AlleleSEQR ®

HLA Sequencing Kit

Instructions for Use

For Research Use Only
HLA-A, B, C, DRB1, DPB1, DQB1

Edition 4 2019/08
6340000
IMPORTANT NOTES AND UPDATES
Updates Edition 4

• In section ‘Volume per tube’, the 100-test MSP kit has been removed, because only a 25-test MSP kit is
available.
• A new section ‘Performance Characteristics has been included, with information on accuracy, precision,
detection limit and specificity.
• The content of section ‘Product use limitations’ has been restructured for clarity and a reference was
included to the GenDx website from where the MSDS can be downloaded. A new warning is included
about the nonamplification of allele HLA-A*03:01:158.
• The content of section ‘Specimen’ includes additional information on the required molecular weight and
purity and extraction method of the specimen.
• In section ‘Test protocol’, step 13 includes an extra sentence to explain to ‘Gently shake the mixture to
produce a homogeneous mixture’.
• Section ‘Ordering information’ now includes both the company name ‘Genome Diagnostics B.V.’ and the
trade name ‘GenDx’.

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CONTENTS

1. Key to symbols 5
2. Kit contents 6
Kit composition common components 6
Kit-specific components 6
Volume per tube 6
3. Catalog numbers 7
4. Shipping and storage 8
5. Technical assistance 8
6. Principle 8
7. Performance characteristics 8
8. Warning and precautions 10
Product application 10
Product use limitations 10
Safety information 10
9. Equipment and reagents 11
10. Specimen 11
11. Test protocol 12
PCR 12
PCR purification 13
PCR dilution 13
Sequencing reactions 13
Ethanol precipitation of sequencing reactions 14
Prepare the samples for electrophoresis 14
Data collection 15
Test interpretation 15
Test validation 15
Ordering information 16

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DISCLAIMER

GenDx has made every effort to ensure that this IFU is accurate. Information in this IFU is subject to change
without notice.

GenDx reserves the right to make improvements to this IFU and/or to the products described in this IFU, at
any time without notice.

If you find information in this manual that is incorrect, misleading, or incomplete, we would appreciate your
comments and suggestions. Please send them to info@gendx.com.

COPYRIGHT

This publication, including all photographs, illustrations, is protected under international copyright laws, with
all rights reserved. Neither this manual, nor any of the material contained herein, may be reproduced
without written consent of the author.

AmpliTaq Gold is a registered trademark of Roche Molecular Systems, Inc.

ABI Prism, Applied Biosystems, BigDye, Hi-Di, POP-6 and POP-7 are trademarks and registered trademarks of
Life Technologies Corporation or its subsidiaries in the U.S. and/or certain other countries.

AlleleSEQR, SBTengine and GenDx are trademarks and registered trademarks of Genome Diagnostics B.V.
All other trademarks are the property of their respective owners.

License and patents

This product or portions thereof is sold under license from Amersham Bioscience Corp under U.S. Patent
Numbers 5,741,676 and 5,756,285 related patents and under license from USB Corporation under U.S.
Patent Numbers 6,379,940 and 6,387,634 related patents.

The purchase of this product allows the purchaser to use it for amplification of nucleic acid sequences and
for detection of nucleic acid sequences for human in vitro diagnostics. No general patent or other license
from any kind other than this specific right of use from purchase is granted hereby. This provision does not
prohibit the resale of this product.

© Copyright 2019

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1. KEY TO SYMBOLS
Material number Taq AmpliTaq Gold
Batch code / Lot number Exo Exonuclease I
Catalogue number SAP Shrimp Alkaline Phosphatase
Consult Instructions For Use Control DNA Control DNA
Contains reagents for N tests NaoAc/EDTA NaOAc/EDTA Buffer
Legal manufacturer
Use-by date
Upper limit of storage temperature
VOL Volume

AlleleSEQR HLA-A AlleleSEQR HLA-C

HLA-A AlleleSEQR HLA-A HLA-C AlleleSEQR HLA-C
A PCR HLA-A PCR Pre-Mix C PCR HLA-C PCR Pre-Mix
A2F A Exon 2 Forward Sequencing Mix C2F C Exon 2 Forward Sequencing Mix
A2R A Exon 2 Reverse Sequencing Mix C2R C Exon 2 Reverse Sequencing Mix
A3F A Exon 3 Forward Sequencing Mix C3F C Exon 3 Forward Sequencing Mix
A3R A Exon 3 Reverse Sequencing Mix C3R C Exon 3 Reverse Sequencing Mix
A4F A Exon 4 Forward Sequencing Mix C4F C Exon 4 Forward Sequencing Mix
A4R A Exon 4 Reverse Sequencing Mix C4R C Exon 4 Reverse Sequencing Mix
AlleleSEQR HLA-A MSP AlleleSEQR HLA-C plus Additional exons
A2F98A A Exon 2 Forward 98A Sequencing Mix C1R C Exon 1 Reverse Sequencing Mix
A2F98T A Exon 2 Forward 98T Sequencing Mix C5F C Exon 5 Forward Sequencing Mix
A2F144A A Exon 2 Forward 144A Sequencing Mix C6F C Exon 6 Forward Sequencing Mix
A2F203G A Exon 2 Forward 203G Sequencing Mix C7F C Exon 7 Forward Sequencing Mix
A2F261C A Exon 2 Forward 261C Sequencing Mix C7R C Exon 7 Reverse Sequencing Mix
A2R311T A Exon 2 Reverse 311T Sequencing Mix AlleleSEQR HLA-C MSP
A3F363A A Exon 3 Forward 363A Sequencing Mix C2F105T C Exon 2 Forward 105T Sequencing Mix
A3F363G A Exon 3 Forward 363G Sequencing Mix C2F142G C Exon 2 Forward 142G Sequencing Mix
A3F414C A Exon 3 Forward 414C Sequencing Mix C2F176G C Exon 2 Forward 176G Sequencing Mix
C3F361T C Exon 3 Forward 361T Sequencing Mix
AlleleSEQR HLA-B C3R368C C Exon 3 Reverse 368C Sequencing Mix
HLA-B AlleleSEQR HLA-B C3R486G C Exon 3 Reverse 486G Sequencing Mix
B PCR HLA-B PCR Pre-Mix C3R539T C Exon 3 Reverse 539T Sequencing Mix
B2F B Exon 2 Forward Sequencing Mix C3R559A C Exon 3 Reverse 559A Sequencing Mix
B2R B Exon 2 Reverse Sequencing Mix AlleleSEQR HLA-DRB1
B3F B Exon 3 Forward Sequencing Mix HLA-DRB1 AlleleSEQR HLA-DRB1
B3R B Exon 3 Reverse Sequencing Mix DRB1 PCR HLA-DRB1 PCR Pre-Mix
B4F B Exon 4 Forward Sequencing Mix R2F DRB1 Exon 2 Forward Sequencing Mix
B4R B Exon 4 Reverse Sequencing Mix R2R DRB1 Exon 2 Reverse Sequencing Mix
AlleleSEQR HLA-B MSP R86 DRB1 Codon 86 Sequencing Mix (GTG)
B2F106A B Exon 2 Forward 106A Sequencing Mix AlleleSEQR HLA-DRB1 MSP
B2F144C B Exon 2 Forward 144C Sequencing Mix R2F124C DRB1 Exon 2 Forward 124C Sequencing Mix (Codon 13)
B2F206C B Exon 2 Forward 206C Sequencing Mix R2F124T DRB1 Exon 2 Forward 124T Sequencing Mix (Codon 13)
B2R311T B Exon 2 Reverse 311T Sequencing Mix R2F197A DRB1 Exon 2 Forward 197A Sequencing Mix (Codon 37)
B3F357C B Exon 3 Forward 357C Sequencing Mix R2R256A DRB1 Exon 2 Reverse 256A Sequencing Mix (Codon 57)
B3F357G B Exon 3 Forward 357G Sequencing Mix R2R286A DRB1 Exon 2 Reverse 286A Sequencing Mix (Codon 67)
B3R559A B Exon 3 Reverse 559A Sequencing Mix
B3R603G B Exon 3 Reverse 603G Sequencing Mix

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 AlleleSEQR HLA-DPB1 AlleleSEQR HLA-DQB1
HLA-DPB1 AlleleSEQR HLA-DPB1 HLA-DQB1 AlleleSEQR HLA-DQB1
DPB1 PCR HLA-DPB1 PCR Pre-Mix QX2 PCR DQB1 Exon 2 PCR Pre-Mix
P2F DPB1 Exon 2 Forward Sequencing Mix QX3 PCR DQB1 Exon 3 PCR Pre-Mix
P2R DPB1 Exon 2 Reverse Sequencing Mix Q2F DQB1 Exon 2 Forward Sequencing Mix
P85 DPB1 Codon 85 Sequencing Mix (GAG) Q2R DQB1 Exon 2 Reverse Sequencing Mix
AlleleSEQR HLA-DPB1 MSP Q3F DQB1 Exon 3 Forward Sequencing Mix
P2F194C DPB1 Exon 2 Forward 194C Sequencing Mix (Codon 36) Q3R DQB1 Exon 3 Reverse Sequencing Mix
P2R251A DPB1 Exon 2 Reverse 251A Sequencing Mix (Codon 55) AlleleSEQR HLA-DQB1 MSP
P2R292A DPB1 Exon 2 Reverse 292A Sequencing Mix (Codon 69) DQB1 Exon 2 Forward 134C Sequencing
Q2F134C
P2R313G DPB1 Exon 2 Reverse 313G Sequencing Mix (Codon 76) Mix (Codon 13)

2. KIT CONTENTS
Kit composition common components

Component Description Volume kit

AmpliTaq Gold Taq DNA Polymerase 1 x 40 µl All Test Kits (except MSP reagents)
1 x 25 µl All 25-Test kits (except MSP reagents)
Exo Exonuclease I
1 x 100 µl All 100-Test kits (except MSP reagents)
1 x 25 µl All 25-Test kits (except MSP reagents)
SAP Shrimp Alkaline Phosphatase
1 x 100 µl All 100-Test kits (except MSP reagents)
Control DNA Control DNA 20 ng/µl 1 x 10 µl All Test Kits (except MSP reagents)
1.5M NaOAc (ph >8.0)
NaOAc/EDTA Buffer 1 x 1.3 ml All Test Kits (except MSP reagents)
250 mM EDTA

Kit-specific components

Component
Gene-specific PCR Pre-Mixes
Exon-specific sequencing mixes
MSP reagents
Description
Gene-specific oligonucleotide primer mixes in a Tris-based PCR buffer
containing dNTPs and MgCl2.
BigDye® Terminator sequencing mix and exon-specific oligonucleotide
primers.
BigDye® Terminator sequencing mix and sequence motif-specific
oligonucleotide primers for heterozygous ambiguity resolution.

Volume per tube

Component Description # Tests Volume

25 tests 1 x 465 µl
Class I
100 tests 1 x 1680 µl
PCR Pre-Mix
25 tests 1 x 220 µl
Class II
100 tests 1 x 875 µl
25 tests 1 x 220 µl
Sequencing Mix
100 tests 1 x 840 µl
MSP 25 tests 1 x 220 µl

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3. CATALOG NUMBERS
Locus Core kit Cat. # # Tests MSP MSP Cat. # # Tests
07K38-01 25 A2F98A 07K38-10 25
07K38-02 100 A2F98T 07K38-13 25
A2F144A 07K38-16 25
A2F261C 07K38-19 25
HLA-A A2R311T 07K38-22 25
A3F363A 07K38-25 25
A3F363G 07K38-28 25
A3F414C 07K38-31 25
A2F203G 07K38-34 25
07K39-01 25 B2F106A 07K39-10 25
07K39-02 100 B2F144C 07K39-13 25
B2F206C 07K39-16 25
B2R311T 07K39-19 25
HLA-B
B3F357C 07K39-22 25
B3F357G 07K39-25 25
B3R559A 07K39-28 25
B3R603G 07K39-31 25
07K40-03 25 C2F105T 07K40-10 25
07K40-04 100 C2F142G 07K40-13 25
C2F176G 07K40-16 25
C3F361T 07K40-19 25
C3R368C 07K40-22 25
C3R486G 07K40-25 25
HLA-C C3R539T 07K40-28 25
C3R559A 07K40-31 25
C1R 07K40-61 25
C5F 07K40-63 25
Additional exons C6F 07K40-65 25
C7F 07K40-67 25
C7R 07K40-69 25
07K41-01 25 R2F124C 07K41-10 25
07K41-02 100 R2F124T 07K41-13 25
HLA-DRB1 R2F197A 07K41-16 25
R2R256A 07K41-19 25
R2R286A 07K41-22 25
07K43-01 25 P2F194C 07K43-10 25
07K43-02 100 P2R251A 07K43-13 25
HLA-DPB1
P2R292A 07K43-16 25
P2R313G 07K43-19 25
07K42-01 25 Q2F134C 07K42-10 25
HLA-DQB1
07K42-02 100

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4. SHIPPING AND STORAGE
• Do not use reagents after the expiration date indicated on the label.
• Store all reagents at -15 °C or lower.
• After first opening, all components are stable until their expiration date if they are stored at or below
-15 °C.
• Reagents can be used successfully after as many as 25 freeze/thaw cycles when stored
between -15 °C and -25 °C.
• Reagents should be returned to recommended storage temperatures immediately after use.
• Changes in the physical appearance of the kit reagents may indicate product deterioration and may
interfere with performance.

5. TECHNICAL ASSISTANCE
For technical assistance and more information:

Email: support@gendx.com
Website: www.gendx.com/support
Phone: +31 (0) 30 252 3799

or contact your local GenDx distributor (www.gendx.com).

6. PRINCIPLE
The principle of these tests relies on two key technologies:
a) PCR amplification
b) Direct automated fluorescent DNA sequencing (the detection method)

The genes of interest are amplified in a PCR amplification mixture using a hot-start DNA polymerase,
AmpliTaq Gold®. The resulting PCR amplicon is treated with exonuclease I and shrimp alkaline phosphatase
to remove unincorporated PCR primers and dNTPs. The treated amplicon serves as the DNA sequencing
template in reactions that use custom sequencing primers in a formulation with BigDye® Terminator
sequencing chemistry. The MSP reagents, ambiguity resolution sequencing mixes, incorporate motif-specific
sequencing primers with BigDye® Terminator in order to generate hemizygous sequences from a
heterozygote PCR product.

All DNA sequences are detected on an automated fluorescent DNA sequencer and the data are processed
with an allele-typing software program (for example, GenDx SBTengine® software).

7. PERFORMANCE CHARACTERISTICS
Note: References to MSP reagents within the Performance Characteristics section refer to products formerly
known as HARP® reagents.

Accuracy
In the UCLA International DNA Exchange proficiency testing program, between June 2000 and August 2003,
internal results with the AlleleSEQR® core kits yielded the results in the table below. The MSP reagents
(formerly HARP® reagent) sequencing mix summary in the following section refers to a separate study used
to determine the accuracy of the MSP reagents only.
Note: These data were generated on an ABI PRISM® 3100 Genetic Analyzer DNA Sequencer using ABI Datta
Collection v1.1 and ABI Sequence Analysis v3.7. The HLA-typing assignments were performed using

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Assign-SBTTM v3.2.7 from Conexio Genomics. The reagents have been optimized for use with the ABI9700
Thermal Cycler and demonstrated to provide acceptable results with ABI Genetic Analyzer models 310, 3100,
3130, and 3730. The Thermal Cycler and DNA sequencer instruments are required materials to run the assay
but are not provided with the AlleleSEQR HLA PCR/Sequencing Kit.

Gene No. Samples Tested Number Mistyped

HLA-A 100 0
HLA-B 100 0
HLA-C 100 0
DRB1 100 0
DQB1 100 0
DPB1 100 0

MSP reagents sequencing mixes

All MSP reagents sequencing mixes were tested against 36 DNA samples representing a total of 1224
sequences. Validation panels for HLA-A, -B, -C, -DRB1, -DPB1 and -DPB1 were designed to include DNAs that
have 0, 1, or 2 allees that match the specificity of each primer; possible sequences results included
hemizygous, heterozygous or no sequence. In 100% of the samples tested, correct sequence data and typing
results were obtained.
Accuracy testing has shown that all MSP reagents sequencing mixex will generate sequence for those alleles
that contains the sequence motif matching the specificity of the resolution primer. In certain allel
combinations the second alle may be detected at a reduced level to the true heterozygous sequence.

Precision
In UCLA DNA Exchange Number 60, 6 DNA samples were typed by 6 different laboratories for HLA-A, -B, -C, -
DRB1 with 100% concordance. Inter-lot reproducibility was established on control panels of 6-12 DNA
samples, depending on the kit, using 3 lots of each kit. The results demonstrated 100% concordance in allele
typings. All MSP reagents were tested agains three DNAs. Each DNA contained a sequence motif that
matched the specificity of the MSP reagents primer. In 100% of the samples tested, correct hemizygous-
typing results were obtained. Lot-to-lot reproducibility was verifified using an approriate panel of DNA
samples. In 100% of the samples tested, correct hemizygous typing results were obtained and all sequences
met established QC specifications.

Detection limit
The recommended amount of input DNA is 40 – 80 ng, but the PCR amplifications have been demonstrated
to work adequately with as little as 10 ng of input DNA and as much as 500 ng.

Specificity
Specificity analysis has shown that each test identifies only alleles defined by the gene-specific PCR Pre-
Mixes. For example, the HLA-A PCR Pre-Mix amplifies only HLA-A alleles and not any other HLA genes or
pseudogenes. In certain heterozygote allele combinations, it is possible to obtain a result with the core
reagents in which more than one allele typing is possible. These ambiguities may be resolved using the MSP
reagents. Specificity analysis of the MSP reagents sequencing mixes has shown that each test identifies
alleles defined by the motif-specific primer. For example, the HLA-A Exon 2 Forward 98A Seuqencing Mix will
efficiently sequence HLA-A alleles that contain an A at position 98. If the samples contain a previously
undefined allele, an indeterminate result may be obtained.

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8. WARNING AND PRECAUTIONS
Product application
For Research Use Only. Not for use in diagnostic procedures. No claim or representation is intended to
provide information for the diagnosis, prevention, or treatment of a disease.

Product use limitations

• To ensure the best performance, use the AlleleSEQR® kits with the materials, reagents and equipment
recommended in section “Equipments and reagents”. Use of materials other than specified, must be
validated by user.
• Do not mix reagents from different batches, except for MSP reagents and HLA-C plus sequencing mixes
for additional exons, which are intended to be used with multiple batches of PCR reagents.
• The AlleleSEQR HLA PCR/Sequencing kits are qualitative assays to identify the genetic variability known
to exist in the HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 genes. For the HLA-A test, non-amplification of the
allele *68:02:01:02 (*68020102) and A*03:01:158 (*0301158) has been observed in testing. For the HLA-
C test, allele C*07:06 (*0706) drop-out has been observed in exons 2 and 3, but it can be detected in
exons 4, 5 and 6. Sequencing results that initially type as homozygous should be reviewed across all
exons. In many cases, the presence of a second allele will not be missed. In general, homozygous HLA
typing results are very rare and should be confirmed by testing the sample with another method.
• The MSP reagents sequencing mixes are qualitative reagents used to resolve cis/trans-typing ambiguities
at the HLA-A, -B, -C, -DRB1, -DPB1, and -DQB1 genes. In certain allele combinations the second allele
may be detected at a reduced level relative to the true heterozygous sequence. For HLA-DQB1, some
cases of allele-preferential amplification or drop-out have been observed in exon 2, although the
heterozygous alleles can be detected in exon 3.

Safety information
• Pre- and post-PCR activities should be separated according to good PCR laboratory procedures.
• The control DNA is extracted from a standardized reference human B-lymphoblastoid cell line from the
International Histocompatibility Working Group inventory. The extracted DNA uses a number of
enzymatic steps to minimize exposure to potentially infectious material.
• Follow standard laboratory procedures to minimize exposure to reagents and to minimize the chance of
reagent contamination; for example, wear suitable personal protective equipment (e.g., safety glasses,
gloves, and protective clothing). For additional handling guidelines, consult the appropriate Material
Safety Data Sheets, available from www.gendx.com/msds.

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9. EQUIPMENT AND REAGENTS
Materials required but not provided

• Thermal cycler. Reagents have been optimized for use with ABI 9700 Thermal Cycler
• ABI Genetic Analyzer. For example, models 310, 3100, 3130, or 3730.
• Centrifuge capable of spinning 96-well trays at 2000 X g.
• Allele-typing software. For example, GenDx SBTengine® Software.
• Pipettors and disposable tips for dispensing volumes in the range of 1 µL - 800 µL.
• Thermal cycling reaction tubes for PCR and sequencing suggested for use are supplied by ABI
(for example, tubes, 96-well trays, caps, full plate covers).
• Ethanol, 100%, non-denatured (Molecular Biology grade)
• Water, sterile, deionized, RNase/DNase-free
• TE Buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0)
• Agarose gel electrophoresis reagents (agarose, ethidium bromide, 10X TBE)
• Hi-Di™ Formamide (ABI)
• Buffer (10X) with EDTA (ABI), for capillary DNA sequencers
• POP-6™ or POP-7™ Polymer (ABI)

10. SPECIMEN
• For PCR, the specimen is human genomic DNA at a concentration of 20 ng/µL in Tris/EDTA buffer,
purified by any method that produces high molecular weight DNA (25 to 100 kb or larger), as defined
by agarose gel electrophoresis, and with an A260/A280 ratio between 1.7 and 2.0.
• The recommended input for PCR is 40 - 80 ng of genomic DNA, but the test has been shown to work
reliably using as little as 10 ng of DNA or as much as 500 ng.
• Blood samples should be collected in ACD or EDTA anticoagulated tubes. Do NOT use heparinized
samples. Heparin has an inhibitory effect on a PCR.
• For MSP reagents, the specimen consists of diluted Exo and SAP-treated PCR products which were
generated with the AlleleSEQR core kit PCR reagents.

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11. TEST PROTOCOL
The following procedure applies to all AlleleSEQR® Class I and Class II kits.

PCR
1. Prepare a solution of PCR/Taq Mix.

2. Add AmpliTaq Gold to the PCR Pre-Mix according to the table below.

The AmpliTaq Gold solution is viscous. Be sure to prepare a sufficient number of reactions so that volumes
less than 1 µL of AmpliTaq Gold are not required.

Note: PCR Pre-Mix combined with AmpliTaq Gold can be stored for up to 3 months at either -15 °C to -25 °C
or +2 °C to +8 °C.

Table 1. AmpliTaq Gold PCR Pre-Mix, Class I (A, B, C)

CLASS I KITS (HLA-A, -B, -C)
# Reactions PCR Pre-Mix AmpliTaq Gold
1 16 µL 0.3 µL
10 160 µL 3.0 µL
25 400 µL 7.5 µL
100 1600 µL 30.0 µL

Table 2. AmpliTaq Gold PCR Pre-Mix Class II (DRB1, DPB1, DQB1)

CLASS II KITS (DRB1, DPB1, DQB1)
# Reactions PCR Pre-Mix AmpliTaq Gold
1 8 µL 0.1 µL
10 80 µL 1.0 µL
25 200 µL 2.5 µL
100 800 µL 10 µL

3. Add the PCR/Taq Mix to each reaction tube:

• Class I – 16 µL
• Cass II – 8 µL

Note: For DQB1, prepare both the Exon 2 and Exon 3 PCR/Taq mixes according to the table above.

4. Add genomic DNA (20 ng/µL) or sterile water (negative control) to reaction tubes containing the
PCR/Taq Mix:
• Class I – 4 µL DNA
• Class II – 2 µL DNA

Note: For DQB1, prepare two PCR amplifications per sample, one for Exon 2 and one for Exon 3.

5. Briefly centrifuge to combine the tube contents.

6. Run the following thermal cycling profile:

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 Table 3. PCR thermal cycling profile
# of Cycles Temperature Time
1 95 °C 10 minutes
96 °C 20 seconds
36 60 °C 30 seconds
72 °C 3 minutes
1 4 °C Infinite

7. Following thermal cycling, run 3 µL of PCR product on a 1% agarose gel containing 0.1 µg/mL
ethidium bromide to evaluate PCR quality. Positive amplicons should appear as discrete bands and
the negative control should not display any amplification product.

PCR purification

8. Add 1 µL of Exo and 1 µl of SAP to each reaction to eliminate residual PCR primers and dNTPs.

9. Centrifuge briefly to combine. Run the following thermal cycling profile.

Note: For DQB1, combine the Exon 2 and Exon 3 PCR products for each sample before treating with Exo and SAP.

Table 4. PCR purification thermal cycling profile.

# of Cycles Temperature Time
1 37 °C 30 minutes
80 °C 15 minutes
1
4 °C Infinite

PCR dilution

Note: This step is important to ensure sufficient PCR template for both core and MSP reagent sequencing
reactions and HLA-C plus sequencing mixes for additional exons.

10. Class I. Dilute Class I PCR product as follows:

Add 1 part PCR product to 1 part TE buffer or sterile deionized water. Mix well.

11. Class II. Dilute Class II PCR product as follows:

Add 1 part PCR product to 2 parts TE buffer or sterile deionized water. Mix well.

Sequencing reactions

The sequencing reactions for all Class I and Class II kits are prepared as follows.

12. Add 8 µL of each sequencing mix to separate reaction tubes.

13. Add 2 µL Exo and SAP treated PCR product to each reaction tube. Gently shake the mixture to
produce a homogeneous mixture and centrifuge briefly.

14. Run the following thermal cycling profile:

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 Table 5. Thermal cycling profile Sequencing

# of Cycles Temperature Time

96 °C 20 seconds
25 50 °C 30 seconds
60 °C 2 minutes
1 4 °C Infinite

Ethanol precipitation of sequencing reactions

Perform the following steps directly in the 0.2 mL sequencing reaction tubes:

15. Add 2 µL of NaOAc/EDTA buffer to each sequencing reaction.

16. Centrifuge briefly to ensure the NaOAc/EDTA buffer is properly combined with the sequencing
reactions (5 seconds at 500 X g).

17. Add 25 µL of 100% EtOH to each sequencing reaction.

18. Vortex the reaction tubes briefly, but vigorously. Note: This step is very important. Incomplete
mixing can reduce the sequence data quality.

19. Centrifuge at 2000 X g for 30 minutes.

20. Remove the supernatant by inverting the tubes/tray onto a paper towel. Centrifuge the inverted
tubes/tray at 50-100 X g for 10 seconds.

21. Add 50 µL of 80% EtOH to each sequencing reaction.

22. Centrifuge at 2000 X g for 5 minutes.

23. Repeat step 20.

Prepare the samples for electrophoresis

24. Prepare the sequencing reactions for loading onto the capillary DNA sequencer by adding 15 μL of
either of the following to each sequencing reaction tube:
• Hi-Di Formamide
• 0.3 mM EDTA in water

25. Centrifuge briefly to collect the solution at the bottom of the tubes.

26. If dissolving in Hi-Di Formamide, denature the sequencing reactions in a thermal cycler for 2 minutes
at 95 °C.

27. If dissolving in 0.3 mM EDTA, DO NOT HEAT DENATURE, simply load the reactions directly on the
instrument.

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Data collection

28. Perform data collection according to the instrument-specific parameters below.

Table 6. Mobility files and run modules

Collection
Instrument Mobility File Run Module Injection Parameters
Parameters
DTPOP6{BDSetAnyPrimer}.mob
ABI 310 SeqPOP 6Rapid (1mL)E 2.0 kV, 10 seconds 15 kV 28 minutes
or DT310POP6{BD}.mob
DT3100POP6{BD}v2.mob or 1.0-1.5 kV, 5-10 Collect for 1800
ABI 3100 RapidSeq 36_POP6_1
KB_3100_POP6_BDTv1.mob seconds seconds
1.0-1.5 kV, 5-10 Collect for 1800
ABI 3130 KB_3130_POP6_BDTv1.mob RapidSeq 36_POP6_1
seconds seconds
1.0-1.5 kV, 5-10 Collect for 800
ABI 3730b KB_3730_POP7_BDTv1.mob RapidSeq 36_POP7_1
seconds seconds
The default run modules can be customized with the recommended injection and collection parameters.
Refer to the sequencing instrument user’s manual for detailed protocols.
POP-7 polymer is not recommended for sequencing HLA-C exons 6 and 7.

Test interpretation

The sequence data are processed with an allele-typing software program to identify the HLA alleles. A software
program that is compatible with these reagents is SBTengine® software (GenDx, Netherlands). Refer to the allele-
typing software user documentation for additional information.

Test validation

Proper reagent performance with the positive control DNA will result in a strong PCR amplification reaction and
correct allele typing as defined in the table below. If a different typing is obtained, the typings of all other samples
should be reviewed as this may indicate a sample mix-up and an invalid test result.

Table 7. HLA Types for Control DNA

Gene Allele 1 Allele 2
HLA-A A*01:01 (0101) A*02:01 (0201)
HLA-B B*07:24 (0724) B*08:01 (0801)
HLA-C C*07:01 (0701) C*07:02(0702)
HLA-DRB1 DRB1*03:01 (0301) DRB1*09:01 (0901)
HLA-DQB1 DQB1*02:01 (0201) DQB1*03:03(0303)
HLA-DPB1 DPB1*04:01 (0401) DPB1*04:02(0402)

The expected result for the negative control is a lack of amplification in the PCR. If there is a positive result,
there may be a contamination problem and other typings should be reviewed carefully.

HLA-typing results by DNA sequencing are, by definition, internally controlled data. Approximately 80% of
HLA gene sequences are conserved across all alleles. Therefore, in any HLA sequence, the majority of data
points (that is, nucleotide bases) are invariant.

IFU AlleleSEQR HLA 6340000 edition 4, 2019-08 RUO 15 of 16

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IFU AlleleSEQR HLA 6340000 edition 4, 2019-08 RUO 16 of 16