GeoMx Digital Spatial Profiler
Software Updates

GeoMx Digital Spatial Profiler v2.1 Instrument Software and Data Analysis Suite Update

GeoMx software version 2.1 is the newest release (Nov 2020) that expands capability in NGS readout to Protein, as well as a host of new features and stability improvements across the instrument software, the GeoMx NGS pipeline, and the Data Analysis Suite.

NanoString recommends that users update to the latest version to take advantage of these enhancements and improvements.

Feature Updates in v2.1:

  • Ability to read Protein content for NGS readout
  • Scan workspace enhancements, including the image overlay feature. This feature allows an external image, such as an H&E stain, to overlay over the GeoMx scan to enhance ROI selection.
  • New user notifications in the Control Center
  • Coordination with the newest version of the GeoMx NGS Pipeline software (v2.0) to automatically process Illumina FASTQ sequencing files to GeoMx readable digital counts (DCC) for input back into the Data Analysis Suite
  • Enhanced Data Analysis Features, including improvements to QC: LOQ calculated separately, negative probe geomean flagged separately, and improvements to the CTA probe QC local outlier algorithm
  • Data Analysis plot performance and display enhancements to support larger studies
  • Cluster Analysis and Pathway Analysis as functions on the toolbar
  • Data Analysis support for multiple CTA probe kits, enabling custom RNA NGS analysis
  • Coordination with most recent version of the GeoMx Auxiliary Server software
  • Bug fixes, security, and usability improvements.
  • Windows 2019 Server with Hyper-V enabled
  • HTTPS (Port 443) open inbound
  • CPU: Intel processor with 64-bit support, 3.40 GHz or faster, 4 cores
  • Memory: 64GB DDR4
  • OS SSD: 512GB
  • Main Storage HD: 2TB

Please make sure that that the version of GeoMx Auxiliary Server installed matches the version of your system software. Mismatched versions will have incompatibility issues.

GeoMx Auxiliary Server v2.0 GeoMx Auxiliary Server v2.1
GeoMx v2.0 Software Compatible Incompatible
GeoMx v2.1 Software Incompatible Compatible and Recommended

geomx-ngs-pipeline.jpg

The GeoMx NGS Pipeline uses a series of novel algorithms to process sequencing files from FASTQ to the Digital Count Conversion (DCC) file format that is 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 resulting in the 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) 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.

Please check the version of the NGS processing pipeline with the version of software installed on your GeoMx system.

GeoMx NGS Pipeline 1.0 GeoMx NGS Pipeline 2.0
GeoMx v2.0 Software Compatible Compatible but not recommended
GeoMx v2.1 Software Incompatible Compatible and recommended

There are two components to the GeoMx NGS Pipeline, the Graphical User Interface (GUI) and the Pipeline which processes the files. There is an installer specific to each Operating System supported for the GUI and each Operating System supported for the Pipeline.

Local and Remote Runs Interface Pipeline Location OS CPU Memory (GB)
Windows GUI Local or Server (Linux/AWS) Windows 10 Intel Core i5-4750 3.20 GHz 16
Mac GUI Local or Server (Linux/AWS) MacOS Catalina V.10.15.5 Intel Core i5 2.60 GHz 16
Linux CLI Server (Linux/AWS) Linux Ubuntu 18.04 AMD Phenom 8650 2.3 MHz 16
AWS instance type: AWS t2.xlarge CLI Server (AWS) AWS Ubuntu vCPU 4 16

For smaller experiments sizes, the GeoMx NGS Pipeline may be run on a local machine and managed through a graphical user interface. Files should be available locally. The specifications below reflect needs of a pipeline run and may be impacted by other programs running on the same machine. These resources are adequate for runs containing up to 96 segments/ROIs (with up to ~50 million reads per segment/ROI).

For Apple OS:

  • Macbook pro with a CPU at 1.4 GHz processor or better
  • 16GB RAM

For Windows OS:

  • Intel® Core™ i5-835OU @ 1.70GHZ 1.90 GHz
  • 16 GB RAM
  • 64-bit Operating System, x64-based processor

For larger experiments sizes or multiple users, the GeoMx NGS Pipeline should be installed on a Linux server or on an AWS instance. Files should be available on the server. The Linux server can be managed from the graphical user interface which can be installed on Windows or Mac as above. The AWS implementation is best managed via the command line interface (CLI). The specifications below reflect needs of a pipeline run and may be impacted by other programs running on the same server. These resources are adequate for runs containing segments/ROI exceeding ~50 million reads per segment/ROI.

The Linux server requirements are as follows:

  • OS: Ubuntu 18 and up
  • 16 GB RAM
  • Adequate storage for data files via EBS, EFS or attached NAS. Recommended at least 2 GB RAM per thread and at least 1 GB of available free memory.
  • Open SSH installed and enabled.

The recommended AWS instance is as follows:

  • Platform: x64 system, capable of running Linux containers (Latest Linux or Ubuntu OS)
  • CPU & RAM: standard Amazon t2.xlarge (4 cores, 16GB RAM)
  • Main storage: 20GB
  • Extra File storage: At least dataset size x 10

Download the GeoMx NGS Pipeline Dataset Package

Goal

These sample FASTQ files can be used to test an installation of the GeoMx NGS Pipeline in a customer’s environment. The corresponding DCC files are also provided to match with outputs from the customer pipeline to ensure the pipeline is functioning properly.

Summary

This sample dataset contains NGS data for three samples (wells). The three wells represent two collected AOI’s (area of illumination) and one NTC (no-template control) from a GeoMx DSP experiment.

These samples are from a dataset prepared by NanoString using an early access version of the Cancer Transcriptome Atlas (CTA). These samples were chosen for this test dataset as they align with the expected typical Region of Interest (ROI) size and sequencing quality.

The defined ROI is roughly 300µm diameter and was segmented using PanCK. Both samples (wells) represented in this dataset were PanCK positive from two different ROIs.

Sequencing saturation was between 70% and 80% ((1 - raw reads/deduplicated reads) x 100)%.

Sample info:

File root/sample name Sample info Area Sequencing saturation
DSP-1001660005876-A01 No template control 0 µm2 -
DSP-1001660005876-A12 PanCK positive auto segment 86118 µm2 77.2%
DSP-1001660005876-C01 PanCK positive auto segment 74208 µm2 70.5%

Files included:

Each sample has eight FASTQ files- data are from four lanes of a NextSeq run using paired-end- so there are forward and reverse reads X 4 lanes for each sample.

File/Folder Description
3sampleAOIs_20201119_GeoMxNGSPipelinev2.ini The configuration file downloaded from the GeoMx DSP instrument. This file is required by the GeoMx NGS Pipeline v2.0 app to process the FASTQ files.
Subfolder: /FASTQ/ Folder containing FASTQ sequencing data from Illumina NextSeq. 8 files per sample (forward & reverse x 4 Lanes), 24 FASTQ files total.
Subfolder: /Run1DCCs/ Contains Summary of output, DCC zip file for upload to DSP DA, and sub-folder with DCC output from NanoString for comparisons.
Subfolder: /Run1DCCs/DCC_Files/ The DCCs files to compare with customer test run
/Run1DCCs/summary.txt Summary of number of raw, trimmed, stitched and aligned reads for each sample
/Run1DCCs/DCC-20201119.zip Sample of the DCC package which would be uploaded back to your GeoMx DSP
Subfolder: /Run1DCCs/logs/ Logs from pipeline
Subfolder: /Run1DCCs/Stats_Files/ Statistics files from the pipeline run for each AOI

Instructions:

Download the FASTQ folder and contents and the config file to a location where your GeoMx NGS Pipeline v2.0 installation can access them. Use “3sampleAOIs_20201119_GeoMxNGSPipelinev2.ini” as the configuration file and the FASTQ folder as the input folder. When your run is complete compare the DCC files in your output folder to those in the provided “Run1DCCs/DCC_Files” folder. You can inspect DCC file contents with a text editor. Sort order of the <Code_Summary> section may differ between your run and the provided DCCs.

You can copy <Code_Summary> sections to a new file and save as a comma separate value file format to compare in a spreadsheet program

Expected results:

The run should complete without warnings. The NTC sample, A01, in this experiment had no reads at all which aligned to our probe kit so aligning will show 0 reads (as observed in the “summary.txt” file). This is often the case for the no template control as no biological sample should be included in that sample well.

In testing, these samples took 3 minutes to process on a locally installed version of the GeoMx NGS Pipeline v2.0 using a system with a 2.30GHz processor, solid state hard drive and 16GB DDR2 RAM running Windows 10. NanoString recommends much larger runs with greater than ~50 million reads per segment/ROI be processed on a server installation of the Pipeline.

Raw reads: The number of reads that pass filter from the flow cell represented in the FASTQ file. GeoMx NGS Pipeline starts with FASTQ files.

Trimmed reads: Remove the adapter sequence

Stitched reads: Create one consensus read from the overlapping sequence of read 1 and read 2

Aligned reads: mapping the RTS-ID to a white list of sequences which represent targets

Deduplicated reads: remove PCR duplicates based on the UMI for digital counting

UMI: Unique Molecular Index. The molecular barcode that uniquely identifies each DNA molecule. During PCR amplification all duplicate molecules contain that barcode.

Please make sure you are on v2.0 prior to upgrading to 2.1. If you are not on 2.0, please go here to download materials and update. Please reach out to GeoMxSupport@nanostring.com for assistance.

GeoMx v2.1 Software Materials

Instructions:

  1. Please review the GeoMx DSP v2.1 Update Instructions prior to updating the software. We recommend printing the instructions out to have it available as you perform the update.
  2. Please have an USB of at least 16GB and an additional laptop or computer (separate from the instrument) available to perform the update.
  3. Please follow the GeoMx DSP v2.1 Update Instructions to perform the update.

GeoMx NGS Pipeline v2.0

GeoMx DSPDA Auxiliary Server v2.1