ImaGene

ImaGene® 9.0 – Leading-Edge Microarray Analysis Software

ImaGene® 9.0 from BioDiscovery is the leading-edge microarray analysis software that offers optimal performance with the broadest and most refined features available today. Through proprietary machine vision technology, ImaGene® 9.0 delivers the highest quality data and most powerful automation to quantify, normalize and analyze your experiments.

Utilizing patented algorithms and processes, ImaGene® 9.0 provides the most reliable data for your microarray experiments. And, the easy to use software interface and elegant navigation makes it a snap to learn and become productive. With just a few mouse clicks, ImaGene® can quantify, normalize and analyze your microarray experiments. ImaGene® 9.0 and our complementary modules for expression analysis and aCGH analysis enable the broadest and most refined microarray data analysis. It’s time to take a closer look at the pioneering and most advanced microarray analysis software used by leading labs from around the globe for over ten years – ImaGene® 9.0.

System Requirements

32-bit Platforms Supported: Windows Win2k/WinXP/Win7   •  Minimum: 1.0 GHz Pentium, 1 GB RAM   •  Recommended: 2.0 GHz or faster, 2 GB RAM 64-bit Platforms Supported: Windows/MAC/Linux – Any platform that supports Java 1.6

Screenshots

Simple User Interface

Advanced Quality Control Parameters

Patented Segmentation and Contamination Removal Algorithm

Cutting Edge Features – Optimal Results

ImaGene® 9.0 offers the latest and most advanced features allowing you to rapidly explore the data from your microarray experiments with the highest level of confidence in its integrity.

Most Advanced Automation

  • Spot Finding & Grid Placement – patented and efficient algorithms automatically thread a grid through each data point, performing thousands of calculations per second ensuring optimal placement and alignment.
  • Spot & Array-Level Quality Control – ImaGene® 9.0 will automatically analyze every spot and the array itself to determine the quality of the array image or individual spots, minimizing the need for laborious manual inspection of tens of thousands of spots, allowing users to have confidence in the reported measurements.

Highest Quantification Accuracy

  • Patented Automated Segmentation – proprietary algorithms and processes are used to automate a precise segmentation process, discriminating between signal, background and contamination, ensuring the highest quality experimental data. This unique feature can even salvage contaminated spots by automatically removing artifacts that statistically do not match the signal or background intensity profiles.

One-Button Batch Processing

  • The optional batch-processing module enables rapid, completely hands-free processing of any number of images. This easy to use feature brings new efficiency to high-throughput environments where large numbers of arrays are processed each day. And, to keep check on workflow, an intuitive progress window shows you where you are along the way.

Elegant Interface and Navigation

  • Easy to learn and use – ImaGene® 9.0 continues a tradition of an intuitive design that is easy to learn to use and is similarly easy to navigate. With tabular formats and sliders, creating and managing settings is as simple as a click of a mouse.
  • Interactive and integrated – with interactive and integrated tabular and graphical data views, ImaGene® makes it is easy for you to see the correlations between images and related data with a wide variety of interactive plots (histogram, box, scatter, GenePie and M-A plots).

Analysis Modules

  • Flexible options in ImaGene® 9.0 can provide: multiple data normalization options including lowess on entire array; sub-grid or control spots; differential regulation analysis and visualization using Nexus Expression-Lite; and statistical aCGH analysis and visualization using Nexus Solo.

System Compatibility

  • ImaGene® 9.0 is platform independent as it is available for Windows, MAC and Linux operating systems; is compatible with broad variety of microarray types (staggered/orange packing, protein, aCGH, etc.) from a large number of manufacturers as well as home spotted arrays.

Additional Features

  • Highly flexible in providing automated features requiring limited user interaction and manual features for asserting more user control when needed
  • Scanner independent – works with all scanners including Tecan, Agilent, GenePix (Axon), Perkin Elmer, Innopysis, and more
  • Ready to use templates for many commercial arrays including Agilent 244K, 4x44K, 44K arrays and Exiqon’s miRCURY LNA™ microRNA Arrays

NEW in ImaGene ® 9.0

The new and improved features in ImaGene® 9 offer substantial performance improvement and higher quality allowing you to get to the results even faster.

  • Improved memory for efficient loading and processing of the latest high density arrays (e.g. 1M probes)
  •  A consolidated and streamlined new grid format containing both grid structure and GeneID information
  • A new design focused on core image quantification and visualization removing downstream analysis tools
  • Ability to add optional modules for further analysis of gene/miRNA expression (Nexus Expression™-Lite) and CGH analysis (Nexus Solo™)

ImaGene ® Full Feature List

Image Analysis

  • Any array: glass, filter, membrane, custom, or commercial – templates available for over 40 commercial arrays
  • Any scanning device, any grid format, any number of spots – Including: Packard, Axon, Agilent, Motorola and more
  • Completely Interactive User Interface
  • The most advanced image segmentation algorithms for contamination removal
  • Rapid quantification processing time for large arrays
  • Background correction and data normalization capability
  • Differential Regulation Analysis
  • Experiment/control and multi-replicate experimental design
  • Multiple channel capability 1, 2, 3, and 4 color channel compatible
  • One-touch contrast/brightness adjustment
  • Support for annotations
  • Saturation Detection
  • Simple and flexible GeneID file column format requirements
  • Measurement cleansing and normalization tools
  • OS independent – Windows, MAC, Linux
  • Automated Quality Control with Automatic Flagging
  • Manual spot flagging Customizable logic for combining flags from multiple channels
  • Ability to reload and review existing quantifications
  • Intuitive multi-spot selection engine for interactive spot analysis throughout plots, measurement table, image and segmentation preview
  • Ability to highlight gene IDs imported from a file as a control type
  • GAL , MAGE-ML import/export for array template info (including batch mode)
  • File formats can be exported in XML and text-based formats
  • Support for TIFF and other popular scanner image formats
  • One-click access to customer support center on our web site
  • Integrated screen capture

Data Analysis

  • Integrated data visualization tools in the core ImaGene® application for quality, trend and regulation assessment
  • M-A, histogram, box, scatter and GenePie Plots

Optional

ImaGene Guide

for Agilent Microarrays Scanned on the NimbleGen MS200 Microarray Scanner

INTRODUCTION

ImaGene Software, from BioDiscovery, provides a simple option for the viewing, grid alignment, and extraction of data from Agilent high-density microarrays that have been scanned on the NimbleGen MS200 Microarray scanner. ImaGene software can be used for primary data analysis or raw data files can be generated and imported into secondary software, such as BioDiscovery’s Nexus Copy Number or Nexus Expression software, for advanced analysis. Here we provide general guidance for the use of ImaGene software to create raw data files from Agilent microarray slides scanned on the NimbleGen MS200 Scanner. This guidance has been tested by Roche NimbleGen and BioDiscovery scientists, with data supplied from several research laboratories and hospitals, specifically on Agilent 1x1M and 4x44k two-color images and 4x44k one-color images. This guidance should also be applicable to other Agilent microarray formats with minimal changes required. To complete the processing of Agilent microarrays, the GAL file associated with the specific Agilent array design is required. The raw data files generated with this guidance were successfully input into BioDiscovery Nexus Copy Number software for the analysis of copy number variation (two-color images) or BioDiscovery Nexus Expression software for analysis of differential gene expression (one-color images). System Requirements With the exception of the 1x1M Agilent format, a 32-bit computer with 1GB RAM is sufficient. For processing 1x1M formats a 64-bit computer with at least 4 to 6GB of RAM is required.

WORKFLOW Array Processing: Arrays should be processed per the recommended protocol supplied by Agilent. NimbleGen MS200 scanning: Array scanning should be performed as described in the NimbleGen MS200 Microarray Scanner Operator’s Manual (v1.0). The array field can be set to “Roche NimbleGen” but a new scan region specific to the Agilent slide format being processed will need to be specified. For ease of feature extraction, it is recommended that full slide scans of multiplex formats be performed. If individual arrays on a multiplex slide are scanned separately, the Agilent GAL file will need to be modified to allow for feature extraction. Images can be saved as either Single or Multi-TIFF for subsequent processing in ImaGene software. ImaGene software processing: Images should be opened and gridded as described in the ImaGene software User Manual. Briefly, the image is loaded and the grid is imported by loading the GAL file associated with the Agilent array design. Once the GAL file is imported, the icons in the Main Control tab can be used to auto adjust the grid, adjust the spots, and compute the spot measures. The raw data can then be viewed and should be saved as “Separate files for each field” and “Separate files for each channel” (for two color images) in order to create individual raw data text files that are ready to use for further analysis.

Figure 1. Workflow icons (above) in the Main Control tab are used sequentially from left to right to process the images in the following order:

Figure 2. After the images are loaded, two tabs appear on the right panel next to the Composite tab containing the images for each channel. Figure 3. After the grid is loaded, aligned, and spots have been adjusted, measurements are made and certain spots are flagged (indicated by colored rings, boxes, or Xs around/on the spots in the “Composite” tab above). The bottom left of the screen shows the quantification tables with the spot intensities as well as quality measures and flag values for each spot. Another set of images can be processed after clicking “Close” on the Raw Data tab and then clicking “Clear All” on the Main tab to enable loading and measurement of another set of images. If a number of similar images are to be processed using the same settings, an optional Batch module can be used to automatically process images. This is quite useful in high-throughput environments where large numbers of arrays are processed daily. The same raw data files are generated by ImaGene whether using the batch mode or regular mode.

Figure 4. The “Save” button on the bottom left of the Raw Data tab allows saving of the quantified data so that it can be loaded into downstream software such as Nexus Copy Number or Nexus Expression for further analysis. Figure 5. When saving the quantified data, mark off both the checkboxes for “Separate files for each field” and “Separate files for each channel.” One tab delimited text file per channel per field (block) will be saved.

Importing into Nexus Copy Number software: For copy number analysis, the raw data files created by ImaGene can be loaded into Nexus Copy Number, following instructions in the Nexus Copy Number User Manual. The files will be loaded using data type “ImaGene”, which will prompt for two files: one for the test/experiment channel and one for the reference/control channel. A probe mapping file is also required for loading ImaGene data into Nexus Copy Number, as described in Appendix B of the Nexus Copy Number User Manual. For many catalog arrays, the probe mapping files are provided in the installer and the type of array needs to be selected in the “Probe Type” dropdown. If an array type is not available in the dropdown, consult Appendix B of the user manual or contact BioDiscovery Support for assistance in creating the probe mapping file. Figure 6. Loading samples in Nexus Copy Number. “ImaGene” is selected in the Type field. As an Agilent Catalog 4x44K array was used in this example, the Probe Type here is “Agilent 44K.” Experiment/Test samples are added to the left column by clicking on the “Add Experiment” button and Control/Reference samples are added to the right column by clicking on the “Add Control” button. Multiple samples can be added by clicking the “Add Experiment” and “Add Control” buttons for each set of samples. Once all samples have been selected, clicking “Done” will start loading the samples into the project. Importing into Nexus Expression software: For differential gene expression analysis, the raw data files created by ImaGene can be loaded into Nexus Expression as described in the Nexus Expression User Manual. The raw data files should be loaded using either:

A.  “ImaGene Single Channel” as the data type for one-color images.

B.  “ImaGene” as the data type for two-color images and if “Separate files for each channel” was checked when saving the quantified data within ImaGene.

C.  “ImaGene Single File” as the data type for two-color images if “Separate files for each channel” was not checked when saving the quantified data within ImaGene.

In case of technical replicates or dye-swaps, a sample descriptor should be loaded pointing to the raw data files (keeping the same sample name for the replicates) and the type of Agilent array as described in Appendix A of the Nexus Expression User Manual. SUMMARY The NimbleGen MS200 Microarray Scanner, when used with BioDiscovery ImaGene, provides an option for the scanning and feature extraction of Agilent microarray slides. ImaGene provides a simple solution for image quantification and easily creates raw data files that can be imported into BioDiscovery’s Nexus Copy Number or Nexus Expression for further analysis.