGA... ... A-- --- --- --- --- --- --- --- --- --- --- C-- --- --- --- --... ... A-- --- --- --- -X- --- --- --- --- --- --- C-- --- --- --- --... ... A-- --- --- --- --- --- --- --- --- --- --- C-- --- --- --- --... ... A-- --- --- --- --- --- --- --- --- --- --- C-- --- --- --- --... ... A-- --- --- --- --- --- --- --- --- --- --- C-- --- --- --- ---->

input
X62106 IGHV1-2*02
X92208 IGHV1-2*03
Z12310 IGHV1-2*04
X07448 IGHV1-2*01
L06612 IGHV1-46*03

55

56

57

CDR2 - IMGT
58 59 60 61<--------------------------62

63

64

65

66

67

68

69

70

71

72

TGG ATC AAC CTT AAG AAT GGT GCC ATC ... ... AGG TAT GCA CAG AGG TTT GAG--- --- --- -C- --C -G- --- -G- -CA ... ... -AC --- --- --- -A- --- C---- --- --- -C- --C -G- --- -G- -CA ... ... -AC --- --- --- -A- --- C---- --- --- -C- --C -G- --- -G- -CA ... ... -AC --- --- --- -A- --- C-C-- --- --- -C- --C -G- --- -G- -CA ... ... -AC --- --- --- -A- --- C-ATA --- --- -C- -GT GG- --- AG- -CA ... ... --C --C --- --- -A- --C C—

IGH Somatic Hypermutation Assay v2.0

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 23 of 24

input
X62106 IGHV1-2*02
X92208 IGHV1-2*03
Z12310 IGHV1-2*04
X07448 IGHV1-2*01
L06612 IGHV1-46*03------------------------ F
73 74 75 76 77 78
79

R
80

3
81

82

I
83

M
84

G
85

T
86

-----------87 88 89... GAC AGG GTC ACC ATG ACC AGT GAC ACG TCC ATT AGC ACA GCC TAC ATG GAG... -G- --- --- --- --- --- --G --- --- --- --C --- --- --- --- --- --... -G- --- --- --- --- --- --G --- --- --- --C --- --- --- --- --- --... -G- T-- --- --- --- --- --G --- --- --- --C --- --- --- --- --- --... -G- --- --- --- -GT --- --G --- --- --- --C --- --- --- --- --- --... -G- --A --- --- --- --- --G --- --- --- -CG --- --- -T- --- --- ---------------------------------------------------------->

input
X62106 IGHV1-2*02
X92208 IGHV1-2*03
Z12310 IGHV1-2*04
X07448 IGHV1-2*01
L06612 IGHV1-46*03

90

91

92

93

94

95

96

97

98

99

______
100 101 102 103 104 105

CTG GGC AGG CTG AAA TCA GAC GAC ACG GCT GTA TAT TAC TGT GCG AGA--- A-- --- --- -G- --T --- --- --- --C --G --- --- --- --- ----- A-- --- --- -G- --T --- --- --- --C --G --- --- --- --- ----- A-- --- --- -G- --T --- --- --- --C --G --- --- --- --- ----- A-- --- --- -G- --T --- --- --- -TC --G --- --- --- --- ----- A-- --C --- -G- --T --G --- --- --C --G --- --- --- --T ---Data from VBase2’s DNAPLOT (not shown) is similar to V Base, however, Vbase2 uses the IMGT delineations for FR
and CDR regions.

12. Technical and Customer Service
Thank you for purchasing our IGH Somatic Hypermutation Assay v2.0. We appreciate your business. We are happy toassist you in the validation of this assay and will provide ongoing technical assistance to keep the assays performing
efficiently in your laboratory. Questions received during business hours usually receive a response within an hour.
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

13. References
1. Miller, JE, Wilson, SS, Jaye, DJ, Kronenberg, M. An automated semiquantiative B and T cell clonality assay. Mol. Diag. (1999)
4(2):101-117.

2. Ghia, P., et al. Geographic patterns and pathogenetic implications of IGHV gene usage in chronic lymphocytic leukemia: thelesson of the IGHV3-21 gene. Blood (2005) 105:1678-1685.

3. Fais, F., et al. Chronic Lymphocytic Leukemia B Cells Express Restricted Sets of Mutated and Unmutated Antigen Receptors.
J. Clin. Invest. (1998) 102(8):1515-1525.
4. Ghia, P., et al. ERIC recommendations on IGHV gene mutational status analysis in chronic lymphocytic leukemia. Leukemia(2007) 21:1-3.
5. Davi, F., et al. Determination of IGHV gene mutational status in chronic lymphocytic leukemia: bioinformatics advances
meet clinical needs. Leukemia (2008) 22:212-214.

IGH Somatic Hypermutation Assay v2.0

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Page 24 of 24

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

Expiration Date

Lot Number

Consult Instructions for Use

Reagent Volume

Manufacturer

Research Use Only

Storage Conditions

15. Legal Notice
Warranty and Liability
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meet or exceed the performance standards described in the Instructions For Use, as to products with such an insert. If a productis covered by product specifications and does not perform as specified, our policy is to replace the product or credit the fullpurchase price. No other warranties of any kind, expressed or implied, are provided by Invivoscribe®. Invivoscribe® liability shallnot exceed the purchase price of the product. Invivoscribe shall have no liability for direct, indirect, consequential or incidental
damages arising from the use, results of use, or inability to use its products; product efficacy under purchaser controlled
conditions in purchaser’s laboratory must be established and continually monitored through purchaser defined and controlled
processes including but not limited to testing of positive, negative, and blank controls every time a sample is tested. Ordering,
acceptance, and use of product constitutes purchaser acceptance of sole responsibility for assuring product efficacy andpurchaser agreement to the limitation of liability set forth in this paragraph.
This product is for Research Use Only; not for use in diagnostic procedures.

Patents and Trademarks
This product is covered by one or more of the following: European Patent Number 1549764, European Patent Number 2418287,
European Patent Number 2460889, Japanese Patent Number 4708029, United States Patent 8859748, and related pending andfuture applications. All of these patents and applications are licensed exclusively to Invivoscribe®. Additional patents licensed
to Invivoscribe covering some of these products apply elsewhere. Many of these products require nucleic acid amplification
methods such as Polymerase Chain Reaction (PCR). No license under these patents to use amplification processes or enzymes
is conveyed expressly or by implication to the purchaser by the purchase of this product.
©2023 Invivoscribe, Inc. All rights reserved. The trademarks mentioned herein are the property of Invivoscribe, Inc. and/or its
affiliates or (as to the trademarks of others used herein) their respective owners.

IGH Somatic Hypermutation Assay v2.0

280287 Rev. F | October 2023
For RESEARCH USE ONLY. Not for use in diagnostic procedures.