Feb 23, 2026

Public workspaceGeoMx NGS Readout Library Preparation & Sequencing

DOI
https://dx.doi.org/10.17504/protocols.io.j8nlk1rowg5r/v1
  • Scott Lindsay-Hewett1,
  • Valentina Stanley1,
  • Kathleen Fisch1,
  • Mana Parast1,
  • Louise C. Laurent1
  • 1University of California San Diego
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: [email protected]
Scott Lindsay-Hewett
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlk1rowg5r/v1
Protocol Citation: Scott Lindsay-Hewett, Valentina Stanley, Kathleen Fisch, Mana Parast, Louise C. Laurent 2026. GeoMx NGS Readout Library Preparation & Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk1rowg5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 22, 2026
Last Modified: February 23, 2026
Protocol Integer ID: 243797
Keywords: GeoMx, Digital Spatial Profiler, NGS Readout, Library Preparation, Quality Control, Spatial Transcriptomics, Illumina Sequencing, GeoMx NGS Pipeline, Data Analysis, geomx digital spatial profiling ng, geomx ngs readout library preparation, files for downstream spatial transcriptomic analysis, downstream spatial transcriptomic analysis, spatial transcriptomic analysis, sequencing, sequencing this protocol, digital count conversion, conversion of fastq data
Funders Acknowledgements:
National Institutes Of Health - Female Reproductive Tissue Mapping Center
Grant ID: U54 HD104393
National Institutes Of Health - Pregnant Female Reproductive Tissue Mapping Center
Grant ID: U54 HD110347
Abstract
This protocol outlines the GeoMx Digital Spatial Profiling NGS readout workflow, including library preparation, quality control, NovaSeq 6000 sequencing, and conversion of FASTQ data into Digital Count Conversion (DCC) files for downstream spatial transcriptomic analysis.
Guidelines
  • Thoroughly treat all surfaces with RNase AWAY prior to use. High-contact areas - specifically those exposed to concentrated RNA probes - must be prioritized to prevent cross-contamination in subsequent runs or downstream library preparation.
  • A 12-channel pipette is recommended to streamline liquid handling, as DSP aspirates are deposited in row-wise format.
  • Ensure that AMPure XP beads are at room temperature and thoroughly vortexed before adding to the pooled PCR product.
Materials
Equipment

Vortex
Picofuge
Plate spinner
Thermal Cycler (BioRad cat # C1000 Touch with 96–Deep Well Reaction Module); others okay
Qubit fluorometer (or similar device for library quantification)
Agilent Bioanalyzer (or similar capillary electrophoresis device for nucleic acid analysis)
Illumina NovaSeq 6000 (other Illumina NGS instruments okay)

Materials/reagents

GeoMx Seq Code Pack A - H (Bruker cat # various depending on number of indexes required)
Pipettes, including 12-channel P10 or P20
Filter tips (DNase/RNase-free)
Microcentrifuge tubes (DNase/RNase-free)
DNA LoBind tubes (for library storage before sequencing) (Eppendorf cat # 022431021)
96-well PCR plates (DNase/RNase free) to match thermal cycler specs
Magnetic stand
AeraSeal (Sigma cat # A9224-50EA)
Adhesive PCR foil sheets (Fisher cat # AB-0626)
PCR 12-strip tubes (Fisher cat # AB-1112)
PCR domed strip caps (Fisher cat # AB-0852)
RNase AWAY (Fisher cat # 21-402-178)
Nuclease-free water
100% ethanol
AMPure XP Beads (Beckman Coulter cat # A63880)
Elution buffer (Tris-HCl 10 mM with 0.05% Tween-20, pH 8.0) (Teknova cat # T1485)
Agilent DNA High Sensitivity Kit (Agilent cat # 5067-4626)
Qubit dsDNA High Sensitivity (HS) Assay Kit (Fisher cat # Q32851)
Illumina Sequencing Reagent Kit
Troubleshooting
Sample Reconstitution
1h 10m
If DSP aspirates are already dry, skip to Step 2. Otherwise, seal the plate with a breathable (permeable) membrane and dry at Temperature65 °C in a thermocycler with the lid open for up to Duration01:00:00 . Monitor periodically; remove immediately once wells are completely dry.
1h
Rehydrate samples with Amount10 µL nuclease-free water. Pipette mix 5 times and incubate at TemperatureRoom temperature for Duration00:10:00 to ensure full recovery. Perform a quick spin to collect the volume at the bottom of the wells.
10m
Library Preparation & Quality Control
5h
Library preparation should be performed according to the manufacturer's instructions. The GeoMx DSP NGS Readout Quick Guide is posted here for reference.

Download QS_MK2550-10_MAN-10133-06-1_GeoMx-NGS_Readout_R3_BSB.pdfQS_MK2550-10_MAN-10133-06-1_GeoMx-NGS_Readout_R3_BSB.pdf

Consult the LabWorksheet.txt from the relevant Readout Package to verify the correct mapping of GeoMx Seq Code index plates to the corresponding DSP collection plates.

Assess library quality using the Agilent Bioanalyzer High Sensitivity DNA assay. The library should appear as a discrete, narrow peak at ~150 bp, without the presence of primer dimers (<100 bp) or high-molecular-weight artifacts.

Determine final library concentration using the Qubit dsDNA High Sensitivity (HS) assay.
Illumina sequencing
Sequencing Depth Calculation: Calculate the required reads by identifying the Total Area (µm2) profiled in the LabWorksheet.txt and multiplying by a factor of 100.

Sample Sheet Generation: Populate the Illumina SampleSheet.csv using the specific i5 and i7 index sequences provided in the SeqCodeIndices.csv file.

NovaSeq 6000 S4 Loading: Load at Concentration250 picomolar (pM) with a 1-5% PhiX spike-in.

Run Parameters:
  • Paired-end
  • Minimal Read Lengths: Read 1 = 27 bp; Read 2 = 27 bp.
  • Index Lengths: Index 1 (i7) = 8 bp; Index 2 (i5) = 8 bp.

Note: While 27 bp is the minimum requirement, standard 100 bp PE configurations are compatible and frequently utilized by core facilities to align with routine flow cell partitioning.
Data Processing & Analysis
Once the sequencing run is complete, the raw FASTQ files must be processed to generate digital count data.

  1. GeoMx NGS Pipeline: Process the FASTQ files using the GeoMx NGS Pipeline. This requires the config.ini file from the relevant Readout Package to map the sequences back to the AOIs.
  2. DCC Generation: The pipeline will output Digital Count Conversion (DCC) files. These files contain the quantified expression counts for each segment.
  3. Data Import: Import the DCC files back into the GeoMx DSP Control Center. This reconnects the count data with the high-resolution images and metadata (ROI/AOI selection) for integrated spatial analysis.

For advanced analysis, data can be exported from the Control Center or processed directly using specialized R-based computational frameworks such as GeomxTools and GeoMxWorkflows.
Protocol references
The quick guide was obtained from the NanoString University website (https://university.nanostring.com). The latest guidance and complete manual is also available on the site, and certain procedures and descriptions are quoted directly from the original manual.