EU - Conexio Genomics

EU - Conexio Genomics
Instructions for Use
Version No: 6.0
Issue Date: August 2015
EC
Conexio Genomics Pty Ltd
2/49 Buckingham Dr
Wangara 6065
Western Australia
Australia
REP
Qarad bvba
Cipalstraat 3
B-2440 Geel
Belgium
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For In-Vitro Diagnostic Use
PRINCIPLE ............................................................................................................................................ 3
INTENDED USE .................................................................................................................................... 3
KIT COMPOSITION ............................................................................................................................ 4
STORAGE REQUIREMENTS ............................................................................................................. 4
MATERIALS, REAGENTS AND EQUIPMENT NOT SUPPLIED ................................................. 4
SAMPLE REQUIREMENTS ................................................................................................................ 5
WARNINGS AND SAFETY PRECAUTIONS ................................................................................... 5
SYMBOLS .............................................................................................................................................. 6
PROCEDURE ......................................................................................................................................... 6
1.
2.
3.
4.
HARPS® SEQUENCING REACTION .............................................................................................. 6
PURIFICATION OF SEQUENCING REACTION PRODUCTS................................................................ 7
DENATURATION & ELECTROPHORESIS OF SEQUENCING REACTION PRODUCTS .......................... 7
EDITING AND ANALYSIS OF ELECTROPHEROGRAMS .................................................................... 8
PERFORMANCE CHARACTERISTICS ........................................................................................... 9
LIMITATIONS AND CAUTIONS ....................................................................................................... 9
BIBLIOGRAPHY................................................................................................................................... 9
TROUBLESHOOTING ......................................................................................................................... 9
RELATED PRODUCTS ...................................................................................................................... 12
SUPPORT AND CONTACT DETAILS............................................................................................. 14
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For In-Vitro Diagnostic Use
Principle
The SBT Resolver™ HARPS® (Heterozygous Ambiguity Resolving Primers) are sequencing
primers developed by Conexio Genomics that are used to resolve heterozygous ambiguities
by producing hemizygous sequence that enable the phase linking of HLA polymorphisms in
locus specific sequencing based HLA typing (SBT). Following SBT with SBT Resolver™
kits1, the data is analysed with Assign™ SBT sequence analysis software 2-4. Once the
software produces a HARPS® report, the PCR product is re-sequenced with the reported
HARP(S)® and the resulting sequence data is analysed with the original data to resolve the
heterozygous ambiguity.
Intended Use
Conexio Genomics’ SBT Resolver™ HARPS® kits are used to assist in the resolution of
heterozygous ambiguities arising from HLA sequencing based typing obtained from the use
of the SBT Resolver™ typing kits. Selection of the appropriate HARP is based on the
analysis of the resultant DNA sequencing data from the typing kits by Conexio Genomics’
Assign™ SBT sequence analysis software.
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For In-Vitro Diagnostic Use
Kit Composition
Each SBT Resolver™ HARPS® product is supplied as a single vial containing a single
HARP® sufficient for 20 tests (44L).
The SBT Resolver™ HARPS® product names are assigned according to the following
nomenclature system: Locus-terminal nucleotides, terminal nucleotide position-HARP
direction. Class I HARPS® contain the “C1” prefix (e.g. C1-TT98-F), while those for Class II
contain either the “RB” (HLA-DRB1), “QB” (HLA-DQB1) or “PB” (HLA-DPB1) prefixes.
For the complete list of available HARPS® please refer “Related Products” section located at
the end of this document.
Storage Requirements
When stored at -20C (temperature range of -15C to -25C is acceptable), the kit components
can be used until the indicated expiry date and can tolerate up to 25 freeze-thaw cycles.
Accelerated stability testing for the HARPS® indicated a shelf life of five years when stored at
-20°C. While confirmatory real-time testing is underway it is strongly recommended that
these HARPS® are NOT to be used beyond their expiry date.
To maintain optimal kit performance, the HARP should be removed from the -20C storage
location and thawed rapidly at room temperature before use. The HARP should then be gently
vortexed to ensure that the components of each tube are appropriately mixed after thawing.
After use, the kits/components should be returned immediately to -20C.
Materials, Reagents and Equipment Not Supplied
NOTE: The use of materials, reagents, equipment or procedures other than those detailed
within this Instructions For Use requires validation by the user prior to use.
1.
Sterile water.
2.
BigDye® Terminator Cycle Sequencing Kit v3.1 or v1.1, Applied Biosystems™ by
Life Technologies™.
3.
5x Sequencing Reaction Buffer (Conexio Genomics, product code SEQ BUF2.0(400) or SEQ BUF-2.0(5000)) or BigDye® Terminator v3.1 or v1.1 5X
Sequencing Buffer, Applied Biosystems™ by Life Technologies™.
4.
Electronic or mechanical pipettes and aerosol-resistant tips.
5.
0.2mL thin-walled thermal cycling reaction tubes (8 well strips or 96 well plates).
Use those recommended for use with your thermal cycler.
6.
Sterile 1.5mL tubes.
7.
Sterile work area.
8.
Table top centrifuge with plate adapters and capacity to reach 2500 x g.
9.
Vortex.
10.
Thermal cycler with heated lid
These HARPS® have been validated using the following thermal cyclers:
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For In-Vitro Diagnostic Use
MJ Research PTC 225 DNA Engine DYAD™, Applied Biosystems™ by Life
Technologies™ Veriti™ Thermal cycler, Gene Amp® PCR System 9700, and
Eppendorf Mastercycler® Pro.
11.
125mM EDTA, pH8.0 (Available for purchase from Conexio Genomics, product
code EDTA-3.0(200) or EDTA-3.0(5000)).
12.
Absolute and 80% Ethanol. Each run requires freshly prepared 80% ethanol
consisting of absolute ethanol and sterile water. DO NOT USE DENATURED
ETHANOL (also known as methylated spirits in some countries).
13.
Hi-Di™ Formamide, Applied Biosystems™ by Life Technologies™, product code
4311320.
14.
Automated DNA Sequencer and accessories (e.g. Applied Biosystems™ by Life
Technologies™ ABI Prism® 3730), including data collection software.
These HARPS® have been tested and validated on the Applied Biosystems™ by Life
Technologies™ 3100, 3730 and 3730xl capillary sequencers and software.
15.
HLA Sequencing Analysis Software (e.g. Assign™ SBT, version 3.6+ or higher,
Conexio Genomics Pty Ltd).
Sample Requirements
Locus-specific, ExoSAP treated amplicons prepared according to the HLA SBT
Resolver™ kit Instructions for Use1.
Warnings and Safety Precautions

This kit must be used by trained and authorized laboratory personnel.

All samples, equipment and reagents must be handled in accordance with good
laboratory practice. In particular, all patient material should be considered as
potentially infectious. The use of gloves and laboratory coats is strongly
recommended. Handle and dispose of all sample material according to local and
national regulatory guidelines.

There are NO dangerous substances contained in any of the SBT Resolver™
HARPS® products. Please refer to the MSDS that is available on the Conexio
Genomics website (http://www.conexio-genomics.com).

Do NOT use reagents beyond their expiration date.

Use of reagents or equipment not listed under “Materials, Reagents and Equipment
Not Supplied in this Kit” is NOT recommended. Such use may affect the
performance of the assay.

Care should be taken to prevent cross-contamination of specimens. Change tips
between samples wherever possible. The use of aerosol-resistant tips is highly
recommended.

Pre- and Post-PCR activities must be strictly physically separated. Use specifically
designated equipment, reagents and laboratory coats.
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For In-Vitro Diagnostic Use
Symbols
The following non-standard symbols have been used:
Symbol
Description
HARP® name
C1-TT98-F
Date of manufacture (required for
non-EU markets).
Procedure
1. HARPS® Sequencing Reaction
1.1. Prepare a fresh solution of sequencing primer mix for each HARP® on ice each time a
sequence reaction is to be performed. The composition and volumes for the mix
indicated below are per sample.
Component
Volume
HARP®
2µL
Sterile water
11.5µL
BigDye® Terminators
1µL
5x Seq Rxn Buffer
3.5µL
1.2. Mix each sequencing reaction mixture gently by pulse vortexing.
1.3. Dispense 18µL of the sequencing reaction mix into each appropriate reaction well.
NOTE: For runs which involve few samples with many different HARPS®, it is
acceptable to dispense the HARP® (2µL) directly into the individual reaction wells. A
master mix may then be created composing of sterile water, BigDye® Terminators and 5x
Seq Rxn Buffer, of which 16µL is to be dispensed into each reaction well. It is strongly
recommended that use of this alternative procedure is validated by the user prior to
implementation.
1.4. Add 2µL of purified PCR product to each appropriate well.
NOTE: Care must be taken to prevent cross-contamination of sequencing reactions.
1.5. Seal the reaction wells, mix gently and centrifuge briefly to ensure that the contents
are located at the base of each reaction well.
1.6. Place the reaction wells into a thermal cycler and run according to the following
profile:
Number of cycles
Temperature and time
25
96°C – 10 sec
50°C – 5 sec
60°C – 2 min
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For In-Vitro Diagnostic Use
Number of cycles
Temperature and time
1
4°C - hold
1.7. Once the program is complete, remove the reaction wells from the thermal cycler and
either proceed directly to purification of the reaction products or store in the dark at
4C until required. It is recommended that samples are purified and run on the DNA
sequencer within 24 hours.
2. Purification of Sequencing Reaction Products
NOTE: Purification of the reaction products may be carried out by procedures other than the
ethanol precipitation method described here. It is strongly recommended that users validate
these procedures before proceeding.
2.1. Briefly centrifuge the reaction wells/plates before proceeding. If reusable lids/caps
have been used during thermal cycling, label the lids/caps to avoid crosscontamination.
2.2. Carefully remove the seals.
2.3. To each reaction well add 5µL of 125mM EDTA, pH8.0. Ensure that the EDTA
reaches the base of the reaction well.
2.4. Add 60µL of 100% ethanol to each reaction well. Seal the wells/plate and vortex
briefly but thoroughly to ensure thorough mixing.
2.5. Pellet the extension products by centrifuging at 2000g for 45 minutes.
IMMEDIATELY PROCEED TO THE NEXT STEP. If this is not possible, recentrifuge for an additional 10 minutes before proceeding.
2.6. Remove the seals to the reaction wells and discard the supernatant by inverting the
reaction wells onto paper towel or tissues.
2.7. Place the inverted reaction wells and paper towel or tissue into the centrifuge.
Centrifuge at 350g for 1 minute to remove any residual supernatant.
2.8. Remove the reaction wells from the centrifuge and replace in an upright position on
the work bench. Discard the paper towel or tissues.
2.9. Prepare fresh solution of 80% ethanol with absolute ethanol and sterile water.
2.10. Add 60µL of 80% ethanol to each reaction well. Reseal the wells and vortex briefly.
2.11. Spin at 2000g for 5 minutes.
2.12. Repeat steps 2.6 and 2.7.
2.13. Remove the reaction wells from the centrifuge and discard the paper towel. Reseal
the reaction wells and proceed to the denaturation step. Otherwise store at -20C in
the dark. It is recommended that the extension products are run on the DNA
sequencer within 24 hours of setting up the sequencing reactions.
3. Denaturation & Electrophoresis of Sequencing Reaction Products
NOTE: The procedure for the denaturation of extension products in Hi-Di™ Formamide
described here may not be necessary if purification procedures other than ethanol
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For In-Vitro Diagnostic Use
precipitation have been used. It is strongly recommended that users validate alternative
procedures before proceeding.
3.1. Add 12µL of Hi-Di™ Formamide to each reaction well. Vortex and centrifuge the
wells/plate briefly.
3.2. Incubate the reaction wells at 98C for 5 minutes. Following incubation, ensure that
the reaction wells are cooled quickly to room temperature (e.g. place on ice or use
the thermal cycler to perform the denaturation and cooling steps) before being placed
on the sequencer. If it is not possible to run the plates immediately, store at 4C until
required.
NOTE: Ensure that there are no air bubbles in the reaction wells. These can enter and
damage the capillary.
3.3. Load the reaction wells/plate onto the automated sequencer and prepare the data
collection file according to the sequencer manufacturer specifications.
3.4. The following instrument parameters have been validated by the manufacturer using
Big Dye® Terminator Sequencing Kit v3.1 and POP-7™. These parameters may
require user validation for other polymers, sequencing chemistries and instruments.
Please refer to the appropriate instrument user’s manual for detailed instructions and
guidance (e.g. ensure that the dye set setting is appropriate for the chemistry used,
for example v1.1 Big Dye® Terminator sequencing chemistry will require a different
dye set).
Parameter
Setting
Dye set
Z_BigDyeV3
Mobility file
KB_3730_POP7_BDTV3
Basecaller
KB.bcp
Run Module
Regular FastSeq50_POP7
Injection time
15 sec
Run time
3000 sec
3.5. Use the instrument’s data collection software to process the raw collected data and
create the sequence files. Please refer to the appropriate instrument user’s manual for
detailed instructions and guidance.
4. Editing and analysis of electropherograms
The SBT Resolver™ HARPS® product range were developed and validated using the
Assign™ SBT and Assign™ ATF software developed by Conexio Genomics Pty Ltd. Users
are recommended to use Assign SBT v3.6+ and higher2-4 as these versions of the software
utilise setting and reference files specifically designed for the SBT Resolver™ typing kits and
HARPS®. For more details in relation to the operation of these software please refer to the
applicable user manuals available for download on the Conexio Genomics website
(http://www.conexio-genomics.com).
For further information regarding the Assign™ reference files to be used for analysis, please
refer to the SBT Resolver™ kit Instructions For Use1.
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Performance Characteristics
Well characterised samples that contained unresolved heterozygous ambiguities were
sequenced using the recommended HARP® reported from Assign™ SBT v3.6+ and higher.
Each HARP produced sufficient hemizygous sequence for the resolution of heterozygous
ambiguities.
Limitations and Cautions

These products are for professional use only.

It is strongly recommended that these products are validated by the user prior to
implementation in the laboratory using samples whose HLA type has been determined by
other molecular based procedures. In particular, any deviations from this procedure (e.g.
the use of alternative DNA sequencing purification procedures) must be validated by the
user prior to implementation.

AssignTM SBT, v3.6+ and higher, calculates the HARP(S)® required to resolve an
ambiguity and includes a score based on the sequence differences at the HARPS®
annealing site. The higher the score, the greater the sequence differences and the highest
probability of producing hemizygous sequence.

For further information, including exceptions and cautions regarding specific HARPS®,
please refer to SBT Resolver HARPS® Technical Notes5 downloadable from the Conexio
Genomics website (http://www.conexio-genomics.com).
Bibliography
1. SBT Resolver™ Typing Kits IFU, Conexio Genomics Pty Ltd.
2. Assign™ SBT v3.6+ Operator Manual, Conexio Genomics Pty Ltd.
3. Assign™ SBT v4.7 Operator Manual, Conexio Genomics Pty Ltd.
4. Assign™ SBT v471 Operator Manual, Conexio Genomics Pty Ltd.
5. SBT Resolver HARPS® Technical Notes, Conexio Genomics Pty Ltd.
6. Current HLA alleles can be found at http://www.ebi.ac.uk/imgt/hla.
Troubleshooting
Problem
Possible cause(s)
Solution
Weak signal intensity of
electropherograms
Weak PCR product
Check
sequence
quality
obtained from the sequencing
primers supplied with the SBT
Resolver™ typing kits.
Ensure sequencing reactions
are set up according to the
manufacturer’s instructions.
Ensure correct addition and
mixture of samples and
sequencing mixture.
Poor sequencing set up.
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For In-Vitro Diagnostic Use
Problem
Signal intensity is too
high (Presence of high
fluorescent peaks –
artefacts)
Noisy baseline (high
background)
Possible cause(s)
Solution
Insufficient reaction products Check sequencer parameters.
applied to sequencer
Injection time and voltage may
need to be increased before
reapplying samples on the
sequencer.
Problems during purification of Ensure there is sufficient
sequencing products
mixing of materials during the
purification procedure.
Use extreme care when
discarding the supernatant as it
may dislodge the pellet.
Too much PCR product
Check
sequence
quality
obtained from the sequencing
primers supplied with the SBT
Resolver™ typing kits.
Consider using a higher
dilution factor following PCR
purification.
Too much reaction products Check instrument parameters.
applied to sequencer.
Consider
reducing
the
injection time and voltage
before reapplying samples on
the sequencer.
Poor PCR purification
Check
sequence
quality
obtained from the sequencing
primers supplied with the SBT
Resolver™ typing kits.
Ensure ExoSAP treatment is
undertaken according to the
SBT Resolver™ typing kit
instructions.
Consider
using
ExoSAP
following the manufacturers
procedure (increasing the
amount of enzyme), or
consider
an
alternative
purification technique.
If
an
alternative
PCR
purification procedure was
used, ensure that the procedure
was undertaken according to
standard operating procedures.
Poor sequencing set up.
Refer to “weak signal intensity
of electropherograms”.
Contaminated sequencing
Ensure that all steps are taken
reactions
to
prevent
cross
contamination. Change pipette
tips wherever possible. Add
liquids at the top of the
reaction
wells.
Prevent
aerosols.
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For In-Vitro Diagnostic Use
Problem
Possible cause(s)
Solution
Contaminated sequencing
reagent(s)
Check
sequence
quality
obtained from the sequencing
primers supplied with the SBT
Resolver™ typing kits.
Check sequence quality of
other samples using the same
reagent batches/aliquots.
Repeat sequencing using fresh
reagents.
Refer to “weak signal intensity
of electropherograms”.
Check spectral calibration and
matrix. Repeat application of
sequencing products.
Refer to “weak signal intensity
of electropherograms”.
Ensure products are washed
sufficiently with 80% ethanol.
Ensure all traces of ethanol are
removed.
Check HARPS® score is above
recommended
threshold.
Check the SBT Resolver™
HARPS® Technical Notes5 for
any comments or exceptions
that may apply to the HARP®.
Ensure that the correct HARP®
is used.
Check sequence data obtained
from SBT Resolver™ typing
kit.
Check sequence data obtained
for other samples that were
sequenced with the same
HARP®.
Repeat sequencing reaction
and ensure that all reagents
and template have been
applied.
Ensure
procedure
is
undertaken according to the
manufacturer’s instructions.
If an alternative purification
procedure was used, ensure
that it was undertaken
according
to
standard
operating procedures.
Refer
to
“Heterozygous
sequence obtained”.
Poor purification of sequencing
products.
Poor or incorrect matrix
Presence of Dye blobs
Poor purification of sequencing
products
Heterozygous sequence
obtained.
Incorrect HARP® selected from
HARPS® report.
Incorrect HARP® used.
No sequence obtained
Random sequencing failure
Poor purification of sequencing
products
Incorrect HARP® selected or
used.
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For In-Vitro Diagnostic Use
Related Products
CE marked IVDs:
XH-PD1.1-2(20)
XH-PD1.1-2(50)
SBT Resolver™ HLA-A kit (20 and 50 tests)
BS-PD2.1-2(20)
BS-PD2.1-2(50)
SBT Resolver™ HLA-B kit (20 and 50 tests)
HH-PD5.2-5(20)
HH-PD5.2-5(50)
LG-PD5.2-7(20)
LG-PD5.2-7(50)
SBT Resolver™ HLA-DRB1 kit (20 and 50 tests)
Product codes:
C1-TT98-F(20)
C1-AC98-F(20)
C1-TC98-F(20)
C1-TA98-F(20)
C1-CA102-F(20)
C1-CT102-F(20)
C1-CC102-F(20)
C1-AG203-F(20)
C1-GT240-F(20)
C1-TT368-F(20)
C1-GG307-R(20)
C1-GT355-R(20)
C1-AT362-F(20)
C1-GG362-AR(20)
C1-GG362-R(20)
C1-GG363-AF(20)
C1-TA363-F(20)
C1-TA368-F(20)
C1-CT423-F(20)
C1-AG453-R(20)
C1-AC497-F(20)
C1-CG570-R(20)
C1-BTA-F(20)
C1-BCG-F(20)
C1-CC144-F(20)
C1-AC206-F(20)
C1-GA206-F(20)
C1-GC209-F(20)
C1-CG285-F(20)
C1-CA309-R(20)
C1-GAA309-R(20)
C1-GAT309-R(20)
C1-CG319-F(20)
C1-AG360-F(20)
C1-AC362-F(20)
C1-GC363-F(20)
C1-GG363-BF(20)
C1-CC387-F(20)
C1-TA420-F(20)
C1-CC486-F(20)
C1-AC559-R(20)
C1-CT559-R(20)
C1-GA559-R(20)
C1-CG572-R(20)
C1-GG572-R(20)
C1-GAG601-R(20)
C1-CT97-F(20)
C1-CT112-F(20)
C1-CG134-F(20)
C1-CA176-F(20)
C1-AG270-F(20)
C1-AC302-R(20)
C1-GC302-R(20)
C1-CC341-R(20)
C1-CA343-F(20)
C1-CG343-F(20)
C1-CC355-F(20)
C1-GA361-F(20)
C1-CT379-R(20)
C1-GG539-R(20)
C1-TG539-R(20)
C1-AG595-R(20)
C1-AA601-R(20)
RB-01-F(20)
RB-04-F(20)
RB-09-F(20)
RB-15-F(20)
RB-52-F(20)
RB-GG125-F(20)
RB-AA197-F(20)
RB-TT197-F(20)
RB-GT196-F(20)
RB-GA196-F(20)
RB-TA164-F(20)
RB-TT227-F(20)
RB-AT258-F(20)
RB-GC258-F(20)
RB-CT257-R(20)
RB-AT257-R(20)
RB-TT321-R(20)
RB-GT344-R(20)
RB-TG344-R(20)
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For In-Vitro Diagnostic Use
Self-certified SBT Resolver™ typing kits:
HH-PD3.2-2(20)
HH-PD3.2-2(50)
SBT Resolver™ HLA-C kit (20 and 50 tests)
PQ-PD6.2-2(20)
PQ-PD6.2-2(50)
AN-PD6.2-3(20)
AN-PD6.2-3(50)
SBT Resolver™ HLA-DQB1 kit (20 and 50 tests)
HH-PD10.1(20)
HH-PD10.1(50)
KD-PD10.2-1(20)
KD-PD10.2-1(50)
SBT Resolver™ HLA-DPB1 kit (20 and 50 tests)
Self-certified HARPS:
QB-TA122-F(20)
QB-GC134-F(20)
QB-CT173-F(20)
QB-TA173-F(20)
QB-GA316-R(20)
QB-CG353-R(20)
QB-GG353-R(20)
QB-GG361-R(20)
PB-GC112-F(20)
PB-TT113-F(20)
PB-TAC121-F(20)
PB-GC194-F(20)
PB-GT313-R(20)
PB-AG341-R(20)
PB-GG341-R(20)
QB-TA185-F(20)
PB-AT251-R(20)
Assign™ SBT software (Self-certified):
Product code: CGX0036+
Product code: CGX00470
Product code: CGX00471
0197
For Research Use Only (except Australia):
AN-PD11.0-0(20)
AN-PD11.0-0(50)
SBT Resolver™ HLA-DRB3 kit (20 and 50 tests)
AN-PD12.0-0(20)
AN-PD12.0-0(50)
SBT Resolver™ HLA-DRB4 kit (20 and 50 tests)
AN-PD13.0-0(20)
AN-PD13.0-0(50)
SBT Resolver™ HLA-DRB5 kit (20 and 50 tests)
LC-PD2.9(20)
LC-PD2.9(50)
SBT Resolver™ HLA-B57 kit (20 and 50 tests)
General Purpose Laboratory Reagents
MgCl2 – 1.0(50)
MgCl2 - 1.0(3000)
2mM MgCl2
SEQ BUF – 2.0(400)
SEQ BUF – 2.0(5000)
5x Seq Rxn Buffer
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For In-Vitro Diagnostic Use
EDTA – 3.0(200)
EDTA – 3.0(5000)
125mM EDTA, pH8.0
Please contact your local distributor for further details.
Support and Contact Details
Conexio Genomics Pty Ltd
PO Box 1294
Fremantle 6959
Western Australia
AUSTRALIA
Tel: +61-08-9336-4212
email: support@conexio-genomics.com
Skype: conexiocgx
Website: www.conexio-genomics.com
Or your local distributor
For ordering details, please refer to the Olerup website (http://www.olerup.com).
Conexio, SBT Resolver, Assign and HARPS are trademarks of Conexio 4 Pty Ltd. HARPS is
a registered trademark in some territories.
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For In-Vitro Diagnostic Use
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