SlideReader V12. Software Manual. System Requirements. Required Privileges

Transcription

1 SlideReader V12 Software Manual System Requirements GHz Processor or higher - Operating System: WIN XP professional or WIN 7 professional, 64-Bit - 1 Gb RAM or more - Minimum 2 Gb free disk space, 20 Gb recommended Required Privileges During installation: - administrative privileges on local machine (Local Administrator) During operation: - administrative privileges on local machine (Local Administrator) CHIPRON GmbH Eresburgstrasse D Berlin Germany

2 SlideReader V12 Manual 2 SYSTEM REQUIREMENTS... 1 REQUIRED PRIVILEGES... 1 INTRODUCTION... 3 GETTING STARTED... 3 LOGIN DIALOG... 4 ADDING NEW CHIP TYPES... 5 GENERAL SETTINGS... 6 UNIQUE IDENTIFIER... 7 SCANNING AN IMAGE... 7 LOADING A TIF IMAGE... 9 ADD SAMPLE INFORMATION... 9 ANALYSIS CUT OFF VALUES FIELD INFORMATION (IMAGE WINDOW) FIELD DETAILS WINDOW EXCEPTIONAL WINDOWS & ERROR MESSAGES FIELD RECOGNITION FAILURE GUIDE DOT RECOGNITION FAILURE REPORT LIST AND SEARCH FOR REPORTS DATA EXPORT DATA STORAGE & SAFETY LOADING AN ANALYSIS CONFIRMATION MODE AUTOMATIC CUT-OFF CALCULATION (KRAS ANALYSIS ONLY) EXAMPLES VIDEO TUTORIAL... 32

3 SlideReader V12 Manual 3 Introduction The LCD SlideReader V12 software package has been developed to provide a fast and easy-to-use analysis tool for the extraction of data from LCD-arrays. The software analyzes one slide of eight arrays within minutes and provides comprehensive data reports in PDF format. Algorithms for pattern recognition are embedded to enable fully automated field and spot identification. Using input masks for samples and slide definitions will ensure that individual data (sample names or origins, slide ID and lot, user name and date of analysis) will be tracked by the analysis, and linked to the final report. This software is designed for optimal functionality in combination with images generated by scanners obtained as part of Chipron s Analysis Package. We can not ensure a flawless function when other scanning devices are used,. Getting Started Make sure the scanner is connected to the PC (USB) and the software protection USB Dongle is connected with a USB slot of the operating PC. USB Dongle (Protection Key) Connected to USB slot Have the slides you want to analyze and the corresponding pattern file ready. Chip type specific pattern files are provided on a CD with each LCD-Array Kit. A selection of standard pattern files can also be found on the SlideReader CD. Double click the SlideReader icon to start the software and the main window will open.

4 SlideReader V12 Manual 4 Four basic operations can be controlled from the main window - Login Dialog - Loading of previous analyses (for more details refer to the Analysis chapter) - Adding new chip types (Pattern Files) - General Settings Login Dialog The SlideReader software can be operated in two Loginn modes: - Free Access Log In is optional, but not required (recommended) - Log In Mandatory The software can only be used after a successful Log In with user name and password. The default setting for the Log In dialog is Free Access. This setting is recommended for standard laboratory use. Only if multiple users operate the software and user-specific separation of generated data is mandatory, the Log In mode should be set to Mandatory. For instructions on how to change the Log In mode refer to the General settings chapter below. How to Log In In the Free Access mode the Log In can either be ignored (User: Default) or a user can log in. To install a new user click the Log In button in the main window and click New in the Log In window. In the new window Create User Account type in your preferred User Name and Password rewrite the password and confirm with the OK button. The new user can now be selected from a drop down menu by clicking the Default button in the Log In window. When running an analysis as a specific user, the scanned images and saved data sets are stored in user specific folders. The PDF reports generated will not be stored in user specific subfolders but in the general Report folder. Note: Although these data are / can be stored in user specific subfolders, they are not protected from access by other users of the PC!

5 SlideReader V12 Manual 5 Adding New Chip Types When starting for the first time, the software has no information about the chip types intended for analysis. All arrays produced at Chipron will be supplied with a chip pattern file, sent on the CD included in the Kit box. Once imported into the software, pattern files will be available for all further analyses at every new start of the software. A new pattern file is imported by clicking the +New Chip Type button in the main window. Click the Add New Chip Type button in the CHIP Type window. An explorer window will open to let you select the destination folder and the chip pattern file to be imported. Be sure to select the pattern file from the folder Pattern File/SlideReader V12 from the CD. Note: Pattern files for SlideReader V9 will not work with SlideReader V12. After confirming the selection in the explorer window with the continue button, the chip pattern file will be imported into the software and will appear on the list of active pattern files. If a chip pattern file has to be replaced by a new version for the same type of chip, follow the instructions above, select Delete Chip Type and delete the respective old file from the explorer window prior to adding the new version. Leave the CHIP type window and confirm your operation by clicking the OK button or press Cancel to abort your operation.

6 SlideReader V12 Manual 6 General Settings The Settings dialog from the main window allows to set some general settings for the operation mode of the SlideReader software. Settings window with default values. Laboratory Your specific laboratory or company name, as it should appear on all generated reports, can be entered in the very first line. Log In Type Select if the SlideReader software should be run in Free Access or Log In Mandatory mode. (See chapter Log In Dialog above for more details). Analysis Confirmation Mode Select whether the automatic analysis of each array field has to be manually confirmed by the operator (active) or not (disabled). For more details about the impact of these selections look up the chapter Confirmation Mode at the end of this manual. Report Setting Select whether Cut Offs used during analysis should also be used for the generated reports (recommended) or if raw data should be used instead (only recommended for experienced users and special applications). CSV table Delimiter At the end of each analysis, the complete data (incl. measured signal values) can be exported as CSV file. Select which delimiter / separator should be used (Tabulator, Semicolon or Comma). Check Dongle By pressing the Check Dongle button, you will receive information about the dongle status, e.g. Unlimited for full version dongles or Limited run time count xy for demo dongles. Note: The settings Log In Type and Analysis Confirmation Mode can only be changed by using the software administrator password which is printed on the original CD. If this password gets lost, contact your supplier or support@chipron.com.

7 SlideReader V12 Manual 7 Unique Identifier All LCD-Array chips carry a label with a unique identifier composed of chip specification number (CsNo.), production lot number and ID-number. In the example given below, this unique identifier is Note: It is strongly recommended to use the unique identifier of each chip to name the image file generated by the scanner during the first step of the analysis procedure. Scanning an Image Open the Image window and click the Init Scanner button. After a short initialization, the scanner is ready to capture the image (green scanner lamp stopped blinking) and the Scan button will be activated.

8 SlideReader V12 Manual 8 Insert the chip in an upright position (chip label face up and directed towards operator) into the slide slot of the scanner, close the inner and outer lids and press the Scan button in the Image window. An explorer window will open where the destination folder (Default path: C:\CHIPRON V12\images\1 ) can be chosen and the image name must be defined. Use the unique identifier of the chip as image name (see above). Confirm your selection by clicking the OK button in the explorer window to start the scan. Due to the high resolution of the captured image, scanning and data processing can take up to 75 seconds. At the end of the scanning process, the captured gray scale image will be displayed in the Image window. In its center the image name (should be the unique identifier) is shown. During this process, the Scan Barcode Or Enter Chip Identifier field has been highlighted in pink. Type the unique identifier into this field (or use a barcode scanner to copy it from the chip label) and press the Confirm button below. Note: Entering the correct chip identifier is essential since the software learns from the first four digits (CsNo.) which chip type will be analyzed (e.g = Fungi 2.1, 0098 = HPV 3.5 etc.).

9 SlideReader V12 Manual 9 Loading a TIF image When using the SlideReader software for routine applications, where more than one chip will be analyzed at a time, it can be time saving to scan all chips prior to doing the analyses. Chip images captured in a batch mode can easily be uploaded with the Load TIF Image button for further analysis. Add Sample Information Prior to analysis, sample names and additional sample information can be assigned to each of the eight fields / tests. Although optional, this step is strongly recommended for later reexamination of the obtained results. If, in rare cases, the step should be omitted, simply proceed with the analysis (next chapter). To add these data to your analysis, open the window Sample Information. Two alternative methods are available: Manual typing of the data sets into the table within the Sample Information window, or importing a previously generated CSV file. For routine applications importing CSV tables is recommended. Manually The sample information mask for direct entering of the data displays eight rows representing the eight fields of one array, according to the field numbering, and six data columns for each field. The first column is predefined for the sample IDs (sample names). The following four columns can be used for defined additional information. Just rename the column headers by overwriting (e.g. overwrite Define 1 with PCR or Operator or Origin etc.). The last column is predefined as Comment and can be filled with special remarks in open text format. If identical information should be entered into one column for all samples, the Fill Column button below each column will copy the content of the first cell of one column into all others cells of that column.

10 SlideReader V12 Manual 10 Import CSV table Alternatively, the complete set of sample information can be imported as a *.csv file generated by an external software (e.g. MS Excel, Open Office, Text Editor) or handed over by a LIMS application. An example of the file structure is given on the SlideReader CD [ Folder: Import Table; File name: Sample Details.csv ]. It is essential to keep the file format exactly as it is given in this example file no other formats can be imported into the software. EMPTY Empty Fields For empty fields which have not been used for hybridization, it is necessary to click on the corresponding blue square in the upper right corner of the sample Information window. The field will turn red and the respective Sample ID field will be named Empty. This step is required to avoid an error message during the analysis due to missing guide dots in empty fields. Analysis Open the Analysis window to start the automatic image analysis process. Once completed, the window will display the analysis results in two different modes. The Image mode will display the real gray scale images of the array fields and the graphic mode will display small graphics indicating which spots have been identified as positive signals. The window will always display the results of four fields at a time, either the upper row of fields (Fields 1-4) or the lower row of fields (Fields 5-8). Using the View buttons or the Row Selector, the operator can choose between all four combinations (see below).

11 SlideReader V12 Manual 11 Example showing the upper fields (Fields 1-4) in Graphic view Example showing the lower fields (Fields 5-8) in Image view Switch quickly back and forth between the Graphic and the Image view to make sure that the software has correctly identified and displays all positive signals in the Graphic view which are clearly visible for the operator in the Image view.

12 SlideReader V12 Manual 12 Cut Off Values The analysis always starts by using a default Cut Off value given in the specific pattern file of the respective chip type. All signals below this cut off value will be ignored during the analysis and report generation (see also chapter General Settings for further options). This value (typically between 1500 and 3000) can be changed by the operator in the main Analysis window. To change the cut off to the same value for all fields, simply enter your new value into the Cut Off box and click Apply to All. The new settings will be active and the recognized, displayed features will change accordingly. Alternatively, the Cut Off can be set to different values for each field (see chapter Field Details below). Setting the Cut Off for all Fields Field Information (Image Window) Field Information (Image Window) Within the main Image window most field-specific data are already displayed. - Field number and sample name (blue arrow head) - Applied Cut Off value (yellow arrow head) - Result table with identified features and signal values (red arrow head). When moving the mouse over a positive signal in the Image view mode, the feature name of this particular spot will be displayed above the array field (green arrow head). This option becomes useful in cases of multiple positive features within one array field. The Details button above each array field (pink arrow head) opens the Field Details window, providing more information and allowing adjustments by the operator if required.

13 SlideReader V12 Manual 13 Field Details Window Once the Field Details window is open, the details for all eight fields can be loaded by clicking on the corresponding blue square in the Field Selector matrix in the upper left corner of the window (1). Three view modes can be selected, providing different options for analysis adjustments (2), individual Cut Off value settings for each field (3) and for changing the search area diameter for positive spots (4). (3) (1) (2) (4) Field Details window in Grid view The Field Details windows allow to control the complete analysis process for each field, to perform adjustments of the automatic grid finding step, to set individual cut off values for each field and to manually set particular, misinterpreted features as positive or negative (if necessary). Inexperienced users are strongly encouraged to open the Field Details window for each field to confirm that the automatic image analysis has worked properly.

14 SlideReader V12 Manual 14 Signal Intensities Switch to the Image view mode to see the detected signal intensities for each positive spot. The table at the right side of the window displays the Mean of both values belonging to one parameter (duplicates). Switch to the Graphic view mode to see which spots have been identified and counted as Positives by the software. Using the information from these two windows can help to identify an optimal cut-off value for each analysis. To change the cut-off value for a single field only, simply enter your preferred value into the small cut-off field above the array field image. Afterwards, click somewhere on the window to let your changes take place. To set the cut-off for all fields to an identical value, use the cut-off function in the main Analysis window (see above).

15 SlideReader V12 Manual 15 Changing the Search Area Size In some exceptional cases it can be helpful to increase or decrease the size of the search area for positve signals (e.g. when automatic spot identification is hampered due to attached dust particles or due to impairment of the spot morphology). The default value for the search area is part of the specific pattern file (GAL-File) for each chip type and depends on the pattern size ( e.g. 35 for a spot pattern of 9 x 9 and 40 for a spot pattern of 7 x 7).

16 SlideReader V12 Manual 16 Excluding Signals from Analysis Once in the View mode, you can choose the Delete option to exclude weak signals, or misinterpreted backgroundor dust particles from the analysis, and your report by clicking into the center of the search area circle for this particular signal. Deleted features will be marked by red crosses and their signal value will be set to zero. To undo this operation, simply choose Detect instead of Delete and click again on the same spot area. To return to the main Analysis window and to confirm new settings and changes you have made, click the OK button in the lower right corner of the Details window. Alternatively, click Cancel to return to the main window without saving any of your changes.

17 SlideReader V12 Manual 17 Exceptional Windows & Error Messages Under standard conditions the Slide Reader software will provide a complete, automatic analysis of the chip image. The underlying algorithms perform a hierarchical analysis in five steps. - identifying the field borders - guide dot localization in each field - grid alignment - spot identification - signal quantification In rare cases external influences like accumulation of dust particles, partial injury of the chip surface by mechanical contact with lab equipment during the protocol steps etc. can hamper this automatic analysis. Therefore, modules have been implemented to allow the user to intervene in such cases. Field Recognition Failure Cases in which a correct localization of all eight fields fails, a Popup window will open displaying the chip image. The image is superposed with eight squares indicating the positions where the software has located the individual fields. Squares displayed in red belong to the fields for which a definite localization was not possible. To adjust the area of analysis, move the mouse over the respective square without clicking and bring the square to its perfect position with the cursor keys of your keypad. Once the square has been placed exactly at the right spot, its color changes into bright green. Continue with the next squares displayed in red, if necessary. Confirm your operations with the OK button. The window will close and the analysis will continue.

18 SlideReader V12 Manual 18 Guide Dot Recognition Failure Three Guide Dots are immobilized in three corners of each array. These dots fulfill two essential functions during the analysis. - Their visibility as clear signals at the end of the protocol indicate that hybridization, labeling and staining has been carried out successfully. - They act as landmarks during the automatic image analysis and support the software during the grid alignment. In very rare cases it may occur that the automatic Guide Dot recognition will not work. The three main causes for such errors are: - you are trying to analyze array fields which have not been used for hybridization (Empty Fields) - the round shape of one or more Guide Dots has been damaged mechanically (e.g. contact with laboratory equipment) - any other disturbances (e.g. dust particles or high back ground) hamper the automatic recognition. In such cases the analysis will continue, but once the main Analysis window opens, the Details button of all fields for which the automatic grid alignment was impaired, will be surrounded by a pink warning rectangle. Perform the following operations to enable the software to perform a correct analysis, depending on the cause of this error.

19 SlideReader V12 Manual 19 Analysis of Empty Fields When empty fields have accidentally been included in the analysis (Fields 7+8 in our example above), simply return to the Sample Information window and click on the blue squares representing the empty fields. The squares will turn red and the fields will be excluded from automatic analysis. Then repeat the analysis by opening the Analysis window again.

20 SlideReader V12 Manual 20 Grid Alignment Failure Should any of the three Guide Dots belonging to an array field not have been detected correctly, the automatic grid alignment might fail (simulated for Fields 1+2 in our example below). For these fields click the Details button to open the Details window and proceed as follows: Simply click on the three guides dots (if visible) or the relative positions where they should be located in the following order: 1) upper left corner, 2) upper right corner and 3) lower right corner. The grid will automatically align, and can be further adjusted by grabbing (click & hold) one of the red guide dot search area circles displayed in the Grid window. Switch to the Image and the Graphic view to make sure that your new grid alignment led to a correct spot identification and to see the detected signal intensities

21 SlideReader V12 Manual 21 Report When all fields have been controlled, proceed with the Report window to generate your preferred report format. The recommended format is to generate a PDF report and to include all Details. Choosing these two options, a report in PDF-Format will be generated consisting of one Summary page displaying an overview of all eight array fields and one page for each field which has been analyzed including all settings, additional sample information (if entered) and a graphical data analysis. To generate this type of report, click Open As PDF, Include Details and the Generate Report button. The reports will be generated by a PDF generator which is part of the SlideReader software package. Displaying the reports requires a PDF viewer installed on your PC (e.g. Acrobat Reader, Adobe). Summary Page Details Page 1 Details Page 2

22 SlideReader V12 Manual 22 Example Report (Details) Analysis Data Chip Data Sample Data Image & Graphics Display Result Table with Signal values and Intensity Bars Explanation of Color Code Registered User Interference

23 SlideReader V12 Manual 23 List and Search for Reports All generated reports can be listed within an explorer window by clicking the List All Reports button. The reports are stored in individual folders named according to the underlying Unique Identifier of the analyzed chip ( in our example below). The complete reports or individual Details pages of single fields can be opened from the explorer window. Alternatively, you can search for particular detailed reports of individual fields by entering the sample name or parts thereof (in our example the sample name prefix K- has been used). All identified reports will be listed and can be opened by a double click a single click will lead to a preview of the respective report within the Report window.

24 SlideReader V12 Manual 24 Data Export Finally, the complete data set of an entire analysis can be exported as *.csv file for further processing in any spreadsheet application (OpenOffice, MS Excel etc.) or be handed over to a connected LIMS. Prior to export you can select if Raw Data should be exported, or filtered data with applied cut-off settings.

25 SlideReader V12 Manual 25 Data Storage & Safety At the first start of the SlideReader software you have selected a destination drive or folder where all data from your LCD-Array analyses should be stored. If no alternative path has been entered, the software has created a folder CHIPRON V12 on drive C:. All primary and secondary data from your analyses will be saved in the respective subfolders at X:\CHIPRON V12, unless you choose alternative destinations during the operation. Scanned images in folder Images Saved data sets in folder Data Reports in folder Reports All pattern files, which have been loaded through the +Chip Type dialog, are stored in the folder GAL Files. All data, which will be generated by operators logged in as Default, will be stored in the subfolders 1. Data generated by specific user logged in with their passwords will be stored in subfolders named by random number codes. For each new user a new specific number code will be generated. Note: These specific folders should solely help to sort the generated data by users. All users of the PC will have access to all folders! It is recommended to make back up copies of the saved images and reports from time to time to make sure that no data is lost in case of a PC break down or a crash of the hard disk.

26 SlideReader V12 Manual 26 Loading an Analysis From the Start window previous analyses can be loaded into the software including the original image, the sample information and any settings which have been changed during the first analysis. The former analysis can be reviewed, changes can be applied and the new set can be transformed into a new report. To load an analysis simply click the Load button in the Start window and choose any of the data sets (*.CHIP files) displayed in the file explorer window. Note: Data sets will only be saved when a report is generated during your analysis! If you close the software without generating a report, the current analysis will be lost. In such cases only the chip image will be saved and can be used for a new analysis. The following pop up window will remind you to generate a report and thereby save your data set before you can close the software.

27 SlideReader V12 Manual 27 Confirmation Mode This function has been implemented to provide more safety when routine analyses is performed with the SlideReader software. Selecting the Confirmation Mode in the Settings menu on the Start window will activate an additional control step for each analysis. This control step requires that any user will have to open the Details dialog for each array field, check the correct grid alignment and spot identification and confirm these steps by clicking the Confirm selector. Since this function has an impact on the general safety level during all analyses, it can only be activated or inactivated with the Admin Password provided with each CD. Note: If you have lost your Admin Password send an to When the Confirmation Mode is active, the Details buttons of all array fields will be surrounded by blue rectangles to indicate that the analyses for these fields have not been confirmed by the operator.

28 SlideReader V12 Manual 28 Open the Details window for each field and check the grid alignment in the Grid view and the spot recognition in the Image view. Finally, confirm the correct data analyses by clicking the Confirm selector for each field. Switch to the next fields by using the Field selector within the Details window.

29 SlideReader V12 Manual 29 When the analyses for all active fields have been confirmed, close the Details window and return to the Analysis window. The Details buttons above all confirmed fields will be surrounded by green rectangles. Only when all fields of a chip (except those which have been flagged Empty ) have been confirmed, a report can be generated. Automatic Cut-Off Calculation (KRAS analysis only) For some LCD-Array tests (e.g. KRAS 1.4 Mutation Analysis) the SlideReader software can perform a calculation of signal intensities and general background signals to provide a recommendation for a specific cut-off value for each array field. The analysis of point mutations in codon 12 and 13 of the kras oncogene from FFPE tissue sections requires the detection of as little as 1% mutated sequences in a wild type background. Due to the intensive amplification (45 PCR cycles) and the hybridization conditions used to reach this target, it can happen that elevated background signals are observed for several mutation specific capture probes. Under such conditions it can therefore be difficult to identify the weak positive signals of mutations present at very low concentrations. To support the user during this operation the new button Automatic appears below the cut-off value in the Analysis window when KRAS 1.4 LCD-Arrays is being analyzed and the respective Chip-Specification number has been entered at the beginning of the analysis ( 0217-Lot-ID for KRAS 1.4 arrays). Apart from the standard functions to define the cut off values which are available for all types of LCD-Arrays - applying the default cut-off value provided with the pattern file to all array fields (7500 for KRAS) - applying an increased or decreased cut off value to all array fields - or defining specific cut off values for each field individually, the Automatic button provides an additional option. When the automatic button is clicked on, the software will first apply the default cut off value (7500 for KRAS) to all array fields. In a second step the values of all detected signals above this cut off will be calculated and, for those fields showing an elevated background a new, higher cut off value will be applied.

30 SlideReader V12 Manual 30 Examples Example 1 Hybridization of the PCR products from a sample containing more than 10 % mutant sequences in a wild typ background. Applying the default cut off (7500) leads to a clear identification of the mutation in codon 13.

31 SlideReader V12 Manual 31 Example 2 Hybridization of the PCR products from a sample containing less than 5 % mutant sequences within a wild type background. When applying the default cut off (7500) to this array field, the interpretation is hampered due to the high background values generated by this sample. Clicking the Automatic button and thereby applying the calculated, higher cut off value to the same array field results in a clear identification.

32 SlideReader V12 Manual 32 Example 3 Hybridization of a sample negative for mutations in codon 12 and 13 of the kras oncogene but with an extremely elevated background due to a high DNA concentration in the DNA extract used as template for PCR amplification. Default cut off (7500) applied: Calculated Automatic cut off (21520) applied: For more information about the KRAS 1.4 LCD-Array kit refer to the product manual. Video Tutorial To watch a video tutorial of the main functions of the SlideReader including an example analysis open the file SRV12Tutorial.swf (interactive) or SRV12Tutorial.mpg on your CD. For any questions regarding this software manual or other products of Chipron GmbH, please contact your local distributor or write an to support@chipron.com