QuantArray(r) Microarray Analysis Software Version 3.00 Release Notes PN 8OM-808 Contents 1. System requirements for host computer. 2. New features in Version 3.00. 3. Bug fixes in Version 3.00. 4. Known problems and workarounds. 1. System Requirements for Host Computer The minimum computer requirements to run QuantArray Microarray Analysis Software Version 3.00 are: 300 MHz Pentium II processor 128 MB RAM for QuantArray; 256 MB RAM for QuantArray and ScanArray 4 GB hard disk, see below CD-ROM 10MB Ethernet network interface card Display capability of 1024 x 768 x 65K colors 17" color monitor Microsoft Windows NT 4.0 with Service Pack 4 or higher Microsoft Internet Explorer 5.0 Sufficient virtual memory, see below Hard Disk Requirements The amount of hard disk space required to store your images will depend on the number and size of the image files stored on the disk at any one time. The size of the image files created by ScanArray is dependent on the scanning resolution. The following chart provides estimates for the amount of hard disk space required for a single microarray and for a set of 50 microarrays. Please note that an instrument with an autoloader can process up to 20 microarrays in a single batch set. #Fluors/ Scan Disk Space for Disk space for Microarray Resolution Single Microarray 50 Microarrays 1 5 microns 125 MB 6 GB 1 10 microns 30 MB 1.5 GB 2 5 microns 250 MB 12 GB 2 10 microns 60 MB 3 GB 3 5 microns 375 MB 18 GB 3 10 microns 90 MB 4.5 GB 4 5 microns 500 MB 25 GB 4 10 microns 120 MB 6 GB 5 5 microns 625 MB 31 GB 5 10 microns 150 MB 7.5 GB Virtual Memory Requirements All computer programs require RAM (Random Access Memory) while they are running to store the results of the operations they perform. The amount of RAM required by a computer program varies, depending on the operations it performs and the system configuration. To achieve optimal performance from a computer program, your workstation should have enough RAM to accommodate the operating system requirements plus those of any application programs that are running concurrently. Most operating systems today (Windows NT included) can accommodate a smaller amount of RAM by using a technique called "swapping", where the operating system moves data between RAM and the hard disk as it is needed. The technique of using hard disk space to extend the amount of RAM available to a program is called virtual memory. Using virtual memory to compensate for insufficient RAM, degrades the performance of the application program and does not remove the requirement for a minimum amount of RAM. It does allow programs to run with a less-than-optimum amount, though. The minimum amount of RAM required to run Windows NT and QuantArray is described earlier in this section. QuantArray uses RAM to save images to display, and perform image processing and data analysis. The following chart provides estimates for the amount of RAM required by QuantArray, depending on how you typically use the software. For your convenience, the chart below contains a column describing the memory requirements if you scan images with ScanArray while you analyze previously-scanned images with QuantArray on the same computer. The memory requirements assume that you are performing a full-slide scan, displaying images while you scan, and all images are left open during the scan and analysis. #Fluors/ Scan QuantArray ScanArray+QuantArray Microarray Resolution Memory Requirments Memory Requirements 1 5 microns 700 MB 887 MB 1 10 microns 180 MB 224 MB 2 5 microns 1,140 MB 1,385 MB 2 10 microns 285 MB 344 MB 3 5 microns 1,450 MB 1,755 MB 3 10 microns 360 MB 434 MB 4 5 microns 1,760 MB 2,125 MB 4 10 microns 435 MB 524 MB 5 5 microns 2,070 MB 2,495 MB 5 10 microns 510 MB 614 MB To determine the total amount of memory required for the host computer, you should add the amount of RAM specified here to the amount of memory required by Windows NT and other applications you run concurrently on the same computer. To specify the amount of disk space that Windows NT uses for virtual memory, perform the following steps: 1. Open the Windows NT Control Panel by clicking "Start", "Settings", "Control Panel". 2. Double click on the System icon. 3. Select the "Performance" tab. 4. Click the "Change" button to change the "Total Paging File Size for all Volumes". 5. Set the initial page file size to the amount of memory required for the programs you run concurrently, using the recommendations above as a starting point. Set the maximum page file size to a value that is 20% greater than the initial page file setting. 2. New Features in Version 3.00 More information on each new feature can be found in the user manual. Save Graph - The software provides the ability to save an exact copy of any of the QuantArray graphs -- scatter plot, bar graph, pie graph, and threshold graph in a BMP format. Link Zoom - The software provides the capability of linking the zoom properties of all image windows within QuantArray. When one image is zoomed, all other images are zoomed the same amount. This option is available for enabling or disabling from the User Preferences dialog box and it will be saved and recalled with all other user preferences. Spot Histogram Display - The software provides the capability of viewing a histogram plot of the pixels within the square area normally known as the spot area or patch. The histogram plot contains the pixel values pertaining to the quantity on the Y axis and the intensity value on the X axis. There is one plot for each image presently loaded in the current experiment, to a maximum of 5. The histogram plot is visible to the user when the user selects the instant quantitation of a spot from the View Reports step. The plot(s) will appear below the spot images currently displayed in this dialog box. Under each plot sub-window shall appear the name of the image and the control designation of C when it is the control image. The software shall take into account the currently selected quantitation method when displaying the histogram plots and adjust the displays according to the requirements for each method below. Histogram: The plot displays draw vertical percentile lines for the signal low and high and the background low and high percentile parameter settings found in the currently loaded protocol. Fixed Circle: There are two plots for each image, one each for the spot histogram and for the background histogram. The plot displays draw vertical percentile lines for the signal low and high and the background low and high percentile parameter settings found in the currently loaded protocol. Adaptive: There are two plots for each image, one each for the spot and for the background. The plot displays draw one vertical percentile line for the threshold calculated and used by the adaptive algorithm to differentiate the signal from the background. Spot Quality Measurements - The software provides the user a set of raw measurements and quality confidence values for diameter, circularity, area, spot uniformity, background uniformity, signal to noise ratio, replicate uniformity, and deviation from nominal position. These values will be reported in the data output file. For each group of replicate spots, the mean and standard deviation of the reported intensity (quantitated by mean, median, mode or total, chosen in the protocol) of the replicate spots are calculated and reported in the output file. A quality confidence (between 0.0 and 1.0) value are calculated for each individual spot and each group of replicate spots and can be used to alert the user to spots that don't meet a user specified set of tolerances. The user is able to set a tolerance range for quality measurements and any spot whose value falls outside the tolerance range will be considered a bad spot. The system calculates a confidence value (between 0.0 and 1.0) for each spot or replicate spots. An overall confidence value, one for each spot, is also calculated from the individual confidence values. The combined value will use the minimum, weighted average or product of the individual measurements. In report views, spots whose measurements are not in tolerance range will be grayed like the filtered spots. The user can turn on or off each tolerance via a checkbox within the information window of the report view. The quality criteria, new measurements, and overall confidence value will be in the export file. In automation mode, the user can set warning and failure criteria by setting the maximum percentage of bad spots per slide and percentage of bad spots per replicate set. Replicate Spot Quantitation - The software provides a feature to intelligently handle microarrays that contain multiple spots with the same gene (replicates). The set of genes will be recognized as those that have the same gene id name. The user can specify the method used to combine multiple intensity values for the replicates into one intensity value, either by mean, median, or maximum. There is another selection indicating whether any spots that do not fall within the tolerance range of any enabled quality measurements should be used in the calculation of the combined intensity value. The combined value of the replicate genes will be exported to the export data file in the measurements section. Since one line will exist for each spot in the microarray, replicate genes will have the same intensity value in this section only. The original data for each spot separately, will appear in the raw data section of the export file. Enable Pie Graph - The software now displays a pie graph of each spot on the microarray. Each pie is comprised of the individual image spot intensity values. Select Ignore Spots - The software provides a feature in which the user can quickly set a spot to be ignored by double clicking on it from within the View Reports step. When multiple spots are simultaneously selected, double clicking on any one of the spots will ignore all of them. Validate File Name when Exporting - The software provides a new feature that automatically checks the string entered by the user in the export dialog box for correctness. Increase Size of Icons in the Graphs - The software increased the size of the icons used to display spot data in the bar, pie, and threshold graphs. This change utilizes the screen space better. Improve Template Import - The software improves the current protocol template import feature available in the protocol creation wizard by now including the import of all parameters included in the edit menu dialog boxes for array pattern, auto adjustable grid, quantitation method, quantitation parameters, and data export. Multiple Spot Selection - The software provides a new tool for a user to select multiple spots quickly with the mouse from the locate spots step. The software will then allow the user to move those spots or ignore them with the methods already available in the software. A new toolbar button is available and when the user presses this button, the user will then be able to click and drag a box around the spots to select all of them. 3. Bug Fixes in Version 3.00 Fix Instant Quantitation bug - The software fixes a problem where the calculation of the ratio and percentage values that appear in the Instant Quantitation dialog box when the "normalize to brightest" option is on, differs from the value reported in the graphs and export file. The software previously tried to normalize all spots by the value of the brightest spot in the array. Since there is only one spot quantitated during instant quantitation, the normalized ratio becomes 1 to 1. To fix this problem, the software temporarily disables the "normalize to brightest" option prior to calculating and displaying the ratio and percentage values during the Instant Quantitation display. Experiment purge parameters - The software now correctly recognizes previous installations of QuantArray and keeps the previous settings for the purge parameters during the update. For new installations, the purge parameters are defaulted to 0 to disable purging experiments. 4. Known Problems and Work-arounds None Issued March 2001