GeoMx DSP Knowledge Base: GeoMx DSP System

Yes, you can scan your slides and select regions of interest (ROI) without collection using the “Scan only” feature. This feature is useful when evaluating tissue staining conditions prior to the experimental run, or practicing/discussing ROI selection prior to collection. When creating a new scan, one can toggle to select “Scan only” on the scan configuration window. Scans which are designated as scan only cannot be sent for ROI collection but can be transferred to Scan and Collect slide at another run using the ROI transfer function.

We do not recommend using TE buffer or any other buffers on the GeoMx DSP instrument, other than those provided in our instrument buffer kit. This is because the different salts could crystallize and clog the fluidic lines. Additionally, our reagents contain antimicrobial agents to prevent microbial growth, which may not be present in other buffers.

The GeoMx DSP instrument will rehydrate your slides every 4 hours, so they will not dry out. One caveat to this if the instrument errors out during collection, the slides will not re-hydrate. Also, the storage conditions inside the GeoMx DSP are not optimal, so we recommend that you remove them as soon as possible and store them according to the slide storage guidance within the GeoMx DSP Slide Preparation User Manuals (MAN-10150 and MAN-10151).

For protein slides, slides can be stored for up to 1 day submerged in 1X TBS-T and stored at 4°C, protected from light. For storage from 1 day to 3 months, the slides can be mounted with mounting media, mounted with coverslip, and stored at 4°C, protected from light.

For RNA slides, the slides can be stored for up to 6 hours submerged in 2X SSC and stored at room temperature, protected from light. Slides can be stored for up to 21 days in 2X SSC, at 4°C, protected from light. It may be necessary to re-fresh morphology marker or nucleic acid stain; please refer to our manual for guidance on this.

For GeoMx DSP Spatial Multiomics (formerly known as Spatial Proteogenomics) assay, store the slides in 1X TBS-T at 4°C, protected from light for up to 7 days.

For best results, minimize storage time between slide preparation and loading on the GeoMx DSP instrument while avoiding tissue from drying out.

Once the GeoMx DSP plate collection is complete, you can proceed to seal it with a permeable membrane and proceed to dry down the plate. To store the plate, seal it with adhesive foil to prevent contamination and store it at 4°C for 24 hours or less. For storage between 1-30 days, it can be stored at -20°C. For longer than 30 days, we recommend storing it at -80°C. Further guidance for this process can be found within the NGS Readout User Manual.

If you encounter a critical error during collection, please reach out to support.spatial@bruker.com to initiate a case. A support representative will send you a link to submit the instrument log files. To download logs, please go to the Administration tab within the GeoMx DSP Control Center on-instrument, followed by Download Logs.  In the custom range, select one day before the issue occurred to one day after, and in the “include images with download” option, choose “none”. You can then proceed to download the logs.

While the Support Team makes our best effort to respond in the same day, it is not always possible for us to do so. Therefore, to protect the samples, we recommend removing the slides from the instrument and safely storing them as outlined below, until the Instrument issue is resolved.

To retrieve your slides from the instrument:

• If you are logged in as an Administrator user, follow the steps below:
  • Under the Administration tab of the GeoMx DSP instrument, please select User Service Actions, select Home All to home instrument hardware. Then, select Control Door Lock, enter desired parameter: False = unlock door and True = lock door.
• If you are logged in as a General user, follow the steps below:
  • Under the Administration tab of the GeoMx DSP instrument, select System Settings, then the System Shutdown tab, then Shutdown System. Once the instrument is turned off, the door will unlock to allow you to remove your slides and collection plates.
  • If you do not have access to the holder at that time, please turn on the machine (this will home all motors) and turn off once again through the System Settings menu (to unlock the door). Allow at least 1 minute between reboots so that the software fully initializes.

Slide storage recommendations are as follows:

Protein – submerged in 1X TBS-T at 4ºC, protected from light.

RNA – submerged in 2X SSC at 4ºC, protected from light.

Spatial Multiomic/Proteogenomic Assay (RNA and Protein Assay on the same slide) – submerged in 1X TBS-T at 4ºC, protected from light.

It is possible to move GeoMx DSP scans and studies to and from any GeoMx DSP instrument using GeoMx instrument’s Data Exchange option. Simply export the GeoMx DSP scans and studies into external drives (NTFS format recommended and not greater than 10 TB) and then import into the other GeoMx DSP instrument using the Data Exchange option under the Administration tab. For detailed instructions on this, please refer to GeoMx DSP instrument user manual.

There are two types of user accounts on the GeoMx DSP instrument, administrator and general. Administrator has access to all the studies and full administration tab options, whereas a general user only has access to their own data. To create a new user account, please go to the Administration tab and select User Management. Here, you may create a new user.

To assign users to a group, go to the Group Management option under Administration tab and click the “Manage” button to create a new group. From there you can check, add, or remove users as needed. Next, create a new folder and move files, scans, and studies into it by dragging and dropping and assign them to this group. To restrict access to the folder; right click on the folder, select “Edit” and then choose the group you want to grant access to the group. Please check the screenshots below for visuals on this:

With software version 3.0 and above, you can delete scans and studies from the GeoMx DSP instrument. This can be done by right clicking on the item’s name and then selecting delete to move this item to the trash folder. Using administrative level access, Scans can also be permanently deleted from the trash folder which will remove it from instrument and the archive. Please note that only scans with status Readout Complete, Scan Only, Error or Aborted can be deleted.

If you are not planning to run your GeoMx DSP instrument for 2 weeks or longer, we recommend running the hibernation protocol on your GeoMx DSP instrument to prevent any crystallization or salt accumulation in the lines. This protocol involves switching the buffers to DEPC-treated water and then flushing them. When you are ready to use the instrument again, it can be returned to operational status by following our protocol. Please refer to the GeoMx DSP Instrument user manual here for detailed instructions.

We do not recommend moving GeoMx DSP instrument on your own, even for a short distance. The reason is that the GeoMx DSP has a leveling process that is critical to its performance. Therefore, we highly recommend having our service engineer on-site to assist with move and post-move validation. Please reach out to customerservice.spatial@bruker.com to assist with this request.

This is possible in some cases; please refer to the table below. Note that this can only be done when the scan status is “Readout Complete.” To proceed, the user will need to complete all the steps including uploading RCC or DCC files. Then, from the scan gallery, click on “Scan Parameters” to update the probe kit files, and save the changes.

For NGS readout, user will need to download a new readout package and re-run the pipeline using the new configuration.ini file. The resulting DCC files can be uploaded to overwrite the original DCC files. For nCounter readout, creating a new study with this scan will show the updated counts.

The IPA panel consists of a core and IPA module. Thus, there are 2 .pkc files associated with this panel and both need to be added. Please note that the IPA Core kit must be selected before the compatible module kits will be available in the dropdown.

Also, configuration files are not pre-loaded on the instrument and must be transferred using a USB drive or remote access over Chrome. Probe kits for all panels can be found on our website here. We recommend uploading new .pkc files under Kit Management in the Administration menu. Once uploaded to the instrument, they are available for selection in the Scan Configuration window.

Windows Defender anti-virus is configured and will receive anti-virus definitions if the instrument has a network route to retrieve updates. In addition to using Windows Defender, we use Windows AppLocker. AppLocker is a whitelisting program that only allows NanoString-approved applications and processes to execute in Windows. As a result, AppLocker also provides robust protection from viruses and malware by blocking execution and enforces a high degree of overall change control of the DSP system state. In contrast to traditional security software that relies on blacklisting, which is inherently vulnerable to zero-day attacks and situations where the blacklist happens to not be updated in a timely manner, AppLocker blocks everything except the software/Apps that has been specifically allowed to run on the instrument. AppLocker allows us to maintain a static, robust, and secure application execution environment and protects the instruments from any unauthorized software or manipulation while at the same time ensuring that the machine remains in an unchanged and securely validated state.

There are three features that require network connectivity:

• Desktop clients access the web interface via the Google Chrome browser for remote ROI selection and data analysis.
• Establishing an automatic data backup system to the archiver server is strongly recommended. If no SMB backup location is established, the instrument will stop working when the hard drive is full. The instrument ships with a 10TB hard drive for OS and GeoMx DSP software installation and storage. On average, 20 GB per scan will be used for storage. At medium throughput, the instrument could run out of hard drive space after 6-8 months if no external storage is established. The effective storage can be expanded by allowing the instrument to connect to a corporate file share via SMB. When connected to external storage, the instrument will backup data to the network share.
• Bruker Spatial Biology includes remote support with the instrument, which allows for faster resolution of issues with the ability for Bruker Support to diagnose, configure, and update components remotely. To accomplish this, the Instrument must have WAN/internet access for Bruker Support personnel to access the instrument.

GeoMx DSP and auxiliary server need to have a static IP or reserved DHCP.

The best way to free up space is to connect to an archive server. This will move data from the instrument to the hard drive so that the instrument always has 2TB of free space. It will move the oldest studies/those that were opened the longest ago to the archive first. Please note that once you do set up an archive, the moving of data process is quite slow and could take several days. For more information on how to set up the archive drive, please refer to the GeoMx DSP Instrument user manual here.

Another way to free up some space is by deleting some scans and studies from the instrument. To delete old scans, you first need to move them to the trash and then an administrator user needs to go into the trash folder and delete them from the trash there. For more details on this, please refer to the GeoMx DSP Instrument user manual.

The maximum modules you can run include the Immune Cell Profiling Core, 6 human IO modules, and a custom module. Since both the immune cell typing and the MAPK module occupy the R4 space, you typically need to drop one human IO module to accommodate the MAPK module and use the substitute probe R for the hybridization step. However, if you prefer to run the immune cell profiling core with all the 7 human IO modules, that is feasible. In this case, MAPK module would need to be shifted to the R8 and R9 space and ordered from an external vendor such as Eurofins, IDT, etc. However, in this case, customers would lose the ability to add any more custom targets.

All nCounter readout products, whether RNA or protein, utilize the same DSP_v1.0 RLF and it should be installed on the Pro/MAX/FLEX or SPRINT during training. If not installed, please reach out to support.spatial@bruker.com to request the file.

The Hyb Code letter must match the Collection row letter. If an incorrect Hyb Code row number is used, we do not have a process to fix this error on a commercial scale. Therefore, it is crucial that the lab process be carried out exactly as described in the lab worksheet. You may collect a dummy ROI into wells and then finalize the row leading to the next collection starting in the next row. For instance, if you only have Hyb Code C on hand, you will need to do this dummy collection for rows A and B so that your actual samples will be collected into row C to match your Hyb Code C. Alternatively, you can order a new Hyb Code Pack for the collection.

This error is due to not uploading the hybcode lot specific files. You can resolve this issue by following these steps:

1. Locate your HybCode Box from -80C freezer, find the lot number written on the box.
2. Click on the 96-well plate icon on the GeoMx DSP screen (or where it says “No Plate”), and input the plate ID. Then, enter that Hyb Code pack lot number and click “update” – visuals and instructions here.
3. Download the calibration files associated with your lot and nCounter system here, leave them zipped!
4. Upload the calibration files to the GeoMx DSP instrument using the “Data Collection” -> “Upload Counts” button.

Once the calibration files are successfully uploaded, you should be able to upload your RCC files.

There could be several reasons to get an error while uploading the RCC files:

• Make sure there is no subfolder folder within the RCC.zip folder.
• Make sure the correct CDF was used for the nCounter run.
• Make sure SampleID in the CDF matches SampleID and CartridgeID in the RCC files.
• Make sure the correct Hyb Code Lot number is associated with the experiment. Check by clicking on the plate icon.
• nCounter data requires a calibration file for each new lot of Hyb Code. Please see the above question for further information on resolving this.

If you are still having issues, please reach out to support.spatial@bruker.com.

If only a few rows of the Seq Code plate were used, then you can use the remaining rows as long as the Seq Code plates are stored correctly. Also, it is important to match the Seq Code row with the collection plate row. Thus, you will need to enter the original collection plate number, so the instrument knows which row to start the collection (you may or may not use the same collection plate).

We do not recommend using the remnants of Seq Code for a second experiment. Because the Seq Code fill volume is 8 µL, it is possible the remaining volume will be less than 4 µL when reused. The extra volume can help salvage this step if a pipetting error occurs; otherwise, the wells contain single-use volume. You may however always use unused wells, please see the above FAQ on how to use the remaining wells of a Seq Code plate.

It is possible to move the collection plates from one readout group to the other one. For this to work, the collection plates must have the same status (eg. Finalized) to be moved to the same readout group.

In addition to this, the following combinations of plates are not allowed in one readout group:

• Single analyte plates using Pro Code indices (IPA) with single analyte plates using Seq Code indices
• Single analyte plates with mixed analyte plates
• Mixed analyte plates using Pro Code indices (IPA) with mixed analyte plates using Seq Code indices.

Steps to move plates from one readout group to another:

• Click on the plate icon on the GeoMx DSP instrument

• Enter the plate barcode number and then click on the readout group name in the window
• You then have the option to select MOVE

• Clicking on move will pull up a screen to select the collection plate to be moved from one readout group to the other.

• Click the Choose button and Readout Group window will appear

• Select the new Readout Group to assign to your plate and select Use Readout Group
• Your plate should be updated with a new Readout Group

For RNA assays with NGS readout, you need to have at least 4 ROI, otherwise the instrument will not allow you to finalize the plate. To resolve this, you can move this plate to a different readout group and then finalize them together. For more information on moving plates, please check the above FAQ How can I move collection plates from one readout group to another?

The readout package is a zipped folder that is downloaded after finalizing the collection plate for NGS readout. It contains 3 files:

• Configuration.ini file: this is the file used by the GeoMx NGS pipeline to assist in converting the Illumina FASTQ files to DCC files.
• Lab worksheet: contains information on the contents of each well of each plate, including ROI and segment details, ROI coordinates, scan details, biological target details, nuclei count, recommended sequencing depth, and more.
• Sample index list (SeqCodeindices.csv): this is the file needed to set up the Illumina sequencing run for demultiplexing the Illumina run (NextSeq 1000/2000 users download a SampleSheet.csv and whitelist.txt instead of an Indices.csv.)

To download the readout package:

• Click the bottom right corner of the screen. It should be a picture of a plate or say, “No Plate”. The Plate Information window will appear.
• Select the tab “Search by Readout Group”
• Click Look Up Readout Group and select the readout group that you want to export
• Enter/change the sequencing parameters and readout group information
• Insert USB drive
• Click “Finalize and Download Readout Package”. If the collection plate was finalized before, then you will see the option to “Re-download Readout Package”

Our recommendation for all Illumina instruments is to run GeoMx libraries at 2 x 27 paired end read strategy. We highly recommend Paired end workflow as it allows higher quality data, typically with no added kit cost.

We recommend assessing library quality on a capillary electrophoresis device such as the Agilent Bioanlayzer or similar to check amplicon size, concentration, and absence of contaminating primers. It is helpful to assess the library at stock concentration, in a dilution and checking the NTC library.

The expected library amplicon size is 162 bp (Seq Code products) or 166 bp (Pro Code products). Using the Bioanalyzer, the library will appear as ~150 bp. Using the TapeStation, the library will appear as ~ 170 bp.

Sequencing kits are sold per cycle. Since we recommend paired-end 2×27 read strategy, a sequencing kit that can accommodate at least 54 cycles (2 x 27 = 54) is required. The minimum number of cycles per kit for most Illumina sequencers is 75 or 100 cycles. In the case where customers are pooling DSP libraries with other Illumina libraries, then they must choose a kit that can accommodate the longest desired read length. Please discuss with your sequencing facility for more information on this.

The FASTQ files should always follow the standard Illumina naming structure:

DSP-PlateNumber, Seq Code plate letter, GeoMx Plate Well Number, sample sheet number, lane, read1/read2, always end in ‘001’

For example: DSP-1001250001985-A-A02_S2_L001_R2_001.fastq.gz The suffix .gz indicates a compressed (gzipped) file.

DSP-1001250001985-A-A02 is the “sample ID” matching that found in the configuration.ini and SeqCodeindices.csv files. A Translator file allows you to run the NGS Pipeline with FASTQ file names that are not an exact match to the configuration file sample name by translating one to the other. However, the translator is only useful for files with a different sample ID in the file name. All other elements – _S2_L001_R2_001.fastq.gz in the example above – must match the expected naming structure. If this is not the case, it will be necessary to rename the FASTQ files or request your sequencer to repeat demultiplexing following the above Illumina naming structure.

To use the translator file, please check here for the example file and instructions on how to use it.

For RNA or protein only assay, there is one fastq.gz file number per ended read (Read 1 “R1” and/or Read 2 “R2”) per flow cell lane (e.g. lane 1 “L1”, lane 2 “L2”, lane 3 “L3”, etc.)

For example, if a customer collects 133 AOI, then with paired end read strategy and run on 1 Flow cell lane, it will generate 266 FASTQ.

For GeoMx DSP Spatial Multiomics (formerly known as Spatial Proteogenomic) assay, there is one fastq.gz file number per ended read (Read 1 “R1” and/or Read 2 “R2”) per flow cell lane (e.g., lane 1 “L1”, lane 2 “L2, lane 3 “L3”, etc..) per analyte.

For example, if a customer collects 133 AOI, then with paired end read strategy and run on 1 Flow cell lane, it will generate 266 FASTQ for RNA and 266 FASTQ for protein, for a total of 532 FASTQ.

Yes, this is possible, but the files need to be merged at the level of the GeoMx NGS pipeline, because uploading another batch of DCCs with the same name to the GeoMx DSP instrument will just overwrite whatever is associated with that scan record.

To combine multiple runs, make sure that each set of FASTQ has a differing lane number (L001, L002, etc.) to allow the GeoMx NGS Pipeline to identify and merge them.

If necessary, it is possible to rename the files from multiple sequencing runs so that no two pairs of FASTQ share the same lane number. These renamed files can be run through a locally installed version of the GeoMx NGS Pipeline. These files cannot be uploaded to Illumina’s Basespace due to upload restrictions surrounding file naming and file metadata matching. It is not necessary to be concerned with whether the samplesheet number (_S4_) matches or not.

For example, we have two runs of a NovaSeq (1 lane) of the same library, which yields two sets of FASTQ:

DSP-1001250002271-A-A04_S4_L001_R1_001.fastq.gz

DSP-1001250002271-A-A04_S4_L001_R2_001.fastq.gz

DSP-1001250002271-A-A04_S32_L001_R1_001.fastq.gz

DSP-1001250002271-A-A04_S32_L001_R2_001.fastq.gz

Rename the second set so that L001 becomes L002, and put all files in the same root folder to process through the pipeline

DSP-1001250002271-A-A04_S4_L001_R1_001.fastq.gz

DSP-1001250002271-A-A04_S4_L001_R2_001.fastq.gz

DSP-1001250002271-A-A04_S32_L002_R1_001.fastq.gz

DSP-1001250002271-A-A04_S32_L002_R2_001.fastq.gz

All 4 files for sample DSP-1001250002271-A-A04 will be processed into one DCC file.

You cannot generate a dummy FASTQ file. However, you can run the standalone NGS pipeline (not Basespace) to generate an empty DCC file for the missing FASTQ. There may be a warning message that it is missing some inputs, but once proceeded the pipeline will create an empty DCC file of size ~ 1kb for the missing FASTQ.

The GeoMx NGS Pipeline uses a series of algorithms to process sequencing files from FASTQ to the Digital Count Conversion (DCC) file format that can be read by the DSP instrument for further analysis. First, raw sequencing files (FASTQ files) are selected for a specific pipeline run. Once selected the reads are processed for quality, the adapters are removed (trimmed) and the paired-end reads are merged (stitched) resulting in a single high quality read. Then, reads are aligned to the Readout Tag Sequence-ID (RTS-ID) barcodes. PCR duplicates are removed by matching on the Unique Molecular Index (UMI) (deduplicated) and the DCC file is generated. The resulting DCC files are presented as a .zip file in a folder which you designate and can then be uploaded into the DSP Control Center for study creation in the DSP Data Analysis Suite.

It can be run two ways:

• Install NGS Pipeline Software on a computer server
• Use BaseSpace Sequencing Hub’s cloud pipeline application, called DRAGEN

Refer to the GeoMx DSP NGS Readout User Manual (MAN-10153) for more information.

The following can be checked to ensure there are no issues while uploading the DCC files into the GeoMx instrument:

• Make sure the folder is zipped (must be .zip).
• Make sure there is not a subfolder within the zipped folder.
• Make sure there is a DCC file for every sample in the readout group

If you are still having issues, please email support.spatial@bruker.com.

This error comes up when the DCC file is missing the umiQ30 field. Usually, when a FASTQ file is missing for one of the AOI/ROI, the pipeline generates an empty DCC file that helps the user to upload and reach readout complete status for their scan with 0 counts for that AOI/ROI. Presently, when a user uses DRAGEN pipeline on Basespace, a bug causes certain header fields to be filled incorrectly. The instrument cannot parse these DCCs at upload and throws an error. You can recognize this issue by looking in the DCCs and checking the <NGS_Processing_Attributes>:

Bad

Good

Just edit the incorrect file, zip together with the other DCC files, and then try to upload the data again. If you are still having issues, please email support.spatial@bruker.com.

You can upload DCC files again and the data will be replaced by the latest uploaded ones. After you upload DCC files, any studies already created will not be updated, but any new study will use the latest counts.