nanoString GeoMx DSP User manual

GeoMx® DSP
Instrument
User Manual
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
© 2022NanoString Technologies, Inc. All rights reserved.
MAN-10152-01 for v2.5 software | March 2022

NanoString, Inc.
530 Fairview Ave N
Seattle, Washington 98109
www.nanostring.com
T: 888.358.6266
F: 206.378.6288
E: [email protected]
Sales Contacts
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[email protected]
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OtherRegions: [email protected]
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NanoString Technologies
Germany Gmbh
Birketweg 31
80639 Munich
Germany
UK Authorized Representative
NanoString Technologies
Europe Limited
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United Kingdom

MAN-10152-01 for software v2.5
Rights, License, & Trademarks
GeoMx DSP Instrument User Manual
Rights, License, & Trademarks
Use
For research use only. Not for use in diagnostic procedures.
Intellectual Property Rights
This GeoMx®Digital Spatial Profiler (DSP) User Manual and its contents are the property of
NanoString Technologies, Inc. (“NanoString”), and are intended solely for use by NanoString
customers, for the purpose of operating the GeoMx DSP System. The GeoMx DSP System
(including both its software and hardware components) and this User Guide and any other
documentation provided to you by NanoString in connection therewith, are subject to patents,
copyright, trade secret rights and other intellectual property rights owned by, or licensed to,
NanoString. No part of the software or hardware may be reproduced, transmitted, transcribed,
stored in a retrieval system, or translated into other languages without the prior written consent of
NanoString. For a list of patents, see www.nanostring.com/company/patents.
Limited License
Subject to the terms and conditions of the GeoMx DSP System contained in the product
quotation, NanoString grants you a limited, non-exclusive, non-transferable, non-sublicensable,
research use only license to use the proprietary GeoMx DSP System only in accordance with the
manual and other written instructions provided by NanoString. Except as expressly set forth in
the terms and conditions, no right or license, whether express, implied or statutory, is granted by
NanoString under any intellectual property right owned by, or licensed to, NanoString by virtue of
the supply of the proprietary GeoMx DSP System. Without limiting the foregoing, no right or
license, whether express, implied or statutory, is granted by NanoString to use the GeoMx DSP
System with any third party product not supplied or licensed to you by NanoString or
recommended for use by NanoString in a manual or other written instruction provided by
NanoString.
Trademarks
NanoString, NanoString Technologies, the NanoString logo, GeoMx, and nCounter are
trademarks or registered trademarks of NanoString Technologies, Inc., in the United States
and/or other countries. All other trademarks and/or service marks not owned by NanoString that
appear in this document are the property of their respective owners.
Copyright
©2022 NanoString Technologies, Inc. All rights reserved.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
3

Table of Contents
GeoMx DSP Instrument User Manual 1
Contacts 2
Rights, License, & Trademarks 3
Changes in This Revision 6
Conventions 7
GeoMx DSP Instrument Introduction 8
Instrument Components 9
Instrument Specifications 13
Fluorescence Channel Specifications 13
Installing and Moving 14
Product Use Limitations 14
Disposal of Electronic Equipment 14
Safety Information (English) 15
Consignes de Sécurité (Français) 16
GeoMx DSP Workflow 17
User Manuals and Resources 19
GeoMx DSPControl Center 20
GeoMx DSP Run 21
Reagents and Consumables 21
Start a new run 22
Identify Slides 28
Scan Slides 34
Select ROIs 36
Segment ROIs (optional) 44
Overlays, ROITransfer, and Export Images 52
Approve ROIs for Collection 57
Unload Plate and Slides 59
Finalize the collection plate 61
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
4
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Uploading Counts 65
Create Slides and Scans outside GeoMx DSPWorkflow 66
Example Scan Parameters 68
Changing a probe kit configuration file after collection 69
Slide and Scan Management 70
Administration Menu 74
GeoMx DSP Instrument Reagents 100
Changing Reagent Bottles 101
GeoMx DSPInstrument Maintenance 103
System Startup & Shutdown 103
Cleaning the Slide Holder 105
Hibernating the GeoMx DSP for Extended Shutdown 106
General Cleaning 109
Replace Air Filter 109
Instrument Best Practices 110
Troubleshooting 111
Symbols & Definitions 115
Final page 116
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
5

Changes in This Revision
This GeoMx DSPInstrument User Manual (MAN- 10152- 01) replaces the GeoMx- NGS
Instrument User Manual (SEV-00087-05) and GeoMx-nCounter Instrument User Manual (SEV-
00065-07).
NGS- specific information is separated from nCounter- specific information with colored text
boxes, as described in Conventions on page 7 and shown below.
Other changes in this manual revision include:
lExtended safe storage guidelines for protein and RNA slides on page60
lAdded instructions to edit a probe kit configuration (.pkc) file after collection on page69
lAdded instructions to synchronize system time with internet time server on page96
lImproved the GeoMx DSPhibernation protocol for instruments that will be inactive for >2
weeks on page106
lAdded an Instrument Best Practices guide on page110
lAdded a Troubleshooting section on page111
For NGS readout:
lAdded guidance for users running standalone
custom RNA-NGSassays without an Atlas
product (such as WTA or CTA) on page39
lAdded instructions to connect the GeoMx
DSP to an Illumina®BaseSpace workgroup to
enable seamless end-to-end run integration,
and to launch the seamless end-to-end
workflow from the GeoMx DSP on page63
lUpdated guidance on i5 index read direction
for Illumina sequencer models and kits on
page64
For nCounter readout:
There are no nCounter-specific updates
to this user manual.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
6
GeoMx DSP Instrument User Manual
Changes in This Revision
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MAN-10152-01 for software v2.5
Conventions
GeoMx DSP Instrument User Manual
Conventions
The following conventions are used throughout this manual and are described for your
reference.
Bold text is typically used to highlight a specific button, keystroke, or menu option. It may also be
used to highlight important text or terms.
Blue underlined text is typically used to highlight links and/or references to other sections of the
manual. It may also be used to highlight references to other manuals or instructional material.
A gray box indicates general information that may be useful for improving assay performance. These
notes aim to clarify other instructions or provide guidance to improve the efficiency of the assay
workflow.
IMPORTANT: This symbol indicates important information that is critical to ensuring a
successful assay. Following these instructions may help improve the quality of your data.
WARNING: This symbol indicates the potential for bodily injury or damage to the instrument if the
instructions are not followed correctly. Always carefully read and follow the instructions
accompanied by this symbol to avoid potential hazards.
For NGS readout: Content in blue
boxes denotes steps or information
specific to NGSreadout of GeoMx DSP.
Follow these instructions if using
Illumina®NGS to read out GeoMx DSP
counts.
For nCounter readout: Content in green boxes
denotes steps or information specific to
nCounter readout of GeoMx DSP. Follow these
instructions if using nCounter®MAX/FLEX, Pro,
or SPRINT to read out GeoMx DSP counts.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
7

GeoMx DSP Instrument Introduction
Figure 1: GeoMx DSP instrument
The GeoMx Digital Spatial Profiler (see Figure 1) is a benchtop instrument designed to spatially
resolve and collect genomic information from distinct, user- defined regions of interest. The
GeoMx DSP instrument is intended for Research Use Only . This GeoMx DSP Instrument
manual is concerned exclusively with the operation of the GeoMx Digital Spatial Profiler. It is
important that you read and comprehend the provided information. Please utilize training
resources in NanoString University at https://university.nanostring.com and contact
[email protected] to address any questions or concerns.
Separate available user manuals provide information on slide preparation, the GeoMx DSP run,
data readout by NGS or nCounter platform, and data analysis. See User Manuals and
Resources on page 19.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
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Instrument Introduction
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Instrument Introduction
GeoMx DSP Instrument User Manual
Instrument Components
Figure 2: GeoMx DSPinstrument exterior diagram
The GeoMx DSP instrument is a benchtop instrument and comes equipped with a 27" color LED
monitor (4K - 8MP, model: LG 27HJ712C-W), as well as a mouse and keyboard.
The front of the instrument has a USBport (USB-A 3.0) and a barcode scanner (see Figure 2).
The main (upper) door opens to the GeoMx DSP instrument stage (see Figure 3). The reagent
bay (lower) door provides access to the buffer bottles and waste container.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
9

Figure 3: GeoMx DSPinstrument interior diagram
To access the GeoMx DSP instrument stage, open the main (upper) door. On the stage a
collection plate is loaded into the plate holder and secured by the plate clamp. When loading a
collection plate before your slides, the system will prompt you to slide the plate cover over the
collection plate until loading is complete. Up to four slides can be loaded into individual slots on
the slide holder in a designated lab space, each is secured with the slide slot clamps, and
covered with buffer. The slide holder is then placed onto the stage of the instrument.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
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Instrument Introduction
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Figure 4: GeoMx DSPinstrument reagent bay diagram
The GeoMx DSP instrument requires the following reagent bottles in the reagent bay (see
Figure 4):
lBuffer S: 1 L bottles in reagent bay slots 1 and 4.
lBuffer H: 250 mL bottles in reagent bay slots 2 and 3.
In addition, the waste bottle, with adequate space to collect waste, must be present in the
position to the right of the reagent bottles.
IMPORTANT: Ensure the instrument is in an idle software state before changing reagents (see
Bring GeoMx DSP Software to an Idle Software State on page 95). In addition, DONOT top
off any reagents. Always replace entire bottles when reagents are low, using the wizard.
For information on replacing reagent bottles, see GeoMx DSP Instrument Reagents on page
100.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
11

Figure 5: GeoMx DSPinstrument backside diagram
The backside of the GeoMx DSP instrument (see Figure 5) has the following ports:
lAn ethernet connection: CAT 6 Network Port with RJ45 connector.
lThree USB connections: USB-A 3.0.
lA power input for the instrument: Power in 100 – 240 VAC, 50/60 Hz, connector C14.
lA power output for the monitor: Power out (same as Power in), connector C13.
lA display port for the monitor.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
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Instrument Introduction
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Instrument Introduction
GeoMx DSP Instrument User Manual
Instrument Specifications
The GeoMx DSP system is a single instrument, intended for Research Use Only. The necessary
cables and a waste bottle are included with each instrument. Do not dispose of the waste
bottle. All other consumables and reagents may be purchased separately. Instrument
specifications are listed below.
Slides per Run 1 - 4 (1x3") slides per run
Weight 210 lbs
Dimensions (W x D x H) 30 in (760 cm) W x 29 in (73 cm) D x 24 in (61 cm) H
Power Requirements 100 - 240 VAC
Fuse 50/60 Hz 10 A
Room Temperature 18–28°C
Room Humidity 30–80% relative humidity (non-condensing)
Table 1: GeoMx instrument specifications
Fluorescence Channel Specifications
The wavelengths and excitation and emission information are listed below for each of the GeoMx
DSP channels.
Channel Excitation (peak/bandwidth) Emission
(peak/bandwidth)
FITC / 525 nm 480 / 28 nm 516 / 23 nm
Cy3 / 568 nm 538 / 19 nm 564 / 15 nm
Texas Red / 615 nm 588 / 19 nm 623 / 30 nm
Cy5 / 666 nm 645 / 19 nm 683 / 30 nm
Table 2: GeoMx fluorescence channel specifications
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
13

Installing and Moving
lIndoor use only. Avoid locating in direct sunlight.
lThe GeoMx DSP instrument may only be installed or moved by appropriately trained
personnel. Contact NanoString for installation or relocation of the instrument. See Safety
Information (English) on page 15.
lPlace the instrument on a flat, stable surface with access to a dedicated power supply.
lEnsure adequate clearance on the backside of the instrument in order to access power
controls and allow at least 3 inches for ventilation.
lEnsure adequate clearance on the front of the instrument to allow opening of the main
instrument door and the reagent bay door.
lAvoid locating near other equipment that may cause vibration, or near large electrical
equipment that may cause interference from noise and/or voltage fluctuation.
lConsult your laboratory guidelines and local regulations for information on reducing hazards
associated with the transport, disposal, or removal of an instrument from use. There are no
hazards unique to the GeoMx DSP instrument that require additional instruction in this
manual, however, some general safety guidelines are provided in Safety Information
(English) on page 15 and in Disposal of Electronic Equipment on page 14.
Product Use Limitations
The GeoMx DSPsystem is intended for research use only. Not for use in diagnostic procedures.
Disposal of Electronic Equipment
Review and follow all laws regarding the safe and proper disposal of
electrical instrumentation. The symbol of a crossed out, wheeled bin on
the product is required in accordance with the Waste Electrical and
Electronic Equipment (WEE Directive of the European Union). The
presence of this marking on the product indicates that:
• The device was put on the European Market after August 13, 2005.
• The device is not to be disposed via the municipal waste collection
system of any member state of the European Union.
For products under the requirement of the WEEE directive, please contact your dealer or local
NanoString office for the proper decontamination information and take-back program, which will
facilitate the proper collection, treatment, recovery, recycling, and safe disposal of the device.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
14
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GeoMx DSP Instrument User Manual
Safety Information (English)
The GeoMx DSP instrument may only be operated by appropriately trained, professional users
for Research Use Only. NanoString recommends that all users read and understand this manual
prior to attempting to operate the system. Keep this manual in close proximity to the instrument
for easy access to instructions and safety information.
If the GeoMx instrument is used in a manner not specified by NanoString, the protection provided
by the equipment may be impaired. Failure to comply with the instructions in this manual may
pose a dangerous risk to the operator and void the manufacturer’s warranty.
Do not attempt to disassemble the equipment. The GeoMx DSP instrument contains no user-
serviceable parts. Service personnel trained by the manufacturer must perform repairs. Do not
modify any part of the equipment as this may cause fire and malfunction, and will void the
manufacturer's warranty. Do not replace detachable main supply cords by inadequately rated
cords.
WARNING: Do not attempt to install, move, or perform maintenance on the instrument. Always
contact NanoString for instruction before installing or moving the instrument. If it is necessary to
move equipment, use extreme caution when lifting items that weigh more than 50 pounds. When
lifting a load heavier than 50 pounds (such as the GeoMx DSP instrument), use two or more
people to lift the load.
WARNING: Do not use this device in close proximity to sources of strong electromagnetic
radiation or vibration, as these may interfere with proper operation.
WARNING: Operate the system using only NanoString reagents in accordance with their
indications for use. Ensure that all consumables are properly inserted prior to starting a run.
WARNING: Connect the instrument power supply to a properly grounded receptacle with
adequate voltage and current (see Instrument Specifications on page 13).
Caution Symbols
ELECTRICAL HAZARD: Do not attempt to disassemble the instrument at any time.
An electric shock can occur if the instrument is operated without its outer case.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
15

Consignes de Sécurité (Français)
Le GeoMx DSP instrument ne peut être utilisé que par une personne ayant reçu une formation
professionnelle appropriée. NanoString recommande que tous les utilisateurs lisent et comprennent ce
manuel avant de tenter de faire fonctionner le système. Conservez ce manuel à proximité de l’instrument
pour faciliter l'accès aux instructions et consignes de sécurité.
Si le GeoMx instrument n’est pas utilisé de la façon spécifiée par Nanostring, la protection fournie par
l’équipement pourrait être altérée. Le non-respect des instructions de ce manuel peut présenter un risque
dangereux pour l'opérateur et peut annuler la garantie du fabricant.
Ne pas tenter de démonter l’équipement. Le GeoMx DSP instrument contient des pièces non réparables
par l’utilisateur. Un réparateur agrée par le fabricant doit faire les réparations. Ne pas modifier les pièces
sous peine de provoquer un incendie ou un mauvais fonctionnement, de plus cela annulera la garantie du
fabricant. Ne pas remplacer les cordons d’alimentation amovibles par des cordons insuffisamment
évalués.
ATTENTION: Ne pas tenter d’installer, de déplacer ou d’effectuer l’entretien de l’instrument. Toujours
contacter NanoString pour instruction avant d’installer ou le déplacer l’instrument. S’il est nécessaire de
déplacer l’instrument, utiliser une extrême prudence pour soulever des objets pesant plus de 23
kilogrammes. Pour soulever une charge plus lourde que 23 kilogrammes (comme pour l’instrument GeoMx
DSP), utiliser au moins deux personnes pour soulever la charge.
ATTENTION: Ne pas utiliser cet appareil à proximité de sources à fortes radiations électromagnétiques
ou vibrations, car elles peuvent interférer avec le bon fonctionnement de l’appareil.
ATTENTION: Faire fonctionner le système en utilisant uniquement des réactifs NanoString conformément
à leurs guides d'utilisation. Assurez-vous que tous les consommables soient correctement insérés avant de
commencer une opérat
ATTENTION: Branchez l'alimentation de l'appareil électrique à une prise correctement mise à la terre
avec une tension et un courant adéquat (voir les spécifications de l'instrument).
Définitions des Étiquettes de Sécurité
RISQUE ÉLECTRIQUE: Ne jamais tenter de démonter l'appareil. Un choc électrique peut se
produire si l'appareil est utilisé sans son enveloppe extérieure. Débranchez l'appareil de la
source d'alimentation avant de remplacer le fi ltre du ventilateur.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
16
GeoMx DSP Instrument User Manual
Instrument Introduction
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MAN-10152-01 for software v2.5
GeoMx DSP Workflow
GeoMx DSP Instrument User Manual
GeoMx DSP Workflow
The GeoMx Digital Spatial Profiler (DSP) is a novel platform developed by NanoString. This
product relies on antibody or nucleic acid probes coupled to photocleavable oligonucleotide tags.
After probes hybridize to targets in slide-mounted tissue sections, the oligonucleotide tags are
released from discrete regions of the tissue via UV exposure. Released tags are quantitated by
nCounter technology or Illumina Next Generation Sequencing (NGS). Counts are mapped back
to tissue location, yielding a spatially resolved digital profile of analyte abundance (see Figure
6).
lDay 1: Slide Staining. Prepare slides and incubate biological targets with UV- cleavable
probes. Prepare manually or using the BOND RX/RXmFully Automated IHC/ISH Stainer from
Leica Biosystems®.
lDay 2: Process Slides on GeoMx DSP. Load prepared slides into the GeoMx DSP
instrument and enter slide/study information. Slides are scanned to capture fluorescent
images used to select regions of interest (ROIs). The instrument collects UV-cleaved oligos
from the ROIs into the wells of a collection plate.
For NGS readout:
Day 3: Transfer the collected aspirates to a
PCR plate and perform Library Prep with
Seq Code primers. Pool and purify the
products, then Sequence on an Illumina
NGS instrument.
Day 4 : Process FASTQ sequencing files
into digital count conversion (DCC) files
using NanoString's GeoMx NGS Pipeline
on Illumina DRAGEN™ accessed via
BaseSpace™ Sequence Hub, or using
GeoMx NGS Pipeline standalone software.
Upload DCC files on to the GeoMx DSP.
For nCounter readout:
Day 2, continued: Transfer the collected
aspirates to a hybridization plate along with
GeoMx Hyb Code reagents. Hybridization
occurs overnight.
Day 3 : Pool wells and Process on an
nCounter MAX/FLEX or Pro Analysis
System or SPRINTProfiler. Upload
reporter count conversion (RCC) files to the
GeoMx DSP.
lDay 4 or 5: Create a Data Analysis study in the Data Analysis suite and perform quality-
control checks and data analysis, and generate analysis plots.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
17

Figure 6: GeoMx DSP workflow summary
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
18
GeoMx DSP Instrument User Manual
GeoMx DSP Workflow
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GeoMx DSP Workflow
GeoMx DSP Instrument User Manual
User Manuals and Resources
The GeoMx DSPworkflow is divided into the following user manuals:
Workflow
Step 1
GeoMx DSP Manual Slide Preparation User Manual
MAN-10150
GeoMx DSPAutomated Slide Preparation User Manual
MAN-10151
Workflow
Step 2
GeoMx DSP Instrument User Manual
MAN-10152
Workflow
Step 3
For NGS readout:
GeoMx DSP NGS Readout
User Manual
MAN-10153
For nCounter readout:
GeoMx DSP nCounter Readout
User Manual
MAN-10089
Workflow
Step 4
GeoMx DSP Data Analysis User Manual
MAN-10154
User manuals and other documents can be found online in the NanoString University Document
Library at https://university.nanostring.com.
Instrument and workflow training courses are available in NanoString University.
For NGS readout:
For documentation specific to the
Illumina platform, see
https://support.illumina.com.
For nCounter readout:
For documentation specific to the nCounter Pro,
MAX/FLEX, and SPRINT instruments, see
https://www.nanostring.com/support/support-
documentation/ or the NanoString University
Document Library at
https://university.nanostring.com.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
19

GeoMx DSPControl Center
The GeoMx DSP Control Center is the main interface of the GeoMx DSP software.
Figure 7: GeoMx DSP Control Center diagram
By clicking the Data Collection button in the GeoMx DSP Control Center header (see Figure
7), you can begin a new run or upload counts or calibration files. The Records button allows you
to create slide records and scan parameters and to add scans to the Data Analysis Queue.
Select the Data Analysis button to build a study from the queue or open a study. The
Administration button is available to administrative users.
The Data Collection Stages, depicted just below the header when Data Collection is selected,
provide you with a visual orientation of the progress of your run.
The Notifications bell will alert you to errors in archiving, the status of readout file upload, and
the status of a data analysis build. These notifications will remain until you click the bell; once
seen, they will persist for 24 hours or until closed with the x (archiving errors may regenerate
hourly).
Slide and Plate Status indicators in the footer indicate the current state of the slides and plate,
respectively.
Reagent Status indicators depict the reagent and waste bottle levels. A locally logged-in user
may use the Abort Run button, if necessary, to halt the run.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
20
GeoMx DSP Instrument User Manual
GeoMx DSPControl Center
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