GeoMx® DSP Software v2.0 Tips & Tricks

GeoMx DSP Software v2.0 Tips & Tricks 8/11/2020

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

NanoString, NanoString Technologies, the NanoString logo, nCounter, and GeoMx are trademarks or registered trademarks

of NanoString Technologies, Inc., in the United States and/or other countries.

The top seven tips and tricks to be aware of are listed on this page (see below). On the following pages, less-

frequent but nonetheless helpful tips and tricks are listed. A section on GeoMx NGS Pipeline is also included.

Top 7 Tips & Tricks

1. In the event of a failed run and with the advice of GeoMx support, it is possible to restart the workflow. To

do so, click the

Assist wrench icon (Figure 1) and select

Restart Data Collection Workflow.

•

Ensure that the instrument door is closed before

restarting data collection.

• If a blank screen appears, refresh the page (see

below).

2. If the screen appears

blank, inaccurate, or unresponsive,

refresh the page. This may be performed remotely or

directly at the instrument. To do this, use keyboard

shortcuts

Ctrl+R or F5 or click the Assist wrench icon (Figure 1) and select Refresh Page. You may also use

Ctrl/Alt/Delete to log out of and then back into Kiosk mode.

Shutdown and restart the GeoMx DSP instrument on a weekly basis.

• When first opening the GeoMx software, click on Assist wrench icon (see Figure 1) and choose

Restart the Workflow Process before starting other tasks or initializing a new run (this will ensure

the instrument door unlocks as a new run is initialized).

4. When downloading logs,

minutes to download; the increased time incurred when remotely connected may result in the system

timing out and interrupting the download.

5. DO NOT open the reagent bay door during Collection.

•

Opening the reagent bay door during a run may cause the run to pause. After closing the reagent

bay door, you may need to manually restart data collection workflow (see above).

6. After finalizing the plate and downloading the CDF and worksheet, DO NOT select Show All from the

downloads bar. This will result in kiosk mode locking. In this case, contact geomxs[email protected]m.

To prevent a critical error, leave the slide tray in the instrument unless you are loading/unloading it or

cleaning/drying it.

Ensure it is in the instrument before shutting down or restarting. If the door is locked, use

the Assist button > Restart Data Collection Workflow to reach the prompt to open the door.

Figure 1: Assist wrench icon to Restart

Data Collection Workflow and Refresh

Page.

GeoMx DSP Software v2.0 Tips & Tricks 8/11/2020

General

• Inadvertently running the GeoMx instrument with a bottle disconnected may result in multiple fluidics error

messages. If the error is not resolved after reconnecting the bottle, there may be air in the lines. In this

case, contact

geomxsuppo[email protected].

• Do not attempt to shut down the GeoMx DSP instrument while a run is in progress or when either door is

open.

• If the GeoMx DSP

Control Center header is missing when in kiosk mode, logout of windows using ctrl-alt-

del, then login again as Kiosk User.

• After changing reagent bottles and closing the reagent bay door, the instrument may erroneously display

Plate Change - Close Door message. Click Next; this will

complete the bottle change process.

•

An error may appear while restarting the GeoMx DSP after

making a change in

File Management under Administration. If

this appears, shutdown and restart the GeoMx instrument as

instructed on page 1.

• An error may occur when

restarting the System Service via

Services (see Figure 2). If this occurs,

shutdown and restart

the GeoMx instrument as instructed on page 1.

• When remotely connected, closing the Chrome browser will automatically log you out of the GeoMx

system.

• When logging into the GeoMx system, the

Cancel button does not cancel the login process as expected.

When logged in directly, use Ctrl Alt Del and then log out, then log back in. If remotely connected, close the

browser and reopen.

•

Runs that are in-progress while DCC files are being uploaded to the system may experience lag times or

failure. To avoid this, do not attempt to load DCC files from more than two NGS plates at a time while a run

is in progress.

• The

Scan Preview in the GeoMx DSP Control Center may disappear after editing Notes. The Scan Preview

may not load if you

click on one preview before all have loaded. View each scan by opening in Scan

Workspace.

• After emptying the waste bottle,

rinse the waste bottle several times, until no foaming is observed.

• Chrome does not connect to DSP on a Mac. To manually connect, while on the Chrome error page, type

'thisisunsafe'. There is no text box, just make sure to click anywhere on the error page and then type. DSP

will auto load.

• Do not click on e-mail or website links in the GeoMx Help documentation while in kiosk mode.

Figure 2: Restart error

GeoMx DSP Software v2.0 Tips & Tricks 8/11/2020

Admin

•

Do not use spaces in usernames. Usernames with spaces may not be able to view backups in the archive.

• Do not enter a phone number when creating a user, as this may cause an error, preventing you from saving

the new user.

•

To download a specific day’s logfile, select a Custom Range that extends at least one day before and one

day after the day desired.

• If deleting a group, that groups’ folders can be transferred to the

All Users group. Right-click on the folder

of interest and select

Edit; the All Users group should be selected. Click Save to make this change go into

effect.

• When local storage is full, the system provides an estimate of the space needed in GB, however the dialog

may read “MB”.

• In order to change a user’s role (Admin to User, for example), that user must be assigned to no more than

one group.

• A critical error state or cascading errors may cause slowness or unresponsiveness in the UI. If no response

is observed for >30 minutes, please contact

geomxsupport@nanostring.com.

• In the Network window, you will see the

Machine Name and the IP address

(see Figure 3). If not, restart the system: shut down from the menu, switch

off the switch in the back, then after 10-20 seconds, switch it back on. Do not

attempt to turn on Archive Settings while a run is in progress.

• If Archiving is working, a file mountverification.txt will be written in the

destination folder almost immediately. Then, initial backup of the GeoMx

DSP files can take up to 2 days to complete.

To troubleshoot archiving:

o Ensure that your GeoMx software is v1.4.2 or later, and your

institute network is set up for SMB3 file sharing.

o Enter the Remote Server path as \\123.45.6.78\\FolderName using

the server IP address and your designated folder. 4 opening

backslashes may be needed.

o Try the username in different formats, such as username,

username@domain, or username@domain.edu

o Do not use these characters in your password: $ ! /

o Confirm that the credentials to access the folder have reading and

writing permission.

o Nested folders may be an issue – try to set the Archive folder in the root of the server as shown in

above example.

o A network load balancer may be an issue – contact GeoMx Support to discuss workarounds.

Figure 3: Network

Settings window

GeoMx DSP Software v2.0 Tips & Tricks 8/11/2020

Run Setup

• The system needs to be in an

idle state (see page 8) in order to be able to change reagents. The reagent

bottle status indicators are managed through the bottle change wizard.

Click on the Reagent Status Indicator

in the Control Center and follow the wizard steps to change a bottle or empty the waste bottle. DO NOT top

off any reagents or pour/pipette reagents out of their bottles.

•

Deviating from the GeoMx loading steps by failing to insert slide holder during the appropriate step or by

loading the collection plate before the UI instructs may result in an error or a stalled UI. In these cases,

restart the workflow (see page 1).

• When

creating a new slide under Records, if the Save slide button is disabled, click the Cancel button and

create a new slide.

• When creating scan parameters, failure to select a Core Kit will result in Scan-only mode (no aspirate

collection).

• The best practice for uploading a config file is to upload it prior to starting the run from the Kit

Management section under Administration. If encountering an error uploading a config file or if not seeing

a newly-uploaded config file in the dropdown when creating a slide record:

o Ensure that the file you are uploading has been unzipped.

o Refresh the page (see page 1) and use the same config file.

o Alternatively (or in addition, if the action above was not successful) you may cancel the creation

of the slide record and start again.

• Once a Core kit is selected in the Scan Parameters window, only module kits compatible with that core kit

will be shown in the probe kit dropdown.

• If doing a plate swap for your NGS platform run, do not use a plate that was previously partially-used for

nCounter platform aspirates. The system will initially accept the plate but will then repeatedly prompt you

to swap the plate again.

GeoMx DSP Software v2.0 Tips & Tricks 8/11/2020

ROIs and Segments

• When

copying and pasting existing ROIs in the Scan Workspace, the GeoMx system does not prevent

against overlapping ROIs. Inspect ROIs to ensure none are overlapping; delete any that overlap.

• Persistent errors may occur while

copying and pasting ROIs on one scan while another scan is in progress.

If this occurs, restart the run.

• Do not attempt to copy and paste an ROI after it has been segmented.

•

Do not rename an ROI after it has been moved (without first saving it in its new location); doing so results

in the ROI moving back to its original location.

• There is currently a defect (to be fixed in 2.1) where importing custom segments may unexpectedly delete

existing segments. To mitigate this, create and upload your custom segments first and wait to do your

auto-segmenting until after that, if possible. If you do import custom segments after auto-segmentation,

please exit the scan workspace without approving ROIs, then re-enter the scan workspace and verify your

auto-segments. If needed, you can re-apply auto-segmentation to restore them before approving your

scan.

•

Resizing the most recently touched polygon may cause unexpected changes in the polygon size and shape.

Create a new polygon, then return to the polygon you wish to alter and it should resize properly.

• Leave the

Override All values at the bottom of the segmentation window set to 0. Change an Override All

value only if you want to override all of the ROI thresholds for that channel.

• If exiting Scan Workspace without approving ROIs, slide status may read Scanned instead of Annotated.

• Channel settings should be modified after scanning only if a re-scan is planned. The Scan Preview in the

GeoMx DSP Control Center may disappear. View each scan by opening in Scan Workspace.

• Names for segments imported from an external masking program will be truncated at 100 characters,

potentially resulting in non-unique names. Using names greater than 175 characters in auto-

segmentation within the Scan Workspace may result in no segmentation.

• The collection order, well order, and name order of segments may differ from each other, as the system

optimizes aspirate collection to reduce complexity.

•

Changing a Channel Thresholds setting to 0 on an ROI will revert the channel threshold back to the default

value.

• When exporting Raw Channel Images, if status stalls at 99%, restart run.

• The annotation in the image export may not reflect the correct settings if they were previously altered

(color mapping was changed or channel was hidden) in Render Settings.

• When changing segment color, if the color assignment in both the ROI tab and in the actual scan do not

change as directed, refresh the page.

• The GeoMx

system may run out of memory if attempting to collect many ROIS on multiple slides.

Internally, this occurred during a run involving nine plate swaps. Restart Data Collection.

Completing the Run/nCounter Preparation

•

Check your plate after collection to ensure the expected wells were collected. The Plate Status Indicator may

display an inaccurate status. If the number or location of filled wells differs from your expectations based

on the number of ROIs and segments collected, contact

geomxsupport@nanostring.com.

GeoMx DSP Software v2.0 Tips & Tricks 8/11/2020

•

After finalizing a row, its wells should appear as green in the plate icon in the lower right of the GeoMx DSP

Control Center. If not, refresh the page.

Data Analysis

• Due to conflicts with Chrome, the

Manage Annotations file may not download when requested. Refresh

(F5) and try again.

•

Check the Data Analysis Queue to be sure that only the intended scans are included before launching Data

Analysis. The Queue may be shared by other users or by someone using the same user login. For this reason,

it is recommended that you create a study immediately after placing scans in the queue.

• Ensure your

scans have unique names, particularly if you want to use the Manage Annotations function.

• If encountering the message, “analysis is locked by another user” check to see if another user is accessing

this study. If not, close the study, wait one minute, refresh the page, then re-open the study.

• The header UI for count file upload will read 1 out of n until the upload is complete.

• P-value filter may be ignored in trendline after toggling between “fold change” and “ratio” selections.

Reset filter after changing selection.

• The

correlation calculation will fail if a segment has the same count for all targets; this may occur if no

targets have been detected and the count=1 for all. Remove these segments from the analysis.

•

Scan names should not have trailing or leading spaces. This can cause segments from that scan to be

unrecognizable in the manage annotations modal of Data Analysis. If spaces are accidentally introduced,

revert to scan workspace, edit the scan name, then create a new Data Analysis study.

• In datasets and within Statistical Tests, when you click on

Trend Line, all targets except the Negative are

automatically

de-selected. Navigate directly back to another plot (such as the volcano plot) and manually

re-select all targets or targets of interest.

•

Off-instrument DSPDA needs to be restarted after a software update.

• If running off-instrument DSPDA and the GeoMx instrument is restarted for any reason, it will be necessary

to restart the NanoString System Service on the off-instrument server, as well. This must be done after the

GeoMX instrument has completed its start up.

• If you receive a service unavailable error when attempting to work with off-instrument Data Analysis, first

restart the GeoMx DSP instrument. Then, restart the off-instrument Data Analysis program via either the

“NanoString System Service” or by restarting the server itself.

• The list of targets in the center pane has a loading defect and sometimes doesn’t display all targets in the

available space. To see to the end of the target list, repeatedly pull the scroll bar down until the last

target in the list appears.

• “Create group from filtered” in target and segment filtering will create a group from those

included rather than excluded in the new dataset. The newly created segment group will not appear in

the segment group list until you refresh the page or close and reopen the study.

• In Manage Annotations, user-created column names should not start with “ROI”, or they will be

incorrectly associated with the ROI_ID and cause an error.

• When you group a bar chart of targets by segment tag, expand the window and display all bars at once if

there are more than the default displayed. If you scroll through the pages only the first page will display

the correct bar heights.

• The bar chart option to set min and max interval for the y-axis does not work.

GeoMx DSP Software v2.0 Tips & Tricks 8/11/2020

GeoMx Pipeline (DND) Software

• Some errors result in empty .dcc files. Check .dcc file sizes and the summary.txt file to ensure files were

processed as expected, especially for AWS batch runs as errors from separate batch jobs are not always

consolidated into logs.

• Ensure that your FASTQ files do not contain underscore characters (_) in the sequence identifier lines, as

this may cause processing to fail, stall, or produce count files with errors. You may have underscores in your

file names if your Illumina instrument name contains an underscore; if this is the case, modify your

instrument name to remove any underscores. In addition, bcl to FASTQ file conversion software may be set

to use custom headers; ensure the software you use to generate FASTQ files does not add any underscores

to the sequence identifier lines.

• Do not modify FASTQ filenames from the

Illumina FASTQ file naming conventions. If possible, retain the

naming from the sample sheets exported from DSP, which incorporate the plate barcode. If you must

change the sample names, ensure you do not add extra underscores, as the GeoMx NGS Pipeline software

uses underscores to parse the Illumina-written portion of the file names to identify well/sample, lane, and

read. In addition, if the sample name portions of your filenames are modified from the default pipeline

workflow, you will need to update the names in your config file accordingly before you begin your NGS

processing pipeline run.

• Submitting jobs from the GUI to a server may result in errors if there is already a run processing on the

server.

• If logs from AWS batch run are not present, you may be able to view them on AWS cloudwatch. See

documentation from AWS for more information.

• The GeoMx NGS Pipeline running on the server only listens on port 5000 and there is no argument to

change this. This may cause a problem if there are other apps on server or network limiting the availability

of that port.

• While the GeoMx NGS Pipeline is processing, it writes many large intermediate files to disc. Therefore, you

will need up to 10x more space than your initial file set size on the computer where processing is occurring

to complete processing. These intermediate files are removed automatically unless you modify the config

file to “keepStepFile=True”.

• If inspecting your .dcc files in a text editor, you may notice lines for “tamperedIni,No” and

“SOMEHASH100000000000”. These are not currently functional and are placeholders for a future feature;

they do not affect your data.

• Processing FASTQ files for only a subset of the ROIs listed in the GeoMx NGS Pipeline (DND) configuration

file will result in empty .dcc files for the missing ROIs. Uploading these empty .dcc files will overwrite any

data that may exist for these segments from a previous DND run. The GeoMx NGS Pipeline configuration

file can be revised to exclude any ROIs for which FASTQ files were previously processed. The resulting .dcc

zip uploaded should then only update segments associated with blank data.

GeoMx DSP Software v2.0 Tips & Tricks 8/11/2020

• When running on AWS batch, some jobs may silently fail, returning .dcc files with zero aligned and

deduplicated reads or not returning some .dcc files at all, even though the AWS job console indicates that

the job succeeded. Always review the summary.txt file to note any unexpected errors or zero reads for any

intermediate steps for ROIs. Logs for a given job ID may be retrieved from AWS cloudwatch in order to

troubleshoot.

• If executing a run from the GUI, any errors or failures will be indicated in the notification menu and listed

by ROI name.

• GeoMx Pipeline runs will fail if Docker Desktop is configured to use WSL2. To get around this issue,

uncheck the Use the WSL2 based engine box on the General tab of the Docker Desktop settings.

Idle State Pages

There are two idle states:

Collection Complete & Replace Plate.

1. Click Data Collection, then

New/Continue Run.

2. Follow on-screen instructions to load

the instrument with a plate and

empty slide holder.

3. Wait for pop-up message “No Slides

Detected”.

4. Click

OK. Now, the instrument is at

the Collection Complete idle state

(Figure 4).

5. Click on reagent icons to change

reagents or click on Administration, then Software Update to perform software update.

6. If you have followed these steps and

the instrument still is not in an idle

state, click

Assist then Restart

Workflow. Pause at “Replace Plate?”

screen (Fig 2). This is an idle state.

7. If the instrument still does not allow

these actions, power cycle by

shutting down through the software,

reboot, start a new run, and pause at

“Replace Plate?” screen (Figure 5).

This is an idle state.

Figure 4: Collection Complete idle state

Figure 5: Replace Plate idle state