Thermo Fisher Scientific Genexus User Manual
Genexus™ Integrated Sequencer
USER GUIDE
Catalog Number A45727
Publication Number MAN0017910
Revision D.0
For Research Use Only. Not for use in diagnostic procedures.
Manufacturer:
Life Technologies Holdings Pte Ltd |
Block 33 |
Marsiling Industrial Estate Road 3 |
#07-06, Singapore 739256
Manufacturer:
Life Technologies Corporation |
200 Oyster Point Blvd |
South San Francisco, CA 94080 | USA
Manufacturer:
Life Technologies Corporation |
7335 Executive Way |
Frederick, MD 21704 | USA
Product:
Genexus™ Integrated Sequencer
Product:
Genexus™ Software
Products:
GX5™ Chip and Genexus™ Coupler
Genexus™ Library Strips 1 and 2‑AS
Genexus™ Library Strips 1 and 2‑HD
Genexus™ Templating Strips 3‑GX5™ and 4
Genexus™ Barcodes 1–96 AS
Genexus™ Barcodes 1–32 HD
Genexus™ Primer Pool Tubes
Genexus™ Pipette Tips
Genexus™ Sequencing Kit
Genexus™ Controls
Genexus™ Conical Bottles
Genexus™ Filter
Genexus™ GX5™ Starter Pack‑AS
Genexus™ GX5™ Starter Pack‑HD
Oncomine™ GX assays
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0017910
Revision Date Description
D.0 25 November
2020
C.0 29 October
2020
Corrected an error in Genexus™ Software version number in “Software compatibility and requirements” on
page 11.
•
Updated the contents of the Genexus™ Installation and Training Kit. See “Genexus™ Integrated Sequencer”.
•
Corrected guidance on reuse of Genexus™ Barcodes plates. See “Guidelines for Genexus™ Integrated
Sequencer operation” on page 26.
•
Added the topic “Guidelines for expired reagents and chips” on page 27.
•
Changed the recommended concentration for manually prepared libraries from 125 pM to 200 pM.
•
Clarified use of "Do Force Clean" checkbox. See “Load the sequencer and start a run” on page 83.
•
Added screenshots for chip verification and leak checking in “Load the sequencer and start a run” on
page 83.
•
Updated “Options for an expired sequencer initialization” and moved to Chapter 7.
•
Updated recommended actions for instrument error and warning messages in “Genexus™ Integrated
Sequencer error and warning messages” on page 140.
•
Performance qualification results table updated with removal of MAPD metric to align with a new version of
the Performance Qualification Assay (v1.4.0). See “Performance Qualification results” on page 135.
B.0 7 July 2020
A.0 13 November
2019
•
Updated for Genexus™ Software 6.2.0.
•
Updated for library and template strip part numbers.
•
Updated the components of the Genexus™ Installation and Training Kit.
•
Added guidance for using inline controls for troubleshooting.
•
Added the troubleshooting section “Genexus™ Integrated Sequencer error and warning messages”.
•
Added the topic “Replace the Genexus™ Conical Bottles” on page 165.
•
Added the section “Library QC Archive: recover library preparations from the Genexus™ Integrated Sequencer
for reuse” on page 172.
New user guide for the Genexus™ Integrated Sequencer
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these
products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Microsoft
and Excel are trademarks of Microsoft Corp. Google and Chrome are trademarks of Google Inc. Ubuntu is a trademark of Canonical
Limited. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. Eppendorf and
LoBind are trademarks of Eppendorf AG. Agencourt and AMPure are trademarks of Beckman Coulter, Inc.
©2020 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■
CHAPTER 1 Product information .................................................. 9
Product description ............................................................. 9
Genexus™ Integrated Sequencer ................................................. 10
Software compatibility and requirements .......................................... 11
Reagents and supplies—Ion AmpliSeq™ library chemistry ........................... 11
Genexus™ Library Strips 1 and 2‑AS ......................................... 11
Genexus™ Barcodes AS .................................................... 12
Genexus™ GX5™ Starter Pack‑AS ............................................ 12
Reagents and supplies—Ion AmpliSeq™ HD library chemistry ........................ 13
Genexus™ Library Strips 1 and 2‑HD ......................................... 13
Genexus™ Barcodes 1–32 HD ............................................... 14
Genexus™ GX5™ Starter Pack‑HD ............................................ 14
Shared reagents and supplies ................................................... 15
Genexus™ Templating Strips 3‑GX5™ and 4 ................................... 15
Genexus™ Primer Pool Tubes and Pipette Tips ................................ 15
GX5™ Chip and Genexus™ Coupler .......................................... 16
Genexus™ Sequencing Kit .................................................. 16
Genexus™ Conical Bottles .................................................. 17
Genexus™ Filter ........................................................... 17
Genexus™ Controls ........................................................ 17
Oncomine™ GX assays ......................................................... 18
Required materials not supplied ................................................. 19
Recommended materials for nucleic acid isolation and quantification ................. 19
Genexus™ Integrated Sequencer components ..................................... 21
Genexus™ Integrated Sequencer deck stations .................................... 22
Genexus™ Integrated Sequencer input and output connections ...................... 23
Workflow ..................................................................... 24
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Genexus™ Integrated Sequencer User Guide
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CHAPTER 2 Before you begin .................................................... 25
Precautions ................................................................... 25
Avoid nucleic acid contamination ............................................ 25
Avoid chip damage ........................................................ 25
Avoid strong electromagnetic radiation ....................................... 25
Protection by equipment .................................................... 25
Guidelines for Genexus™ Integrated Sequencer operation ........................... 26
Guidelines for expired reagents and chips ........................................ 27
Power the Genexus™ Integrated Sequencer on or o ............................... 28
Power on ................................................................. 28
Power o ................................................................. 28
Get started with Genexus™ Software ............................................. 29
About the Genexus™ Software user interface ................................. 29
User-access levels ......................................................... 30
System tracking ........................................................... 30
Request and sign in to a new account ........................................ 30
Sign in ................................................................... 31
Contents
■
CHAPTER 3 Create and manage assays (manager/administrator) ............ 32
About assays in Genexus™ Software ............................................. 32
System-installed assays ........................................................ 33
Manage assays (manager/administrator) .......................................... 33
Create a new assay (manager/administrator) ....................................... 35
Copy an assay (manager/administrator) ........................................... 43
Import an assay (manager/administrator) .......................................... 44
■
CHAPTER 4 Enter samples and libraries ........................................ 46
Create a new sample ........................................................... 46
System-installed sample attributes .......................................... 47
Create a custom sample attribute (manager/ administrator) ...................... 48
Import samples ................................................................ 49
Download an example samples file .......................................... 50
Manage samples ............................................................... 51
Sort, search, and filter samples .............................................. 51
Export samples ............................................................ 52
View notes or add a note to a sample ........................................ 52
Edit a sample (manager/ administrator) ....................................... 52
Edit a sample and amend a report after a run (manager/ administrator) ............ 53
Genexus™ Integrated Sequencer User Guide
5
Contents
■
■
Review sample edit history .................................................. 54
Delete samples ............................................................ 54
Prepare or import a library batch ................................................. 55
Prepare a library batch ..................................................... 55
Import a library batch ...................................................... 58
CHAPTER 5 Plan and manage runs ............................................. 61
Plan a sample run .............................................................. 62
Plan a library run ............................................................... 70
CHAPTER 6 Dilute the samples and load the sample plate ................... 75
Guidelines for nucleic acid isolation and quantification—sample runs ................. 75
Dilute or concentrate the samples (if needed) and load the sample plate—sample run .. 76
Guidelines for library quantification—library runs ................................... 77
Dilute and pool libraries, and load the sample plate—library run ...................... 78
■
CHAPTER 7 Load the sequencer and start a run ............................... 79
Before you begin ............................................................... 79
Fill Genexus™ Primer Pool Tubes (custom assays only) .............................. 81
Load the sequencer and start a run .............................................. 83
Clear the instrument deck and perform a UV Clean ................................. 90
Options for an expired sequencer initialization ..................................... 93
■
CHAPTER 8 Monitor the run ...................................................... 94
View run progress on the instrument .............................................. 94
■
CHAPTER 9 Review data and results ............................................ 95
Review sample results .......................................................... 96
Review run results ............................................................. 97
Assign PCR Plate .......................................................... 98
Lab Report .................................................................... 99
Download the Lab Report ................................................. 100
View sequencing results and assay metrics ...................................... 100
Summary of the Sample Results ............................................ 102
Assay metrics ............................................................ 103
QC results ............................................................... 107
View SNV/INDEL results ................................................... 112
View Fusion results ....................................................... 114
View CNV results ......................................................... 119
View annotation sources ................................................... 121
6
Genexus™ Integrated Sequencer User Guide
Create and assign variant classifications ..................................... 121
Filter results .............................................................. 122
View Oncomine™ TCR Beta‑LR Assay GX run results .......................... 124
Reanalyze a run ............................................................... 126
Sign o on the run results (manager/ administrator) ................................ 127
Generate customized reports ................................................... 128
Results files .................................................................. 130
Review coverageAnalysis plugin results .......................................... 132
Upload sample results files to Ion Reporter™ Software ............................. 132
Verification runs .............................................................. 133
View and sign o on the verification run results ............................... 134
■
APPENDIX A Troubleshooting .................................................. 137
Troubleshoot Genexus™ Integrated Sequencer performance with CF-1 and
inline controls .............................................................. 137
Genexus™ Integrated Sequencer—general and QC troubleshooting ................. 138
Genexus™ Integrated Sequencer error and warning messages ...................... 140
Genexus™ Software ........................................................... 144
Contents
■
APPENDIX B Touchscreen reference ........................................... 147
Touchscreen icons ............................................................ 147
Settings ..................................................................... 149
Network Settings ......................................................... 149
Perform a Clean instrument procedure ...................................... 152
System Tools ............................................................ 154
Data Management ........................................................ 158
Instrument settings ....................................................... 160
■
APPENDIX C Supplemental information ....................................... 163
Maintain the sequencer ........................................................ 163
Materials required ........................................................ 163
Clean or decontaminate the sequencer ...................................... 164
Replace the Genexus™ Filter ............................................... 164
Replace the Genexus™ Conical Bottles ...................................... 165
Genexus™ Integrated Sequencer power o and power on before and after a
long-term shutdown .................................................... 169
Quantify FFPE DNA with the Qubit™ Fluorometer .................................. 171
Library QC Archive: recover library preparations from the Genexus™ Integrated
Sequencer for reuse ......................................................... 172
Required materials and equipment .......................................... 172
Recover libraries from the sequencer and purify .............................. 172
Quantify the purified libraries ............................................... 175
Genexus™ Integrated Sequencer User Guide
7
Contents
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■
Combine libraries ......................................................... 175
Store libraries ............................................................ 175
Guidelines for using custom assays with the Genexus™ Integrated Sequencer ........ 176
Planning sequencing runs for ecient use of consumables ......................... 178
Configure an Ion Reporter™ Server account (administrator) ......................... 179
Tag an Ion Reporter™ Software analysis workflow for use with the
IonReporterUploader plugin .............................................. 181
Configure Thermo Fisher Accounts in Genexus™ Software (administrator) ............ 182
APPENDIX D coverageAnalysis plugin in Genexus™ Software .............. 184
Reads statistics .............................................................. 184
Example Coverage Analysis Report ............................................. 187
Example charts generated by the coverageAnalysis plugin ......................... 189
Output files generated by the coverageAnalysis plugin ............................. 190
APPENDIX E Safety .............................................................. 193
Symbols on this instrument .................................................... 193
Standard safety symbols .................................................. 193
Additional safety symbols ................................................. 194
Location of safety labels ................................................... 195
Control and connection symbols ........................................... 196
Conformity symbols ...................................................... 196
Instrument safety ............................................................. 197
General ................................................................. 197
Physical injury ............................................................ 197
Electrical safety .......................................................... 198
Cleaning and decontamination ............................................. 199
Instrument component and accessory disposal .............................. 199
Safety and electromagnetic compatibility (EMC) standards ......................... 200
Safety standards ......................................................... 200
EMC standards ........................................................... 200
Environmental design standards ............................................ 201
Radio compliance standards ............................................... 201
Chemical safety .............................................................. 203
Biological hazard safety ....................................................... 205
■
APPENDIX F Documentation and support ..................................... 206
Related documentation ........................................................ 206
Customer and technical support ................................................ 206
Limited product warranty ...................................................... 207
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Genexus™ Integrated Sequencer User Guide
1
Product information
Product description .................................................................... 9
■
Genexus™ Integrated Sequencer ....................................................... 10
■
Software compatibility and requirements ................................................ 11
■
Reagents and supplies—Ion AmpliSeq™ library chemistry ................................. 11
■
Reagents and supplies—Ion AmpliSeq™ HD library chemistry .............................. 13
■
Shared reagents and supplies .......................................................... 15
■
Oncomine™ GX assays ............................................................... 18
■
Required materials not supplied ........................................................ 19
■
Recommended materials for nucleic acid isolation and quantification ....................... 19
■
Genexus™ Integrated Sequencer components ........................................... 21
■
Genexus™ Integrated Sequencer deck stations .......................................... 22
■
Genexus™ Integrated Sequencer input and output connections ............................ 23
■
Workflow ............................................................................ 24
■
IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.
Product description
The Ion Torrent™ Genexus™ Integrated Sequencer is a next-generation (NGS) sequencing system that
integrates library preparation, template preparation, and sequencing into a single-day, single-instrument
automated run. The instrument supports both sample-to-results and library-to-results sequencing runs
(up to 32 DNA or RNA samples/run). Ion Torrent™ Genexus™ Software streamlines the NGS workflow by
integrating the setup-to-report workflow within a single software system. Key features include:
•
Go from nucleic acid to report in a single day
•
Flexible and cost-eective run planning making use of a multi-lane, multi-run sequencing chip: the
Ion Torrent™ GX5™ Chip
•
Automated library preparation, including cDNA synthesis, for up to 400 base-read libraries using
either standard Ion AmpliSeq™ or Ion AmpliSeq™ HD library chemistry
•
Automated Ion Sphere™ Particle (ISP)-loading and template preparation with on-chip amplification
•
Support for up to four compatible assays on a GX5™ Chip in a single run, with an output of 12–
15 million reads from each lane.
•
As little as five minutes total hands-on time required per run
•
Real-time consumables tracking by the instrument to guide consumable loading during run setup
Genexus™ Integrated Sequencer User Guide
9
Chapter 1 Product information
1
Genexus™ Integrated Sequencer
•
Consumables that are usable for up to 14 days after loading
•
On-instrument sequencing data analysis requiring no external server
Genexus™ Integrated Sequencer
The Genexus™ Integrated Sequencer includes the following components.
Components Cat. No.
Genexus™ Integrated Sequencer A45727
Genexus™ Installation and Training Kit A40278
[1]
N
ot available for separate purchase.
[1]
The Ion Torrent™ Genexus™ Installation and Training Kit (Cat. No. A40278) is available to first-time
owners of a Genexus™ Integrated Sequencer and is shipped with the instrument. The kit contains the
following reagents, supplies, and controls that are used during the installation, training, and operation of
the instrument.
Genexus™ Installation and Training Kit
Contents Part No. Quantity Storage
Genexus™ Controls A40267 1 kit −30°C to −10°C
Genexus™ Strip 1 A46812 8 strips 2°C to 8°C
Genexus™ Strip 2‑AS A46813 8 strips −30°C to −10°C
Genexus™ Barcodes 1–32 AS A40258 1 plate 15°C to 30°C
Genexus™ Strip 3‑GX5
Genexus™ Strip 4 A46816 8 strips −30°C to −10°C
Genexus™ Cartridge A40272 2 cartridges −30°C to −10°C
™
A46815 8 strips 2°C to 8°C
10
Genexus™ Bottle 2 A40273 4 bottles
Genexus™ Bottles 1 and 3 A40274 2 bottles each
Genexus™ Pipette Tips A40266 12 racks
Genexus™ Conical Bottles A40275 2 sets of 5 bottles
Genexus™ Filter A40302 1 filter
GX5™ Chip and Genexus™ Coupler A40269 2 each
Adhesive PCR Plate Foils AB0626 1 box (100 foils)
Genexus™ Integrated Sequencer User Guide
15°C to 30°C
Chapter 1 Product information
Software compatibility and requirements
Software compatibility and requirements
The procedures in this guide are designed for use with Genexus™ Software 6.2.0 or later. Versionspecific information is provided in the software release notes for your version of the software. An
administrator-level user can view the software version in the (Settings) / Software Updates screen.
Genexus™ Software is supported on Google™ Chrome™ browser version 64 and later and is best
viewed with 1440 × 900 screen resolution. Google™ Chrome™ browser is recommended for use with the
software.
The operating system of the sequencer is Ubuntu™ 18.041.
For more information on using the software, see the online Genexus™ Software 6.2 Help.
Reagents and supplies—Ion AmpliSeq™ library chemistry
Genexus™ Integrated Sequencer reagents and supplies can be ordered in convenient combo kits and
starter packs, but most consumables can also be ordered individually as your needs require. The
following tables provide information on the various ordering options that are available for Ion AmpliSeq
library chemistry.
1
™
Note:
Consumables that have catalog numbers are orderable. Components that have part numbers cannot
·
be ordered individually.
Reagents that are specific to Ion AmpliSeq™ library chemistry have an AS sux.
·
Genexus™ Library Strips 1 and 2‑AS
Ion Torrent™ Genexus™ Library Strips 1 and 2‑AS (Cat. No. A40252) for standard Ion AmpliSeq™ librarybased chemistry are ordered as kits with eight pairs of strips/kit.
Note: Genexus™ Library Strips 1 and 2‑AS with part numbers listed in the following table are
compatible only with Genexus™ Software 6.2.0 and later.
Component Carrier color Part No. Quantity per kit Storage
Genexus™ Strip 1 Light red A46812 8 strips 2°C to 8°C
Genexus™ S
trip 2‑AS
Light blue A46813 8 strips −30°C to −10°C
Genexus™ Integrated Sequencer User Guide
11
Chapter 1 Product information
1
Reagents and supplies—Ion AmpliSeq™ library chemistry
Genexus™ Barcodes AS
Ion Torrent™ Genexus™ Barcodes AS are supplied in plates containing 32 dual barcodes per plate. The
barcodes can be ordered as a set of three plates (Cat. No. A40257), or ordered individually.
Item Label color Cat. No. Quantity Storage
Genexus™ Barcodes 1–96 AS Blue A40257 3 plates
Genexus™ Barcodes 1–32 AS Blue A40258 1 plate
Genexus™ Barcodes 33–64 AS Blue A40259 1 plate
Genexus™ Barcodes 65–96 AS Blue A40260 1 plate
Genexus™ GX5™ Starter Pack‑AS
Ion Torrent™ Genexus™ GX5™ Starter Pack‑AS (Cat. No. A40279) supplies the following components for
Ion AmpliSeq™ library preparation and sequencing using a Genexus™-ready assay.
Note: For custom assays, Genexus™ Primer Pool Tubes (Cat. No. A40262) must be ordered separately.
15°C to 30°C
Component Part or Cat. No. Quantity Storage
Genexus™ Strip 1 A46812 8 strips 2°C to 8°C
Genexus™ Strip 2‑AS A46813 8 strips −30°C to −10°C
Genexus™ Strip 3‑GX5
Genexus™ Strip 4 A46816 8 strips −30°C to −10°C
Genexus™ Barcodes 1–32 AS A40258 1 plate 15°C to 30°C
Genexus™ Pipette Tips A40266 12 racks
Genexus™ Cartridge A40272 2 cartridges −30°C to −10°C
Genexus™ Bottle 2 A40273 4 bottles 15°C to 30°C
Genexus™ Bottles 1 and 3 A40274 2 bottles each
™
A46815 8 strips 2°C to 8°C
12
Genexus™ Integrated Sequencer User Guide
Chapter 1 Product information
Reagents and supplies—Ion AmpliSeq™ HD library chemistry
Reagents and supplies—Ion AmpliSeq™ HD library
chemistry
Genexus™ Integrated Sequencer reagents and supplies can be ordered in convenient combo kits and
starter packs, but most consumables can also be ordered individually as your needs require. The
following tables provide information on the various ordering options that are available for Ion AmpliSeq
HD library chemistry.
Note:
Consumables that have catalog numbers are orderable. Components that have part numbers cannot
·
be ordered individually.
Reagents that are specific to Ion AmpliSeq™ HD library chemistry have an HD sux.
·
Genexus™ Library Strips 1 and 2‑HD
Ion Torrent™ Genexus™ Library Strips 1 and 2‑HD (Cat. No. A40255) for Ion AmpliSeq™ HD library-based
chemistry are ordered as kits with eight pairs of strips/kit.
1
™
Note: Genexus™ Library Strips 1 and 2‑HD with part numbers listed in the following table are
compatible only with Genexus™ Software 6.2.0 and later.
Component Carrier color Part No. Quantity per kit Storage
Genexus™ Strip 1 Light red A46812 8 strips 2°C to 8°C
Genexus™ S
trip 2‑HD
Violet A46814 8 strips −30°C to −10°C
Genexus™ Integrated Sequencer User Guide
13
Chapter 1 Product information
1
Reagents and supplies—Ion AmpliSeq™ HD library chemistry
Genexus™ Barcodes 1–32 HD
Ion Torrent™ Genexus™ Barcodes 1–32 HD are supplied in a plate containing 32 dual barcodes.
Item Label color Cat. No. Quantity Storage
Genexus™ Barcodes 1–32 HD Purple A40261 1 plate 15°C to 30°C
Genexus™ GX5™ Starter Pack‑HD
Ion Torrent™ Genexus™ GX5™ Starter Pack‑HD (Cat. No. A40280) supplies the following components for
Ion AmpliSeq™ HD library preparation and sequencing using a Genexus™-ready assay.
Note: For custom assays, Genexus™ Primer Pool Tubes (Cat. No. A40262) must be ordered separately.
Component Part or Cat. No. Quantity Storage
Genexus™ Strip 1 A46812 8 strips 2°C to 8°C
Genexus™ Strip 2‑HD A46814 8 strips −30°C to −10°C
Genexus™ Strip 3‑GX5
™
A46815 8 strips 2°C to 8°C
Genexus™ Strip 4 A46816 8 strips −30°C to −10°C
Genexus™ Barcodes 1–32 HD A40261 1 plate 15°C to 30°C
Genexus™ Pipette Tips A40266 12 racks
Genexus™ Cartridge A40272 2 cartridges −30°C to −10°C
Genexus™ Bottle 2 A40273 4 bottles 15°C to 30°C
Genexus™ Bottles 1 and 3 A40274 2 bottles each
14
Genexus™ Integrated Sequencer User Guide
Shared reagents and supplies
The following reagents and supplies are used in both Ion AmpliSeq™ library chemistry and Ion
AmpliSeq™ HD library chemistry runs.
Note: Consumables that have catalog numbers are orderable. Components that have part numbers
cannot be ordered individually.
Genexus™ Templating Strips 3‑GX5™ and 4
Ion Torrent™ Genexus™ Templating Strips 3‑GX5™ and 4 (Cat. No. A40263) are ordered as kits with eight
pairs of strips/kit.
Note: Genexus™ Templating Strips 3‑GX5™ and 4 with part numbers listed in the following table are
compatible only with Genexus™ Software 6.2.0 and later.
Component Carrier color Part No. Quantity per kit Storage
Genexus™ Strip 3‑GX5
™
Brown A46815 8 strips 2°C to 8°C
Chapter 1 Product information
Shared reagents and supplies
1
Genexus™ S
Genexus™ P
Genexus™ Primer Pool Tubes and Pipette Tips can be ordered individually. Genexus™ Primer Pool Tubes
are required for custom assays.
Genexus™ P
Genexus™ P
trip 4
Yellow A46816 8 strips −30°C to −10°C
rimer Pool Tubes and Pipette Tips
Item Cat. No. Quantity Storage
rimer Pool Tubes
ipette Tips A40266 12 racks
A40262 50 assemblies
(2 tubes/assembly)
Bag of 100 caps
15°C to 30°C
Genexus™ Integrated Sequencer User Guide
15
Chapter 1 Product information
1
Shared reagents and supplies
GX5™ Chip and Genexus™ Coupler
The GX5™ Chip and Genexus™ Coupler (Cat. No. A40269) are ordered as a set that contains two chips
and two couplers, sucient for up to eight sequencing runs.
Component Part No. Quantity Storage
GX5™ Chip 100081364 2 chips 15°C to 30°C
Genexus™ Coupler 100081252 2 couplers
Genexus™ Sequencing Kit
xus™ Sequencing Kit is ordered as a combo (Cat. No. A40271). Each combo kit is
16
The Ion Torrent™ Gene
sucient to sequence up to 2 full chips. The components of the combo are also orderable as listed.
Component Cat. No. Quantity Storage
Genexus™ C
Genexus™ Bottle 2 A40273 4 bottles 15°C to 30°C
Genexus™ Bottles 1 and 3 A40274 2 bottles each
artridge
A40272 2 cartridges −30°C to −10°C
(4 bottles t
Genexus™ Integrated Sequencer User Guide
otal)
Genexus™ Conical Bottles
Genexus™ Conical Bottles (Cat. No. A40275) are installed in the sequencing reagents bay and serve as
reservoirs for nucleotide reagent dilutions. For information on when and how to replace the bottles, see
“Replace the Genexus™ Conical Bottles” on page 165.
Component Quantity Storage
Genexus™ Conical Bottles 5 bottles 15°C to 30°C
Genexus™ Filter
Chapter 1 Product information
Shared reagents and supplies
1
The Genexus™ Filter (Cat. No. A40302) is installed in the liquid waste disposal port on the instrument
deck to prevent liquid waste line blockage. For information on installation, see “Replace the Genexus
Filter” on page 164.
Component Quantity Storage
Genexus™ Filter 2 filters 15°C to 30°C
Genexus™ Controls
The Ion Torrent™ Genexus™ Controls kit (Cat. No. A40267) provides sucient Genexus™ Control
Library‑AS to perform four library runs. The kit also provides sucient Genexus™ Control Panel‑AS
and Genexus™ DNA Control to perform eight sample runs.
IMPORTANT! Gene
information, see “Genexus™ Library Strips 1 and 2‑AS” on page 11.
Note: The Gene
Genexus™ Contr
xus™ Control Library‑AS is barcoded with IonCode™ 0101.
Component Quantity Storage
ol Library‑AS 1 tube
™
xus™ Strip 2‑AS is required for sequencing Genexus™ Controls. For ordering
Genexus™ DNA Control 2 tubes
Genexus™ Integrated Sequencer User Guide
−30°C to −10°CGenexus™ Control Panel‑AS 8 carriers (white)
17
Chapter 1 Product information
1
Oncomine™ GX assays
Oncomine™ GX assays
Ion Torrent™ Oncomine™ GX assays are Genexus™-ready assays sucient for 32 reactions, and
are supplied in pre-measured ready‑to‑load Genexus™ Primer Pool Tubes. Assays are provided with
Genexus™ Library Strips 1 and 2‑AS (Cat. No. A40252) or Genexus™ Library Strips 1 and 2‑HD (Cat. No.
A40255) in the amount listed.
Contents
Oncomine™ Comprehensive Assay v3 GX (Cat. No. A46296)
Oncomine™ Comprehensive Assay v3 DNA GX Magenta DNA Pool 1 4 A40281 −30°C to
Oncomine™ Comprehensive Assay v3 RNA GX Pale orange RNA Pool 1 4 A44351
Genexus™ Strip 1 Light red — 2 × 8 A46812 2°C to 8°C
Genexus™ Strip 2‑AS Light blue — 2 × 8 A46813 −30°C to
Oncomine™ Precision Assay GX (Cat. No. A46291)
Oncomine™ Precision Assay GX (panel only) Magenta OPA Pool 1
Genexus™ Strip 1 Light red — 8 A46812 2°C to 8°C
Genexus™ Strip 2‑HD Violet — 8 A46814 −30°C to
Carrier
color
Pale green DNA Pool 2 4
Blue RNA Pool 2 4
Pool
(FWD and
REV primers)
Carriers
per kit
8 A44350 −30°C to
Part No. Storage
−10°C
−10°C
−10°C
−10°C
Oncomine™ TCR Beta‑LR Assay GX (Cat. No. A46297)
Oncomine™ TCR Beta‑LR Assay GX (panel only) Magenta RNA Pool 1 8 A40282 −30°C to
−10°C
Genexus™ Strip 1 Light red — 8 A46812 2°C to 8°C
Genexus™ Strip 2‑AS Light blue — 8 A46813 −30°C to
−10°C
Note:
Assays using Ion AmpliSeq™ chemistry have primer pools that are loaded in capped tubes in
·
position 1 of the primer pool tube carriers. Empty uncapped tubes are loaded in position 2.
Assays using Ion AmpliSeq™ HD chemistry have FWD and REV primer pools that are loaded in
·
capped tubes in both positions of the primer pool tube carrier.
18
Genexus™ Integrated Sequencer User Guide
Required materials not supplied
Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates
that the material is available from fisherscientific.com or another major laboratory supplier. Catalog
numbers that appear as links open the web pages for those products.
Item Source
Chapter 1 Product information
Required materials not supplied
1
MicroAmp™ EnduraPlate™ Optical 96-Well Clear Reaction Plates with
Barcode
Adhesive PCR Plate Foils AB0626
20‑, 200‑, and 1,000‑µL pipettors and appropriate filtered tips MLS
Microcentrifuge tubes, 1.5-mL or 1.7-mL (low retention for nucleic acids) MLS
Vortex mixer with a rubber platform MLS
Gloves, powder-free nitrile MLS
Ice buckets and ice —
Nuclease-free water, molecular biology grade AM9932
Isopropyl alcohol, 70% solution MLS
Wipes, disposable lint-free MLS
(Optional) Uninterruptible Power Supply (UPS)
[1]
F
or laboratories that experience frequent power outages or line voltage fluctuations, we recommend that you use an uninterruptible
power supply that is compatible with 2500 W output or higher.
[1]
4483352, 4483354
MLS
Recommended materials for nucleic acid isolation and
quantification
Unless otherwise indicated, all materials are available through thermofisher.com. Catalog numbers
that appear as links open the web pages for those products.
Item Source
Nucleic acid isolation
Ion AmpliSeq™ Direct FFPE DNA Kit A31133, A31136
RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE AM1975
RecoverAll™ Multi-Sample RNA/DNA Workflow A26069
MagMAX™ FFPE DNA/RNA Ultra Kit A31881
PureLink™ Genomic DNA Mini Kit K1820-00
MagMAX™ Cell‑Free DNA Isolation Kit A29319
Genexus™ Integrated Sequencer User Guide
19
Chapter 1 P
1
Recommended materials for nucleic acid isolation and quantification
roduct information
(continued)
Item Source
MagMAX™ Cell-Free Total Nucleic Acid Isolation Kit A36716
RNaseZap™ RNase Decontamination Solution AM9780
TaqMan™ RNase P Detection Reagents Kit (Recommended for DNA only) 4316831
Nucleic acid quantification
Qubit™ 4 Fluorometer
One or more of the following kits for use with the Qubit™ 4 Fluorometer:
· Qubit™ dsDNA HS Assay Kit (High-sensitivity DNA)
· Qubit™ dsDNA BR Assay Kit (Broad range DNA)
· Qubit™ RNA HS Assay Kit (High-sensitivity RNA)
· Qubit™ RNA BR Assay Kit (Broad range RNA)
[1]
Q33238
Q32851, Q32854
Q32850, Q32853
Q32852, Q32855
Q10210, Q10211
Library quantification (library runs only)
Ion Library TaqMan™ Quantitation Kit 4468802
[1]
Q
ubit™ 2.0 Fluorometer and later are supported.
20
Genexus™ Integrated Sequencer User Guide
2
10
1
3
4
7
8
9
5 6
11 11
Chapter 1 Product information
Genexus™ Integrated Sequencer components
Genexus™ Integrated Sequencer components
1
Major features and components of the exterior and sequencing reagents bay of the Genexus™ Int
Door to deck chamber. The door is locked in the closed
1
position during an instrument run.
Power button
2
Genexus™ Bottle 1 (Chemical W
3
Genexus™ Conical Bottles (Reusable conical bottles for
4
xus™ Cartridge reagent dilution)
Gene
Genexus™ Bottle 2 (Sequencing Solution)
5
aste)
Genexus™ Bottle 3 (Cleaning Solution)
6
Waste Carboy
7
Waste pipette tip bin
8
Genexus™ C
9
Touchscreen
10
Sequencing reagents bay door. Doors are locked in the
11
closed position during an instrument run.
artridge
egrated Sequencer
Genexus™ Integrated Sequencer User Guide
21
3
13
7
1
6
2
4
5
15
14
16
12
8
11
9 10
Chapter 1 Product information
1
Genexus™ Integrated Sequencer deck stations
Genexus™ Integrated Sequencer deck stations
Interior Genexus™ Int
PCR amplification station
1
Microcentrifuge
2
Genexus™ Bar
3
Genexus™ P
4
Genexus™ Coupler station
5
Chip install station
6
Zone 1 station (Genexus™ S
7
Zone 2 station (Genexus™ S
8
Strip 2‑HD, depending on your assay,)
egrated Sequencer deck components and stations
codes station
rimer Pool Tube station
trip 1)
trip 2‑AS or Genexus
™
Zone 3 station (Genexus™ S
9
Zone 4 station (Genexus™ S
10
Enrichment plate station
11
Liquid waste disposal port
12
Waste pipette tip disposal port
13
Sample plate station
14
Genexus™ P
15
Robotic pipettor
16
ipette Tips station
trip 3‑GX5™)
trip 4)
22
Genexus™ Integrated Sequencer User Guide
1
5
2
3
4
Chapter 1 P
Genexus™ Integrated Sequencer input and output connections
roduct information
Genexus™ Integrated Sequencer input and output
connections
The connection panel, power port, and an on/o switch are located on the right side of the rear panel of
the instrument.
1
Power port—100–240VAC port that provides power to the instrument.
1
On/o switch—P
2
USB ports—Connects a USB device to the instrument.
3
Ethernet port—An RJ45 port that provides Ethernet (Gigabit) communication between the sequencer and a local area
4
network.
Ethernet port—An RJ45 port that provides Ethernet (Gigabit) communication between the sequencer and an
5
accessor
y instrument.
ower switch, where the states are on ( | ) or o ( O ).
Genexus™ Integrated Sequencer User Guide
23
Chapter 1 Product information
1
Workflow
Workflow
Sample-to-results sequencing run using the Genexus™ Integrated Sequencer
Create an assay (page 32)
ystem installed assays that are specifically configured for each sample type are available in Genexus
S
Software. You can use the system-installed assays in your run without change. If you want to modify any
assay settings, copy the system-installed assay that best represents your experiment, then edit the assay
settings as needed.
Enter samples (page 46)
er samples in Genexus™ Software to assign sample names and provide other information such as
Ent
sample collection date, gender, type, and disease category.
Plan a sample run (page 62)
™
Runs planned in Gene
xus™ Software contain all of the settings that are used in library preparation,
templating, sequencing, and analysis, including sample information and plate location, assays, and
barcodes.
Quantify and dilut
e your nucleic acid samples, then load the sample plate.
Dilute the samples and load the sample plate (page 75)
Load the sequencer and start a run (page 79)
ollow the step-by-step instructions on the sequencer touch screen to load the sample plate and
F
consumables in the Genexus™ Integrated Sequencer.
Monitor the run (page 94)
or the run in Genexus™ Software in real time.
Monit
Review data and results in Genexus™ Softwar
e (page 95)
Review data and results in Genexus™ Software, or analyze data in Ion Reporter™ Software using an Ion
Reporter™ analysis workflow.
24
Genexus™ Integrated Sequencer User Guide
2
Precautions .......................................................................... 25
■
Guidelines for Genexus™ Integrated Sequencer operation ................................. 26
■
Guidelines for expired reagents and chips ............................................... 27
■
Power the Genexus™ Integrated Sequencer on or o ..................................... 28
■
Get started with Genexus™ Software ................................................... 29
■
Precautions
Avoid nucleic acid contamination
IMPORTANT! A primary source of contamination is spurious DNA fragments from previous sample
processing steps. Do not introduce amplified DNA into the work area where the instrument is located.
Before you begin
Avoid chip damage
IMPORTANT! To avoid possible damage due to electrostatic discharge, ground yourself before picking
up a chip or placing a chip on a surface such as a lab bench. For example, touch the deck surface or
one of the metal deck stations.
Avoid strong electromagnetic radiation
WARNING! Do not use the instrument in close proximity to sources of strong electromagnetic
radiation (for example, unshielded intentional RF sources), as these sources can interfere with proper
operation.
Protection by equipment
WARNING! The protection that is provided by the equipment can be impaired if the instrument is
operated outside the environment and use specifications, the user provides inadequate maintenance,
or the equipment is used in a manner that is not specified by the manufacturer (Thermo Fisher
Scientific).
Genexus™ Integrated Sequencer User Guide
25
Chapter 2 Befor
2
Guidelines for Genexus™ Integrated Sequencer operation
e you begin
Guidelines for Genexus™ Integrated Sequencer operation
•
Follow guidance that is provided by Genexus™ Software when you plan a run to determine which
consumables must be loaded and which consumables can be reused from a previous run.
•
Follow guidance that is provided by the software when you plan a run to determine how many
samples can be run with a given assay or assays in an instrument run. The number of samples that
can be included in a sequencing run depends on multiple factors.
Limiting factor Description
The number of available
barcodes in the barcode
plate
Maximum number of target
amplification reactions per
run
The number of primer pools
per assay
The number of unused
lanes on an installed chip
The minimum read count
per sample for an assay
•
T
wo assays cannot share a chip lane, so a maximum of 4 assays can be run per chip.
•
The assays that are used in a single run must use the same chemistry (Ion AmpliSeq™ or Ion
The maximum number of available barcodes per run is 32.
IMPORTANT! When libraries are prepared on the Genexus
Sequencer, each target amplification reaction for a sample requires a
unique barcode.
One library strip pair has the reagents necessary for 4 target
amplification reactions, or 4 barcodes. With a maximum of 8 library strip
pairs loaded, a maximum of 32 samples can be run using an assay with
one primer pool.
Given the limits of 32 target amplification reactions, and 32 available
barcodes, the number of samples in a run multiplied by the total number
of primer pools in the assays that are used in a run cannot exceed 32.
For one single‑pool assay, a maximum of 32 samples can be run on a
single chip. If you are using 2 assays with two primer pools each, you
can sequence a maximum of 8 samples in a run. Similarly, for one assay
with 4 primer pools, you can sequence a maximum of 8 samples in a run,
if the minimum read count per sample allows it.
A maximum of 4 lanes are available on a single GX5™ Chip.
The minimum read count per sample parameter is set during assay
creation.
™
Integrated
AmpliSeq™ HD), and have compatible cycling parameters to allow amplification in the instrument
thermal cycler. The thermal cycler has two independently controlled heating zones. After you select
an assay, Genexus™ Software restricts the list of available assays to use in the run to those that are
compatible with the selected assay or assays.
•
One library strip pair is required for each primer tube position 1–8 that is filled in a run.
•
One template strip pair is required for every chip lane that is used in a run.
•
Consumables are configured to support sample batch sizes in multiples of four samples. The most
ecient use of consumables occurs when samples are run in multiples of four.
•
If a chip installed in a sequencer has unused lanes, do not remove it unless you are sure that
you want to replace it with a new chip. After a partially used chip has been removed from the
sequencer, it cannot be reinserted and reused. The sequencer cannot track lane usage after chip
removal.
26
Genexus™ Integrated Sequencer User Guide
Chapter 2 Before you begin
Guidelines for expired reagents and chips
•
You can remove a chip in one of the following situations.
–
After all the lanes of a chip are used in a run, the chip shuttles to the install position and you
are asked to remove the used chip.
–
When you select a run plan that requires more lanes than are available on the installed chip,
you are asked to remove the partially used chip, and the sequencer performs a post-chip
clean. In addition, you must clear consumables from the lower sequencing reagents bay, even
if only a single lane of the chip was used.
•
The Genexus™ Integrated Sequencer can track used and unused barcodes in barcode plates in
Genexus™ Software 6.2.0 and later, enabling you to swap plates between runs as needed, and
reload a partially used barcode plate for a run if a sucient number of barcodes are available on
the plate.
•
After loading in the sequencer, reusable consumables, such as barcodes, chips, and sequencing
reagents bay components, must be used within 14 days for optimal results.
•
An assay that is selected in a library run cannot include library batches that share one or more
samples. However, two dierent assays in a run can include shared samples, because assays are
run in separate lanes of a chip.
2
Guidelines for expired reagents and chips
Follow these guidelines for using reagents and sequencing chips that are at or near expiration. We
do not recommend using components past the expiration date, but under certain circumstances, a
sequencer warning for expired reagents can be overridden to allow a sequencing run to proceed.
•
For all reagents except for Genexus™ barcode plates, the instrument bypasses an expired reagent
warning after you tap the Help button in the sequencer screen.
•
If a barcode plate has expired, a run cannot proceed, even if you tap Help.
•
If a GX5™ Chip is expiring in a given month, ensure that you use up all lanes of the chip within
that calendar month. If you start a run in the next calendar month with the same chip installed,
the sequencer does not allow the run to proceed. The sequencer prompts you to perform a Clean
instrument procedure and install a new chip and sequencing consumables, even if the initialization
has not expired. For further information, see “Perform a Clean instrument procedure” on page 152.
Genexus™ Integrated Sequencer User Guide
27
Chapter 2 Before you begin
2
Power the Genexus™ Integrated Sequencer on or o
Power the Genexus™ Integrated Sequencer on or o
Note: If the Genexus™ Integrated Sequencer is powered on, and the touchscreen is blank, touch the
screen to "wake" the touchscreen.
Power on
If the touchscreen is unresponsive, check the power switch on the back of the instrument to ensure that
the switch is in the on ( | ) position. If the power switch is in the o ( O ) position, proceed with step 1. If
the power switch is already in the on position, proceed to step 2.
1.
Turn the power switch on the back of the instrument to the on ( | ) position.
2.
Press the power button on the front of the instrument.
The button illuminates.
3.
In the Sign In screen, enter the username and password created by the field service engineer
when the instrument was set up.
When the instrument home screen appears, the instrument is ready for use.
Power o
It is not necessary to power o the instrument overnight or over the week
Genexus™ Software will not be used for more than 3 days, power o the instrument as follows:
1.
2.
3.
IMPORTANT! Do not pr
a run can result in sequencing run failure and loss of sample.
end. If the instrument or
In the home screen, tap Settings4System Tools4Shut down.
Select either Shutdown or Reboot.
If you select Shutdown, a confirmation message appears. Select Yes to power o the instrument.
Note: If you power o the instrument with a partially used chip installed, the chip and
consumables status is saved. When you power back on, the saved chip and consumable
information enables you to use the chip for up to 14 days after the chip was installed.
ess the power button during a run. Interrupting power to the instrument during
28
Genexus™ Integrated Sequencer User Guide
Get started with Genexus™ Software
1 2 3 4 5 6 7
About the Genexus™ Software user interface
Chapter 2 Befor
Get started with Genexus™ Software
e you begin
2
The Genexus™ Software user interface provides menus to help you add, select, and manage samples,
libraries, runs, and assays. You can also view and manage your sequencing results, monitor Genexus
Integrated Sequencer runs in progress, and manage software settings.
Samples: add new samples, impor
1
Runs: plan a run star
2
Monitor: view a sequencer run in pr
3
Results: view sample r
4
Assays: manage, cr
5
Settings: access audit r
6
Profile: access Help, manage and edit user pr
7
ting from a sample (Sample Run), or library (Library Run). View, edit, and manage runs.
esults, run results, and verification results.
eate, and import assays. Manage assay preset parameters and panels.
ecords and run logs, configure network settings, and manage data archiving, disk space, and users.
t samples, prepare library batches, import library batches and manage attributes.
ogress.
ofile settings and SSH key, sign out.
™
Genexus™ Integrated Sequencer User Guide
29
Chapter 2 Befor
2
Get started with Genexus™ Software
e you begin
User-access levels
Users at this level...
•
Operator
Manager Operator functions plus:
Administrator Operator and manager functions plus:
Add and import samples
•
Prepare library batches
•
Plan and save runs
•
Monitor runs
•
View results and reports
•
Create and edit sample attributes
•
Delete runs
•
Create and import assays
•
Manage reference sequences and panel, hotspot, and other sequence files
•
Access services information
•
View, export, and print audit records
•
Configure network settings
•
View and manage software updates
•
Configure data archive and storage settings
•
Manage instrument and software log files
•
Add and manage users
Can...
System tracking
The system tracks and checks user, sample, workflow
If the software detects an error at any step—for example, a scanned barcode is inconsistent with the
information given for the run—the software alerts the user and does not proceed with the run.
Request and sign in to a new account
Only administrator-level users can create user accounts.
After account creation, the Genexus™ Integrated Sequencer automatically sends an email to the new
user with the username and password information.
•
To request a new account, contact your local administrator.
•
To sign in to a new account for the first time:
a.
Open the Genexus™ Software, then enter your username and password.
b.
Press Enter, or click Sign In.
c.
Click Accept to accept the End User Software License Agreement.
30
, reagents and QC metrics for auditable records.
Genexus™ Integrated Sequencer User Guide
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