Qiagen PyroMark Q96 ID User manual

Version 5 January 2016
Sample & Assay Technologies
PyroMark®Q96 ID User Manual

Contents
PyroMark Q96 ID User Manual 01/2016 Contents-1
Contents
1Safety Information 1-1
1.1 Proper use 1-1
1.2 Electrical safety 1-2
1.3 Chemical hazards 1-3
1.4 Mechanical hazards 1-3
1.5 Heat hazards 1-4
1.6 Symbols used on the PyroMark Q96 ID 1-4
2Introduction 2-1
2.1 About this user manual 2-1
2.2 General information 2-2
2.2.1 Technical assistance 2-2
2.2.2 Policy statement 2-2
2.2.3 Version management 2-2
2.3 Intended use of the PyroMark Q96 ID 2-3
2.3.1 Requirements for PyroMark Q96 ID users 2-4
3General Description 3-1
3.1 PyroMark Q96 ID definitions 3-1
3.2 Pyrosequencing principle 3-1
3.3 PyroMark Q96 ID principle 3-3
3.4 External features of the PyroMark Q96 ID 3-5
3.5 Internal features of the PyroMark Q96 ID 3-6
3.6 Process chamber 3-6
3.7 Dispensing unit 3-6
3.8 Controls and indicators 3-7
3.8.1 Front panel 3-7

Contents
Contents-2 PyroMark Q96 ID User Manual 01/2016
3.8.2 Rear panel 3-7
3.9 Computer and software 3-8
4Installation Procedures 4-1
4.1 Instrument delivery and installation 4-1
4.2 Requirements 4-1
4.3 Moving the PyroMark Q96 ID 4-3
4.4 Reinstalling the PyroMark Q96 ID: Stand alone 4-4
4.5 Reinstalling the PyroMark Q96 ID: Network installation 4-4
4.6 Connecting mains power 4-5
4.7 Setting up network properties 4-5
4.7.1 Used IP addresses f or instrument and computer 4-5
5Operating Procedures 5-1
5.1 Required items 5-1
5.2 Starting the system 5-1
5.2.1 Starting the PyroMark Q96 ID 5-1
5.2.2 Starting the PyroMark Q96 Software 5-2
5.3 Preparing a run 5-2
5.3.1 Setting up an assay 5-2
5.3.2 Setting up a run 5-3
5.3.3 Managing instrument parameters 5-4
5.4 Sample preparation 5-6
5.4.1 Assembly of the PyroMark Q96 Vacuum Workstation 5-7
5.4.2 DNA amplification 5-7
5.4.3 Immobilization of the PCR product to beads 5-8
5.4.4 Strand separation 5-9
5.4.5 Annealing of sequencing primer to samples 5-14
5.4.6 Reagent preparation 5-14
5.5 Run 5-15
5.5.1 Loading the PyroMark Q96 Plate Low 5-15

Contents
PyroMark Q96 ID User Manual 01/2016 Contents-3
5.5.2 Loading the reagent cartridge 5-16
5.5.3 Starting the run 5-17
5.5.4 Monitoring the run 5-17
5.6 Post-run procedures 5-18
5.6.1 Analyzing the run 5-18
5.6.2 Viewing the analysis results 5-19
5.6.3 Analysis reports 5-22
5.7 Finishing work 5-23
5.7.1 After a run is finished 5-23
5.7.2Leaving the instrument on 5-23
5.7.3 Exiting PyroMark Q96 Software 5-24
5.7.4 Cleaning the instrument 5-24
5.7.5 Emptying the waste container and troughs 5-24
6PyroMark Q96 Software 6-1
6.1 System requirements 6-1
6.1.1 Operator’s and office computers 6-1
6.1.2 Printer 6-1
6.1.3 Data storage device 6-1
6.2 Installing PyroMark Q96 Software 6-1
6.3 Uninstalling PyroMark Q96 Software 6-2
6.4 Upgr ading from PyroMark Q96 ID Sof tware 6-2
6.4.1 Installing the PyroMark Data Converter Tool 6-3
6.4.2 Using the PyroMark Data Converter Tool 6-3
6.5 Data backup 6-5
6.6 Installation testing 6-5
7Maintenance 7-1
7.1 Shutdown procedure 7-2
7.2 Routine maintenance of the PyroMark Q96 ID 7-2
7.3 Cleaning the lens array 7-3

Contents
Contents-4 PyroMark Q96 ID User Manual 01/2016
7.4 Cleaning the heating block 7-3
7.5 Changing the fuses 7-4
7.6 Cleaning and testing the reagent cartridge 7-5
7.6.1 Standard cleaning procedure 7-5
7.6.2 Clogged cartridge cleaning procedure 7-6
7.6.3 Cartridge function check 7-6
7.7 Cleaning the PyroMark Q96 Vacuum Workstation 7-8
7.8 Testing and replacing the filter probes 7-9
7.8.1 Function test for filter probes 7-9
7.8.2 Replacing filter probes 7-10
7.9 Replacing the tubing 7-10
8Troubleshooting 8-1
8.1 Unexpected results 8-1
8.2 Sequence errors 8-2
8.3 Peak errors 8-3
8.4 Instrument-related errors 8-5
8.5 Vacuum workstation-related errors 8-6
9Glossary 9-1
Appendix A A-1
Technical data A-1
Environmental conditions A-1
Waste Electrical and Electronic Equipment (WEEE) A-4
FCC declaration A-5
Appendix B B-1
Assay design and validation B-1

Contents
PyroMark Q96 ID User Manual 01/2016 Contents-5
Appendix C C-1
Liability clause C-1
Appendix D D-1
Informations de sécurité D-1
Appendix E E-1
Sicherheitshinweise E-1
Index Index-1

Contents
Contents-6 PyroMark Q96 ID User Manual 01/2016
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Safety Information
PyroMark Q96 ID User Manual 01/2016 1-1
1Safety Information
Before using the PyroMark Q96 ID, it is essential that you
read this user manual carefully and pay particular attention
to the safety information. The instructions and safety
information in the user manual must be followed to ensure
safe operation of the instrument and to maintain the
instrument in a safe condition.
The following types of safety information appear throughout
this manual.
WARNING
The term WARNING is used to inform you about situations
that could result in personal injury to you or other
persons.
Details about these circumstances are given in a box like
this one.
CAUTION
The term CAUTION is used to inform you about situations
that could result in damage to the instrument or other
equipment.
Details about these circumstances are given in a box like
this one.
The advice given in this manual is intended to supplement,
not supersede, the normal safety requirements prevailing in
the user’s country.
1.1 Proper use
WARNING/
CAUTION
Risk of personal injury and material damage
[W1]
Improper use of the PyroMark Q96 ID may cause personal
injuries or damage to the instrument.
The PyroMark Q96 ID must only be operated by qualified
personnel who have been appropriately trained.
Servicing of the PyroMark Q96 ID must only be performed
by QIAGEN Field Service Specialists.

Safety Information
1-2 PyroMark Q96 ID User Manual 01/2016
WARNING/
CAUTION
Risk of personal injury and material damage
[W2]
The PyroMark Q96 ID is too heavy to be lifted by one
person. To avoid personal injury or damage to the
instrument, do not lift the instrument alone.
Perform the maintenance as described in Section 7. QIAGEN
charges for repairs that are required due to incorrect
maintenance.
1.2 Electrical safety
Disconnect the line power cord from the power outlet before
servicing.
WARNING
Electrical hazard
[W3]
Any interruption of the protective conductor (earth/ground
lead) inside or outside the instrument or disconnection of
the protective conductor terminal is likely to make the
instrument dangerous.
Intentional interruption is prohibited.
Lethal voltages inside the instrument
When the instrument is connected to line power, terminals
may be live, and opening covers or removing parts is likely
to expose live parts.
To ensure satisfactory and safe operation of the PyroMark
Q96 ID and PyroMark Q96 Vacuum Workstation, follow the
advice below:
The line power cord must be connected to a line power
outlet that has a protective conductor (earth/ground)
Keep mains plugs easily accessible in case the
equipment needs to be disconnected quickly from mains
power
Use only power supplies and cords supplied with the
instrument
When replacing the mains fuse, replace only with the
type and current rating specified on the rating label

Safety Information
PyroMark Q96 ID User Manual 01/2016 1-3
1.3 Chemical hazards
WARNING
Hazardous chemicals
[W4]
The Denaturation Solution used with the Vacuum
Workstation contains sodium hydroxide, which is irritating
to eyes and skin.
Always wear safety glasses, gloves, and a lab coat.
The responsible body (e.g., laboratory manager) must take
the necessary precautions to ensure that the surrounding
workplace is safe and that the instrument operators are not
exposed to hazardous levels of toxic substances (chemical
or biological) as defined in the applicable Safety Data
Sheets (SDSs) or OSHA*, ACGIH†, or COSHH‡documents.
For more information, visit www.qiagen.com/safety.
Venting for fumes and disposal of wastes must be in
accordance with all national, state, and local health and
safety regulations and laws.
* OSHA: Occupational Safety and Health Administration (United States of America).
†ACGIH: American Conference of Government IndustrialHygienists (United States of America).
‡COSHH: Controlof Substances Hazardous to Health (United Kingdom).
1.4 Mechanical hazards
The lid of the PyroMark Q96 ID must remain closed during
operation of the instrument.
WARNING
Moving parts
[W5]
To avoid contact with moving parts during operation of the
PyroMark Q96 ID, the instrument must be operated with
the lid closed.
Do not remove the cover panels since there are no user-
serviceable parts inside. If there is a problem with the
PyroMark Q96 ID, contact QIAGEN Technical Services
immediately.
WARNING
Pinch and impact hazards
[W6]
The dispensing unit, process chamber lid, and robot may
move at any time.

Safety Information
1-4 PyroMark Q96 ID User Manual 01/2016
WARNING
Sharp needles
[W7]
Do not touch the sharp needles at the bottom of the
reagent cartridge. Handle the needles with care. Small
particles and fibers may obstruct the needles.
1.5 Heat hazards
WARNING
Hot surface
[W8]
The external heating block can reach temperatures of up
to 80°C (176°F). Avoid touching them when they are hot.
CAUTION
Risk of overheating
[C1]
To ensure proper ventilation, maintain a minimum
clearance of 10 cm (4 in.) at the sides and rear of the
PyroMark Q96 ID.
Slits and openings that ensure the ventilation of the
PyroMark Q96 ID must not be covered.
1.6 Symbols used on the PyroMark Q96 ID
Symbol
Location
Language
Description
Type plate on
the back of
the instrument
EN
CE mark
All products
EN
Catalog or recorder
number
All products
EN
Temperature limitation
Type plate on
the back of
the instrument
and all other
products
EN
Legal manufacturer

Safety Information
PyroMark Q96 ID User Manual 01/2016 1-5
Symbol
Location
Language
Description
Type plate on
the back of
the instrument
EN
Warning, consult user
manual
Type plate on
the back of
the instrument
and box
EN
Serial number
Type plate on
the back of
the instrument
EN
Waste Electrical and
Electronic Equipment
(WEEE)
Type plate on
the back of
the instrument
EN
FCC mark of the United
States Federal
Communications
Commission
Type plate on
the back of
the instrument
EN
RCM mark for Australia
(supplier identification
N17965/N15128)
Type plate on
the back of
the instrument
EN
RoHS mark for China (the
restriction of the use of
certain hazardous
substances in electrical and
electronic equipment)
Type plate on
the back of
the instrument
EN
CSA listing mark for
Canada and the USA

Safety Information
1-6 PyroMark Q96 ID User Manual 01/2016
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Introduction
PyroMark Q96 ID User Manual 01/2016 2-1
2Introduction
Thank you for choosing the PyroMark Q96 ID. We are
confident it will become an integral part of your laboratory.
Before using the PyroMark Q96 ID, it is essential that you
read this user manual carefully and pay particular attention
to the safety information. The instructions and safety
information in the user manual must be followed to ensure
safe operation of the instrument and to maintain the
instrument in a safe condition. A French and German
translation of the safety information can be found in
Appendices D and E, respectively.
2.1 About this user manual
This user manual provides information about the PyroMark
Q96 ID in the following sections:
1. Safety Information
2. Introduction
3. General Description
4. Installation Procedures
5. Operating Procedures
6. PyroMark Q96 Software
7. Maintenance
8. Troubleshooting
9. Glossary
Appendices
The appendices include:
Technical data
Environmental conditions
WEEE recycling requirements
Assay design and validation
Warranty terms
French and German translations of safety information

Introduction
2-2 PyroMark Q96 ID User Manual 01/2016
2.2 General information
2.2.1 Technical assistance
At QIAGEN we pride ourselves on the quality and availability
of our technical support. Our Technical Services Departments
are staffed by experienced scientists with extensive practical
and theoretical expertise in sample and assay technologies
and the use of QIAGEN®products. If you have any questions
or experience any difficulties regarding the PyroMark Q96 ID
or QIAGEN products in general, do not hesitate to contact us.
QIAGEN customers are a major source of information
regarding advanced or specialized uses of our products. This
information is helpful to other scientists as well as to the
researchers at QIAGEN. We therefore encourage you to
contact us if you have any suggestions about product
performance or new applications and techniques.
For technical assistance and more information, please see
our Technical Support Center at
www.qiagen.com/goto/TechSupportCenter or call one of the
QIAGEN Technical Service Departments or local distributors
(see back cover or visit www.qiagen.com).
2.2.2 Policy statement
It is the policy of QIAGEN to improve products as new
techniques and components become available. QIAGEN
reserves the right to change specifications at any time.
In an effort to produce useful and appropriate
documentation, we appreciate your comments on this user
manual. Please contact QIAGEN Technical Services.
2.2.3 Version management
This document is the PyroMark Q96 ID User Manual, version
5.0.

Introduction
PyroMark Q96 ID User Manual 01/2016 2-3
2.3 Intended use of the PyroMark Q96 ID
The PyroMark Q96 ID System detects changes in specified
variable positions in, or base-calling of, DNA prepared from
biological samples.
The PyroMark Q96 ID Instrument and PyroMark Q96
Vacuum Workstation are intended to be used only in
combination with QIAGEN kits indicated for use with the
PyroMark Q96 ID Instrument for the applications described
in the kit handbooks.
If the PyroMark Q96 ID Instrument and PyroMark Q96
Vacuum Workstation are used with kits other than QIAGEN
kits, it is the user’s responsibility to validate the performance
of such product combination for any particular application.
The PyroMark Q96 ID System is intended for use by
professional users, such as technicians and physicians
trained in molecular biological techniques and the operation
of the PyroMark Q96 ID System.

Introduction
2-4 PyroMark Q96 ID User Manual 01/2016
2.3.1 Requirements for PyroMark Q96 ID users
The table below covers the general level of competence and
training necessary for transportation, installation, use,
maintenance, and servicing of the PyroMark Q96 ID.
Task
Personnel
Training and experience
Delivery
No special requirements
No special requirements
Installation
QIAGEN Field Service
Specialists only
Routine use
(running
protocols)
Laboratory technicians or
equivalent
Appropriately trained and
experienced personnel
familiar with use of
computers and automation
in general
Preventive
maintenance
Laboratory technicians or
equivalent
Appropriately trained and
experienced personnel
familiar with use of
computers and automation
in general
Servicing and
annual preventive
maintenance
QIAGEN Field Service
Specialists only

General Description
PyroMark Q96 ID User Manual 01/2016 3-1
3General Description
The PyroMark Q96 ID is a multi-application sequence
analysis system which uses Pyrosequencing®technology for
the real-time analysis of specific DNA sequences, including
single nucleotide polymorphisms (SNPs), mutation, and
methylation analysis. The instrument can analyze 96 samples
simultaneously. An easy-to-use protocol is used to prepare
samples.
Enclosed in the light-proof instrument housing, a light
detection system with cooled charged coupled device (CCD)
camera detects light emitted from reactions in the wells of the
PyroMark Q96 Plate Low. The instrument includes an
embedded computer module with hard disk and network
interface.
3.1 PyroMark Q96 ID definitions
PyroMark Q96 ID Instrument: Instrument only
PyroMark Q96 Software: Software only
PyroMark Q96 Vacuum Workstation: Vacuum
Workstation only
PyroMark Q96 ID: Instrument, software, and installation
PyroMark Q96 ID System: All of the above, plus any
PyroMark kits
3.2 Pyrosequencing principle
Pyrosequencing uses sequencing by synthesis for accurate
and quantitative analysis of DNA sequences.
1. A sequencing primer is hybridized to a single-stranded,
PCR-amplified DNA template.
2. The template is incubated with enzymes and substrates.
3. The first of four nucleotides is added to the reaction. If
the nucleotide is complementary to the base in the
template strand it will be incorporated into the DNA
strand by the DNA polymerase.

General Description
3-2 PyroMark Q96 ID User Manual 01/2016
4. Each incorporation event is accompanied by release of
pyrophosphate (PPi) in an equimolar quantity to the
amount of nucleotide incorporated.
5. ATP sulfurylase quantitatively converts PPi to ATP in the
presence of adenosine 5' phosphosulfate.
6. This drives the conversion of luciferin to oxyluciferin by
luciferase, generating visible light in amounts
proportional to the amount of ATP. Light is detected
using charged coupled devices (CCDs) and seen as a
peak in the Pyrogram®. Each light signal is proportional
to the number of nucleotides incorporated.
7. Apyrase, a nucleotide-degrading enzyme, continuously
degrades unincorporated nucleotides and ATP. When
degradation is complete, another nucleotide is added.
8. Nucleotides are added one at a time.
Note: Deoxyadenosine alfa-thio triphosphate (dATPαS)
is used instead of natural deoxyadenosine triphosphate
(dATP) since it is used efficiently by the DNA polymerase,
but not recognized by the luciferase.