GeoMx Digital Spatial Profiler Software Updates v2.4

GeoMx Digital Spatial Profiler v2.4 Software Update

Introduction

The GeoMx DSP technology expands its functionality through the release of GeoMx DSP software version 2.4 in October 2021. For additional support or to report an issue with the software, email GeoMxSupport@nanoString.com.

If you are using or plan to use the Next Generation Sequencing (NGS) readout assays, Cancer Transcriptome Atlas or Whole Transcriptome Atlas, you will need to download the GeoMx NGS Pipeline to process the FASTQ files to DCC files which are then uploaded for data analysis. There were no updates to the pipeline software in this release cycle so please download and install version 2.3 GeoMx NGS Pipeline to use with v2.4 of the instrument software.

The GeoMx DSP software v2.4 offers the following new features:

  • Startup checklist on screen, notifying you of the system’s progress.
  • Pressure Sensor Delta error recovery, resulting in fewer errors of this type.
  • Fine alignment tool in Scan Workspace overlays panel.
  • Data Exchange in Administration, allowing you to export and import GeoMx files from one instrument to another via USB.
  • Performance Monitoring option in Administration.
  • Sample sheet output from GeoMx for NextSeq 2000 users.
  • Thumbnail mode for Data Analysis studies with numerous scans.
  • LMM analysis time optimized to be faster in Data Analysis.
  • New Custom Scripts environment in Data Analysis, GeoMx Set.
  • Search bar in pathway volcano plot and scatter plot, allowing you to search for x- and y- targets.
  • Improved versatility of software updates – ability to update to v2.4 from either v2.3 or v2.2.
  • Copy/paste field to create custom target group in Data Analysis.
  • Bug fixes, security, and usability improvements.
GeoMx Auxiliary Server
GeoMx NGS Pipeline
NGS Pipeline Dataset
Glossary of Terms
Download Material
GeoMx Auxiliary Server

System Requirements for GeoMx Auxiliary Server – Data Analysis Server for NGS

  • 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 the version of GeoMx Auxiliary Server installed matches the version of your system software. Mismatched versions will have incompatibility issues. Instrument software must be updated before Auxiliary server software.

GeoMx Auxiliary Server v2.2 GeoMx Auxiliary Server v2.3 GeoMx Auxiliary Server v2.4
GeoMx v2.2 Software Compatible and Recommended Incompatible Incompatible
GeoMx v2.3 Software Incompatible Compatible and Recommended Incompatible
GeoMx v2.4 Software Incompatible Incompatible Compatible and Recommended
GeoMx NGS Pipeline

System Requirements for GeoMx NGS Pipeline

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. Instrument software must be updated before Pipeline software.

GeoMx NGS Pipeline 1.0 GeoMx NGS Pipeline 2.0 GeoMx NGS Pipeline 2.2 GeoMx NGS Pipeline 2.3
(*last major release)
GeoMx v2.0 Compatible Compatible but not recommended Compatible but not recommended Compatible but not recommended
GeoMx v2.1 Incompatible Compatible Compatible but not recommended Compatible but not recommended
GeoMx v2.2 Incompatible Compatible but not recommended Compatible Compatible but not recommended
GeoMx v2.3 Incompatible Compatible but not recommended Compatible but not recommended Compatible
GeoMx v2.4 Incompatible Compatible but not recommended Compatible but not recommended Compatible

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
NGS Pipeline Dataset

Dataset for Testing GeoMx NGS Pipeline

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_20210224_GeoMxNGSPipelinev2.2.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_20210224_GeoMxNGSPipelinev2.2.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 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 2 minutes to process on a locally installed version of the GeoMx NGS Pipeline v2.2 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.

Glossary of Terms

Glossary of Terms

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.

Download Material

Download Material

If you are not on v2.2, please reach out to GeoMxSupport@nanostring.com for assistance with your upgrade.

For all GeoMx v2.4 software update materials on the Instrument Software, NGS Pipeline, and Auxiliary Server, please access here.

Please perform the Instrument Software Update before proceeding to update the NGS Pipeline and the Auxiliary Server.

Instructions:

  1. Please review the GeoMx DSP v2.4 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 v2.4 Update Instructions to perform the update.