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Fragment Analysis

1. User Name Image Filename Administrator c data tutor tutorl ds Experiment Form File c data tutor samplet frm 8 23 1994 1 04pm Prepared On 2 3 4 5 6 Experiment Date 7 8 9 K SERAKAHERKEAHARRREnIA s Of fant Am e Bl 2 a Sle sl e6 EAC Al 4 Band RF Report A B a D E F G H 1 J Kjt 4 Band RF Ln Report generated on 8 23 1994 1 07pm Ready Image File c data tutor tutor1 ds Scanned on 1993 06 14 10 41 17 10 Units bp 11 12 Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane6 Lane 7 Lane 8 Lane 9 13 14 0 023 0 866 0 466 0 868 0 021 0 866 0 463 0 705 0 028 15 0 081 0 953 0 954 0 078 0 954 0 775 0 086 16 0 142 0 14 0 147 17 0 233 0 231 0 238 18 0 296 0 295 0 301 19 0 357 0 355 0 362 20 0 446 0 445 0 453 fal TETP Sheen z ae Figure 1 3 Transferring results to a spreadsheet pl 4 Fragment Analysis User s Guide Chapter 1 The Fragment Analysis workflow Se 1 4 Automating the analysis After you create a form for a typical sample you can use the form to analyze similar samples automatically You do this by loading the form selecting the steps you want to automate figure 1 4 and then loading the image After you load the image Fragment Analysis can perform a complete analysis without intervention on your part unless a problem ar
2. 4 12 4 7 Automating the analysis 0 00 0 ccc 4 14 4 7 1 Accessing the Automation Options window 4 14 4 7 2 Selecting the steps to automate 00005 4 14 4 8 Saving the experiment analysis 00 00 c eee e eee 4 16 4 8 1 The save procedure cece 4 16 4 8 2 Retrieving the saved experiment analysis 4 16 4 9 Closing the Fragment Analysis software 0 c eee eee eae 4 18 4 9 1 Closing the Image window 00 0 c eee eee 4 18 4 9 2 Closing the Report window 0 cee eee 4 18 4 9 3 Closing Fragment Analysis 00 0000 eee eee ee 4 18 Fragment Analysis User s Guide v Table of contents vi 5 1 5 2 5 3 5 4 5 5 5 6 5 7 5 8 5 9 Chapter5 Dual channel image analysis What is a dual channel image 0 00 e eee eee eee 5 1 How to create a dual channel image 000 eee eee 5 3 Examples of two channel experiments 00 0 cee eeu 5 3 Setting up for dual channel image analysis 5 5 5 Running a dual channel image analysis 0 0005 5 7 Dual channel reporting 0 00 0 c eects 5 7 Automating dual channel image analysis 0 000 5 8 Saving a dual channel image experiment analysis 5 8 Loading a saved dual channel image experiment analysis 5 8 Parttwo Reference Chapter 6 File menu 6 1 6 2 6 3 Experiment F OM estas ca Cann aks Sa aaa
3. 6 15 6 17 table 4 6 6 10 6 15 Report window 4 6 Reset command in zoom area 8 3 Returning 3 18 Rf in band statistics 3 24 9 15 rotate image 3 14 9 4 S saving analysis 4 16 6 12 9 16 analysis parameters 4 12 Experiment Form 3 8 6 11 specifications 4 12 scale factor parameter 9 12 scope in band finding 3 22 9 13 scrolling image 3 12 Select command 8 2 select lanes in band finding 9 13 semiauto in band statistics 9 15 Show command Experiment Form 4 12 6 2 Show Toolbar command 10 2 Side by Side Grey Color command 7 9 Index Side by Side Two Color command 7 8 size standards 3 6 6 6 9 9 size standards creating 9 9 size in band statistics 3 24 9 15 source in band statistics 3 24 9 15 standards amount 3 6 identifying lanes 3 6 3 18 6 7 pKI 3 6 size 3 6 verifying 9 13 standards files amount 6 6 9 9 creating 3 20 9 9 deleting 9 10 editing 3 20 9 10 loading 3 20 6 6 9 8 9 9 pKI 6 6 9 9 saving 3 20 size 6 6 9 9 types 6 6 9 9 using multiple 9 10 Standards Files in Experiment Form 3 4 Standards Setup inspector 3 20 9 8 status bar 2 1 Synchronize command 7 9 T tab separated delimiter 6 11 6 16 table report 4 6 6 10 6 15 Tile command 10 2 Tile Horizontal command 10 2 Tile Vertical command 10 3 toolbar about 2 3 Tools menu 8 1 tutorial image file 3 1 using 3 1 Fragment Analysis User s Guide Index 5 Index U unassigned in band statistics 3 24 Use Lane
4. Actual Size Full Size Zoom In Zoom Out Magnification Map Dual Channel Display Grey Color Adjust F9 Shift F9 gt F8 The View menu includes commands for modifying the image display In this chapter View menu options that are also available from the toolbar are identified with the appropriate toolbar icon The topics in this chapter are e Actual Size section 7 1 Full Size section 7 2 Zoom In section 7 3 e Zoom Out section 7 4 e Magnification section 7 5 e Grey Color Adjust section 7 6 e Map section 7 7 Dual Channel section 7 8 e Display section 7 9 Fragment Analysis User s Guide p7 1 Parttwo Reference 7 1 Actual Size The Actual Size command displays the image at 100 which is the actual size of the sample To display the image at the actual size of the sample click the Actual Size command Note The 100 actual size applies to 17 inch monitors with 1024 by 768 pixel array or 13 inch monitors with 640 by 480 pixel array For monitors that vary from these specifications the display may vary slightly from the actual sample size 7 2 Full Size The Full Size command scales the image to fit within the mage window boundaries To display the full image click the Full Size command 7 3 Zoom In The Zoom In command doubles the size of the image The center point of the Image window is maintained To enlarge the image click the Zoom In command 7
5. Click No unless you forgot to save the report and want to do so 4 9 3 Closing Fragment Analysis To close the Fragment Analysis software double click the control menu button E in the application main menu or select Exit from the File menu p4 18 Fragment Analysis User s Guide Chapter 4 Post analysis operations step by step FragmeNT Analysis File View Tools Analysis Window _____e detatuontuort ds Eo Band Characteristics Lane Number Lane Name 00 00 o o Lane Type gop aio Band Number qug 0p jilo Band Name Size 09 00 Alo Rf OYy 00 gilo Distance uy 0p milo Amount Volume OY 00 Amilo Source Percent c data tutor samplel fm Figure 4 9 Closing the saved analysis To try it now 1 Doubleclick the control menu button E of the Image window When a message appears asking if you want to save the experiment analysis click No Both the Image window and inspector close 2 Doubleclick the control menu button E of the Fragment Analysis main menu The Fragment Analysis window closes The tutorial is concluded For additional information on the features and functions discussed in chapters 3 and 4 see the Reference part of this guide Fragment Analysis User s Guide p4 19 Chapter 5 Dual channel image analysis The Fragment Analysis software can analyze two channel dual channel images Two channel data collection allows you to increase eff
6. File Name Directories _ds gel c data Ca List Files of Type File Name Directories MD Files ds gel tutorl ds c data tutor TS tutor2 ds G tutorl dir tutor2 dir List Files of Type MD Files ds _gel Network Figure 3 5 Selecting the file to process To try it now 1 2 On the main menu bar click File to display the File menu Click O pen Image The O pen Image window appears figure 3 5 In the Directories box double click the tutor directory located under the data directory Two file names appear in the file names list box Double click tutor1 ds A progress bar appears followed by the image and the Image Setup inspector The N umber of Lanes Experiment Type and Interpolation parameters specified in the Experiment Form appear in the inspector figure 3 6 Go to section 3 6 Fragment Analysis User s Guide p3 11 Partone Basics 3 6 Adjusting the image display The Fragment Analysis software includes many features that allow you to adjust the screen display Below are some examples of these features For a complete discussion see chapter 7 View menu reference and chapter 8 Tools menu reference 3 6 1 Moving and hiding the inspectors To move an inspector place the pointer on the inspector title bar and drag the inspector to a new location To hide an inspector double click the inspector control menu button 2 To show the inspector o
7. WOND a aN e Figure 5 6 The Lane Setup window 4 Complete the Lane Setup form for your channel 1 data and then click OK The window closes 5 In the Experiment Form click the down arrow next to the Channel box and then select 2 from the list 6 Set the analysis parameters for the channel 2 data if different from the channel 1 parameters 7 Click Lane Setup The Lane Setup window appears again with the lanes for channel 2 listed in the Lane List box and a 2 displayed in the Channel box 8 Complete the Lane Setup form for your channel 2 image and then click OK The window closes 9 In the Experiment Form window click Save or click Save As if you are modifying an existing form The Save Form File window appears 10 In the Save Form File window type a name for your form and then click OK N ote For detailed information on completing the Experiment Form see sections 3 1 through 3 4 and section 6 1 p5 6 Fragment Analysis User s Guide Chapter 5 Dual channel image analysis 5 5 Running a dual channel image analysis Running a dual channel image analysis is the same as running two single channel image analyses in sequence except that you perform the first two steps Image Setup and Lane Finding only once The parameters you set in those two inspectors affect both images After you prepare the Experiment Form and load a dual channel image perform the analysis as follows 1 With t
8. a SE ea a aa ae xii Partone Basics Chapter 1 The Fragment Analysis workflow 1 1 Setting up the experiment 0 2 ee 1 2 1 2 Performing the analysis 0 00 0 cece cee ee 1 3 1 3 Creatingareport 0 cece 1 4 1 4 Automating the analysis 2 0 00 cece 1 5 Chapter 2 Getting started 2 1 Starting the Fragment Analysis software 00 0 eee eee 2 1 2 2 Understanding the toolbar 1 20 0 eee 2 3 2 3 USING INSPECIOIS ses coca ete chew eee al ees E ae ee 2 4 2 4 Quitting the Fragment Analysis software 0 0 eee eee 2 5 Chapter 3 Fragment Analysis step by step 3 1 Accessing the Experiment Form 00 0 0 cece eee eee 3 2 3 1 1 Creatinganewform 00 eee 3 2 3 1 2 Three ways to use a form 0 ee 3 2 3 1 3 Working without a form 1 0 ee 3 3 3 2 Setting up the experiment 02 0 eee 3 4 3 2 1 Entering the experiment setup information 3 4 3 2 2 Entering the lane identification information 3 4 3 2 3 Automating the analysis 0 00 c eee eee 3 4 3 2 4 For more information 000 0 cee eee 3 4 3 3 Entering the lane setup information 00 0 cee eee eee 3 6 3 3 1 Identifying the standards 0 0 cece eee eee 3 6 3 3 2 Changing the information 0 0 0 0 eee 3 6 Fragment Analysis User s Guide iii Table of contents 3 4 3 5 3 6 3 7 3 8 3 9 Saving the Experiment Form 2 0 0 c cec
9. and then click Apply followed by Close Profiles indicating the intensity of each band appear on the image 6 Go to section 4 2 Fragment Analysis User s Guide p4 3 Partone Basics 4 2 Editing the bands Special band editing buttons on the toolbar or their counterpart commands on the Tools menu allow you to identify bands not found by Fragment Analysis or to remove bands from the analysis For example if the system identified two close bands as a single band you can delete the band marker and then add two new bands 4 2 1 Using the band editing buttons Three band editing tools are available For manually creating a band Click the Draw Band M anual button E and then move the pointer to the area where you want to create a band Drag the pointer diagonally to create a rectangle that surrounds the band figure 4 2 When you release the mouse button a band marker is inserted in the center of the rectangle you created a For finding a band semiautomatically Click the Draw Band Semiautomatic button and then move the pointer to the area in which you want the system to determine the band position Drag the pointer diagonally to create a rectangle that defines the band size When you release the mouse button a band marker is inserted in the area of maximum pixel intensity For deleting a band marker Click a band or bands by holding down x the SHIFT key and then click the Delete Band button or press the DELETE ke
10. available from the Experiment Form submenu serve different purposes e Use the Open command to load the form you want to use for the analysis or to modify an existing form e Use the Show command at the end of the analysis session to view the currently loaded form Because Fragment Analysis retains changes you make during the analysis you can use the Show command to retrieve the form and save the changes to update your form p3 8 Fragment Analysis User s Guide Chapter 3 Fragment Analysis step by step Save Form File File Name Directories A foe _Cancet_ data gt tutor tutor1 dir 5 tutor2 dir a Save File as Type Form Files frm Figure 3 4 Saving the Experiment Form To try it now 1 In the Experiment Form click Save The Save Form File window appears figure 3 4 In the File N ame box type sample and then click OK The window closes Fragment Analysis adds the frm file extension to the file name and replaces Form1 frm on the title bar of the form with the name of the file In the Experiment Form window click Close The window closes 4 Go to section 3 5 Fragment Analysis User s Guide p3 9 Partone Basics 3 5 Loading the image After setting up the experiment you select and load an image file Loading the image begins the analysis session If you did not load or create an Experiment Form you enter the experiment information in the inspectors as you proceed thr
11. 10 3 Cascade section 10 4 Tile section 10 5 Tile H orizontal section 10 6 Tile Vertical section 10 7 Arrange Icons section 10 8 Close All section 10 9 List of O pen Windows section 10 10 Fragment Analysis User s Guide p10 1 Parttwo Reference 10 1 Show Toolbar The Show Toolbar command displays or hides the toolbar W hen the toolbar is displayed a check mark appears next to the Show Toolbar command To show or hide the toolbar select the Show Toolbar command 10 2 Attach Toolbar The Attach Toolbar command attaches or detaches the toolbar to or from the right side of the screen When attached a check mark appears next to the Attach Toolbar command When detached you can move the toolbar To toggle between attach and detach select the Attach Toolbar command To move the toolbar first detach it and then move the pointer into the title bar of the toolbar Drag the toolbar to a new location and then release the mouse button 10 3 Show Status The Show Status command displays or hides the status bar at the bottom of the Fragment Analysis main window W hen the status bar is displayed a check mark appears next to the Show Status command To show or hide the status bar select the Show Status command 10 4 Cascade The Cascade command arranges multiple Image windows in an overlapping manner The title bar for each window is displayed and accessible To arrange windows in cascade fashion select the Ca
12. 3 Adjusting brightness and contrast Three buttons allow you to adjust brightness and contrast Adjustments you make with the buttons are temporary They are not saved with the image and they do not affect the data To adjust brightness and contrast click one of the buttons described below and then move the pointer to the intensity profile area hold down the mouse button and move the line Adjusts brightness M ove the line up to increase the brightness pixel intensity and down to decrease the brightness Repositioning the square bend point on the intensity profile changes the center of rotation Use the brightness button to move the square and then use the contrast button to change the contrast Adjusts contrast M oving the line closer to vertical increases the P contrast Fragment Analysis User s Guide p7 5 Parttwo Reference Adjusts brightness and contrast together M oving the pointer up and to the left increases the contrast at the lower range of intensity values and decreases the contrast at the upper range of intensity values logarithmic display M oving the pointer down and to the right increases the contrast at the higher intensity values and decreases the contrast at the lower intensity values exponential display Click Reset to return to the original display values 7 6 4 Adding color to the displayed image To add color to the displayed image click the Palette box A list of color choices appears Cl
13. 7 Automating dual channel image analysis You automate dual channel image analysis by setting the automation options first for the channel 1 image and then for the channel 2 image as part of setting up a dual channel experiment If you want the analysis to be completely automated image 1 analysis followed by image 2 analysis select all automation options for the channel 1 setup including Export as well as all automation options for the channel 2 setup Export optional If you want to omit Export you will need to double click image 2 after the image 1 analysis is complete to start the image 2 automated analysis 5 8 Saving a dual channel image experiment analysis To save a dual channel image experiment analysis 1 Complete the analysis for both the channel 1 image and the channel 2 image 2 When you reach the Band Statistics inspector for the channel 2 image click the Save Analysis button or open the File menu select Experiment Analysis to display a submenu and then select Save The Save Analysis File window appears 3 In the FileNamebox typea name for the experiment analysis file and then click OK Fragment Analysis adds the exp extension 5 9 Loading a saved dual channel image experiment analysis To load a saved experiment analysis 1 Load the image from which the experiment was saved 2 Open the Fragment Analysis File menu and select Experiment Analysis and then select O pen from the submenu The O pen
14. A 8 2 B22 SPAN hats aeneae Sala each ow Davies Mae ee aa a Sea whe aa alana 8 2 8 35 Pixel Locator seeds etna Ve ae Mee A Ae Alaa Awan Waa we LM 8 2 Fragment Analysis User s Guide vii Table of contents 8 4 Pixel Distance 6 eee 8 2 8255 ar Enla Eren re e E a Psy eu ee ean 8 3 8 6 Red CEr oski eepiig pi aep oa siaha naa eh A ip tes 8 3 to AT AET 010 a0 1 a 1 EEEE A EE EAE A AET ET need TE 8 3 8 8 Magnifier ean era E EEA O E AI E A 8 4 8 9 Add Band Manual 0 0 a 8 4 8 10 Add Band Semiautomatic auaa a 8 4 8211 Delete Bandiera aiora a a EE a E a OEA A 8 5 Chapter 9 Analysis menu 9 1 9 2 9 3 9 4 9 5 mage Setup ccc ace ee eee ede ee ed eee nun niis 9 2 9 1 1 Selecting the experiment type 00 0 2 e eee eee 9 3 9 1 2 Specifying the number of lanes 0 000 00s 9 3 9 1 3 Entering information for your records optional 9 3 9 1 4 Selecting an interpolation method 0 0 000 9 3 9 1 5 Rotating the image 0 cee ee 9 4 Lane Finders sceiedes ne mend oe be Mea ed bei Saeko 9 5 9 2 1 Initiating lane finding 0 e eee 9 6 9 2 2 Editing lane markers 0 00 0 cece 9 6 Lane Sep osi eens ede kee ea aed Pee Rae wee eee 9 6 9 3 1 Identifying lanes 0 0 c cece 9 7 9 3 2 Adding descriptive information 0 0 000 9 8 Standards Setup 2a ecede er Ries eae gated ede eek eae s 9 8 9 4 1 Three types of standards file
15. Band Statistics inspector click the Report button The Report window appears figure 4 3 In the Report window ensure that the Band Report button is selected Click the down arrow next to the Band Report box A list of band characteristics appears Click Rf Click the Report button The Report window closes and a different window the Fragment Analysis Report window appears showing the results data Double click in the Fragment Analysis Report window Excel starts with your data in a worksheet figure 4 4 Go to section 4 4 Fragment Analysis User s Guide p4 7 Partone Basics 4 4 Printing the results You can print the data report from Excel After returning to the Fragment Analysis application window you can also print the image as well as the Experiment Form Printing the report from Excel To print the report from Excel open the Excel File menu and then select Print figure 4 4 The Print window appears In the Print window select the print options or accept the defaults and then click OK A message appears confirming that the report is being printed Printing the image from Fragment Analysis To print the image from Fragment Analysis open the File menu and select Print Image T he Print O ptions window appears To accept the default settings click OK A second Print window appears To accept the default settings and print the image click OK Printing the Experiment Form from Fragment Analysis
16. Copy the ds file to the data directory 6 3 4 Changing drives The Drives box displays the current drive To change drives click the Drives box to display the list of drives and then click the option you want The list of files stored on that drive appears in the files list box 6 3 5 Connecting to shared directories The N etwork button displays the Connect N etwork Drive window Use this window for connecting to shared directories on the network For detailed instructions on using this window click the H elp button located in the window 6 4 Export Results The Export Results command displays the Export window figure 6 9 Use this window to specify the contents and format of a report and save it for export to a word processing or database application Reports you create contain the results of an analysis session p6 14 Fragment Analysis User s Guide Chapter 6 File menu Report Type Bend Report lt Otome Repor i Table Database Compatible Field Delimiter Comma Tab Figure 6 9 The Export window After selecting the format and contents sections 6 4 1 and 6 4 2 click SaveAs to open the Save Export File window 6 4 1 Selecting the format and contents You can choose among three report formats To select one of the formats click the appropriate button e Band Report Shows information about a selected band characteristic such as size amount or distance for all lanes To s
17. Excel File menu 4 5 1 Saving your report To save your Excel worksheet open the File menu and select Save C opy As The Save As window appears figure 4 5 In the File Name box type a name for the report Excel adds the xls extension and then click OK A Summary Information window appears Click OK unless you want to add information about the report 4 5 2 Closing the worksheet To close the worksheet double click the control menu button E of the worksheet 4 5 3 Exiting Excel To exit Excel you can either close the application reduce it to an icon or move it to the bottom of the window stack Close To close the application double click the control menu button E located on the Excel application window not the worksheet window e Reduceto an icon To reduce Excel to an icon click the minimize button E near the upper right corner of the Excel window This action leaves Excel running in the background and speeds processing when you again transfer data from Fragment Analysis to Excel e Hide the Excel worksheet To bring Fragment Analysis to the front of the window stack hold down the ALT key and press TAB until the Fragment Analysis window appears Repeat to display Excel p4 10 Fragment Analysis User s Guide Chapter 4 Post analysis operations step by step Save As FAENA Name Directories Lox worksheet in worksheet in fragm c excel test_xls fees excel Save File as Type Micr
18. Factor 0 100 Scope All Lanes Size Amount Std Figure 9 6 The Band Finder inspector 9 5 1 Finding bands To initiate band finding click the Find Bands button Fragment A nalysis e Locates the bands in all selected lanes e Marks each found band with a boundary marker and band position marker If Fragment Analysis incorrectly identifies artifacts as bands or overlooks bands use the editing tools to make corrections sections 8 9 through 8 11 and or adjust the N oise Factor or Scale Factor section 9 5 2 to change the band finding parameters and then initiate band finding again N ote If you want to see a profile representing the pixel intensity of the bands open the View menu select Display and then click in the Lane Profile check box Fragment Analysis User s Guide p9 11 Parttwo Reference 9 5 2 Adjusting the band finding parameters The N oise Factor and Scale Factor boxes allow you to fine tune the parameters that govern band finding By changing the values in the boxes you can instruct Fragment Analysis to find either more or fewer bands N ote You can apply different band finding parameters to the different groups of lanes selectable from the Scope buttons For example you can apply one set of parameters to Size standards select the Size button and a different set to Unknowns select the Unknown button You can also apply different parameters to individual lanes or groups of lanes you se
19. Positions in Experiment Form 6 8 V View menu 7 1 volume in band statistics 3 24 9 15 W Windows menu 10 1 windows list of open 10 3 workflow for analysis 1 3 Z Zoom Area command 8 3 Zoom In command 7 2 Zoom Out command 7 2 Index 6 Fragment Analysis User s Guide
20. Reference 9 2 1 Initiating lane finding To initiate automatic lane finding click the Find Lanes button Fragment Analysis finds the number of lanes you specified in the Experiment Form or in the N umber of Lanes box of the Image Setup inspector displays the number of lanes found and identifies the found lanes with rectangular markers 9 2 2 Editing lane markers If the lane markers do not accurately define the lanes you can move resize add or delete them Moving lane markers To move a lane marker click on it to select it then place the pointer inside the lane hold down the mouse button and drag the marker to a new location Resizing Lane Markers To resize a marker click on the marker you want to move Place the pointer on one of the edges and drag the edge in or out N ote that you can change the width but not the height of the lanes The height is determined by the size of the region of interest you define in the Image Setup step Adding and deleting lane markers You can use the buttons in the Lane Editor box to add and delete lane markers f Adds a selected lane marker Click the marker you want to duplicate l and then click the duplicate button Fragment Analysis creates a lane marker to the right of the selected lane Deletes lane markers Click the lane you want to omit from analysis W and then click on the delete button You can also delete the selected lane marker by pressing the DELETE key 9 3 Lane
21. Setup The Lane Setup command displays the Lane Setup inspector figure 9 4 Use this inspector to indicate which of the lanes on the image are standards lanes and to add additional identifying information regarding the lanes If you entered this information in the currently loaded Experiment Form the information will appear in the Lane Setup inspector Unless you want to change the information you need only click Done p9 6 Fragment Analysis User s Guide Chapter 9 Analysis menu Inspector Panes Lane Setup H Lane Identification Tape nan UO Description Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown 1 2 3 4 5 6 7 8 9 Figure 9 4 The Lane Setup inspector 9 3 1 Identifying lanes Lane identification is a three step process 1 Indicate the lane type by clicking anywhere on the Type box and then selecting the type from the drop down list Size Std or pKI Std Size A mt Std both size and amount standards in the same lane Amt Std Unknown Unused 2 Select the lane s from the lane list box or from the Image window To select one lane at a time click the lane To select more than one lane in the lane list box such as lane 1 and lane 6 CTRL 4click each lane number To select contiguous lanes in the Image window SHIFT 4click the first and last lane To select a group of contiguous lanes in the lane list box drag through the group 3 After you have
22. To print the current Experiment Form from Fragment Analysis open the File menu and select Print Form The Experiment Form is printed p4 8 Fragment Analysis User s Guide Chapter 4 Post analysis operations step by step Microsoft Excel Worksheet in FragmeNT Analysis Save Copy As ga Edit View Insert Format Tools Data Window Help New Ctrl N al U Ghose WE BARRREnIA Rn Al Update Save Workspace g 1 Find File 2 3 Summary Info 4 Page Setup ds 5 Print Preview ble1 frm E Print Ctrl P 7 Print Report 8 d Send Ti Add Routing Slip 1 CADATA TUTOR TUTORI CS Lane5 Lane6 Lane Lane8 Lane9 i 0 866 0 466 0 868 0 021 0 866 0 463 0 705 0 028 0 081 0 953 0 954 0 078 0 954 0 775 0 086 0 142 0 14 0 147 0 233 0 231 0 238 0 296 0 295 0 301 0 357 0 355 0 362 0 445 0 453 Prints document Figure 4 4 To try it now Printing the report from Excel Click File in the Excel main window The File menu appears Click Print The Print window appears In the Print window click OK The window closes and the report is sent to 1 2 3 the default printer 4 Go to section 4 5 Fragment Analysis User s Guide p4 9 Partone Basics ee 4 5 Exiting from Excel Before leaving Excel you might want to save your report To retrievea file saved in Excel use the O pen command from the
23. Understanding the rectangles The two rectangles on the image serve the following purposes figure 3 7 Small rectangle D efines the approximate band size and lane width for the analysis M ove and resize this rectangle to surround the largest band M ake sure that the rectangle closely outlines the band edges Large rectangle Defines the region you want to analyze Resize this rectangle to exclude from the analysis unwanted areas of the image such as the wells at the top of the image Set the top boundary at the bottom edge of the wells Set the right and left boundaries within half a lane width of the outermost bands the rectangle edges should be close to but not touching the edges of the outermost bands 3 7 2 Moving and resizing the rectangles To move or resize a rectangle you must first select it Place the pointer within one of the rectangles and click the mouse button Small square handles appear at the corners To move the rectangle place the pointer inside the rectangle and drag the rectangle to a new position To resize a rectangle place the pointer on one of the corner handles and drag the corner to a new position 3 7 3 Rotating the image To rotate an image that is not squarely aligned in the Image window click the image rotation button A red line with black handles at both ends appears on the image Drag the handle to indicate the degree of rotation and then click on Rotate The image rotate
24. and then click OK 3 In the Experiment Form click Save and then click Close The Experiment Form closes 4 In the Band Statistics inspector click in the Panes window to display a list of inspectors and then click Image Setup The Image Setup inspector appears with the rectangles located where you placed them 5 Click Done Fragment Analysis performs the complete analysis 6 After the analysis is complete click the Show Form button on the Band Statistics inspector When the Experiment Form appears click the Automation button to open the Automation O ptions window N ext deselect all automation options and click OK and then click Save and Close in the Experiment Form 7 Go to section 4 8 Fragment Analysis User s Guide p4 15 Partone Basics 4 8 Saving the experiment analysis Before exiting Fragment Analysis you can save the final results of the analysis just the way it appears at the end of a session You can then retrieve the results the objects and the data they represent for further work 4 8 1 The save procedure To save the experiment analysis click the Save Analysis button in the Band Statistics inspector or open the File menu select Experiment Analysis to display a submenu and then select Save The Save Analysis File window appears figure 4 8 In the File Name box type a name for the analysis file and then click OK Fragment Analysis adds the exp extension 4 8 2 Retrieving the saved experi
25. appears on the left side of the status bar After you release the button the information shifts to the right side of the status bar The information includes the Po pixel coordinates at point of origin the Pf pixel coordinates at the final point and the distance in mm between the two points The line remains on the image until you return to the Select mode p8 2 Fragment Analysis User s Guide Chapter 8 Tools menu To add the distance in pixels to the status bar display CT RL drag the pointer across the area you want to measure If you mark theimage with both the Pixel Locator and the Pixel Distance tools you can switch the information on the status bar from one type to the other by clicking the Pixel Locator or Pixel Distance toolbar button 8 5 Enlarge The Enlarge command doubles the image magnification To enlarge an image select Enlarge from the menu and then click the image area you want to enlarge The point at which you placed the pointer becomes the center of the image Click Select from the menu or toolbar N to end the Enlarge mode 8 6 Reduce The Reduce command halves the image magnification To reduce an image select Reduce from the menu and then click the area you want reduced The point at which you placed the pointer becomes the center of the image Click Select from the menu or toolbar N to end the R educe mode 8 7 Zoom Area The Zoom Area command displays a menu for creating and viewing a stack o
26. descriptions to lanes You can assign identifying names to the lanes and or add descriptive comments regarding the lanes To do this first type the name in the Lane N ame box and the description in the Description box N ext in the lane list box click the lane to which you want to assign the name and or description To select multiple lanes CTRL click each lane Finally click Set The names and descriptions are added to the lane list box Editing the lane list To remove a line from the lane list click the lane and then click Remove To add an entry to the list first make sure that the information about the new lane such as type and lane name is displayed in the Lane Setup window N ext click the lane name below which you want to insert a new lane and then click Insert Fragment Analysis renumbers the lanes Saving the changes to the lane positions The Use Lane Positions check box allows you to save the adjustments you make to the lane positions after Fragment Analysis has marked them For example if during the Lane Finder step you move one of the lane markers you can retain the new positions for subsequent analyses O therwise Fragment Analysis recalculates the lane positions each time you perform an analysis N ote that the Use Lane Positions box appears only after you have completed an analysis session 6 1 9 Selecting automation options Click the Automation button to obtain a list of automation options figure 6
27. el 6 2 6 1 1 About the Experiment Form 0000 cece 6 2 6 1 2 Entering information for your records 005 6 3 6 1 3 Entering the number of lanes 0 00 00 eee eee 6 3 6 1 4 Selecting 1 or 2 channels 0 0 00 eee eee eee 6 4 6 1 5 Selecting the experiment type 0 0 00 eee ee 6 4 6 1 6 Selecting interpolation methods 00005 6 5 6 1 7 Loading the standards file 0 0 c eee eee ee 6 6 6 1 8 Entering the lane setup information 6 6 6 1 9 Selecting automation options 0 0 eee 6 8 6 1 10 Saving the Experiment Form 00000 eee eee 6 11 6 1 11 Saving the form toanewname 000 00 eee 6 12 6 1 12 Closing the Window 00 0 c eee 6 12 Experiment Analysis 0 00 c cece eet tees 6 12 Open MMAGe svi aoa eb oe ea ee a Con aes 6 13 6 3 1 Locating the file you want 0 0 0 c eee eee 6 13 6 3 2 Selecting a file type 6 ee 6 14 6 3 3 Changing the directory aasan a 6 14 6 3 4 Changing drives aaan 6 14 6 3 5 Connecting to shared directories aaau asaan 6 14 Fragment Analysis User s Guide Table of contents 6 4 Export Results 0 0 ete 6 14 6 4 1 Selecting the format and contents sasaaa aaaea 6 15 6 4 2 Selecting the field delimiter aaa aa 6 16 6 4 3 Naming the report 0 0 e eee eee 6 16 6 5 Generate REP Ort sssri isor i aie aE tae i eae die eee 6 17 6 5 1 Selecting
28. file information e Thelocation of the standards and unknown lanes Band and lane names and descriptions e Thenoise and scale factor parameters Some parameters such as noise and scale factor are not displayed on the form Nevertheless they are saved 4 6 2 The save procedure To save the specifications click the Show Form button in the Band Statistics inspector or open the File menu click Experiment Form and then click Show from the submenu The current Experiment Form appears Click the Save button to add the new specifications to the form If you want to save the lane positions make sure the Use Lane Positions check box Lane Setup window figure 4 6 has an X in it 4 6 3 Creating a new form based on the current form To save the specifications under a new name create a new Experiment Form click the Save As button When the Save As window appears typea new name in the File N ame box and then click OK The new parameters are saved to a new Experiment Form and the original Experiment Form is retained p4 12 Fragment Analysis User s Guide Chapter 4 Post analysis operations step by step positions Click here to save lane CONDON AUN Description Insert uo te Benovo Tape Lone Name _Chomet 1 Size Std HMN Unknown Unknown Unknown Size Std HMN Unknown Unknown Unknown Size Std HMN h My Figure 4 6 Saving your specifications T
29. for an isoelectric focusing experiment type left and a DNA protein experiment type right If you specified a standards filename in the currently loaded Experiment Form the standards information will bein place You need only click Done p9 8 Fragment Analysis User s Guide Chapter 9 Analysis menu 9 4 1 Three types of standards files The analysis software handles three types of standards size standards amount quantity standards and pK1 standards Size standards files C ontain the sizes molecular weights of the standards bands Size standards file names include a siz extension Amount standards files Contain the amount of material in units such as nanograms in each standard band Amount standards file names include a mas extension pKI standards files Contain the isoelectric point values of the standards bands The pKI standards file names include a siz extension 9 4 2 Creating a standards file To create a standards file you specify a value size amount or isoelectric point for each band in the standards lane 1 2 Oyo Oy Click one of the Type buttons Size Std pKI Std or Amount Std In the Unit box type the unit such as bp Select the sorting order by clicking one of the Order buttons Ascending Descending or M anual If you select M anual use the Before and After buttons to specify where you want the value to be inserted In the Value box type the value for the first b
30. in tabular form You cannot save or print from this window Fragment Analysis User s Guide p6 17 Parttwo Reference SSS 6 5 2 Transferring the results to Excel To transfer the results to Excel double click the Fragment Analysis Report window Excel starts and your report data appears in a worksheet For information about saving and printing the report from within Excel see sections 4 3 through 4 5 6 6 Delete The Delete command displays the Delete window The delete window is nearly identical to the O pen Image window section 6 3 To delete a file or dataset locate the file name and click it The name appears in the File N ame box Click OK A message asks you to confirm the deletion The Fragment Analysis file type extensions are e gel Single image file e ds Image dataset file A ds file is actually a pointer file Selecting a ds file instructs Fragment Analysis to retrieve the image and associated files from the special directory dir directory in which they are stored e frm Experiment form file e exp Analyzed image file e siz Size standards file or pKI standards file e mas M ass amount standards file e txt Tab separated report file e csv Comma separated report file N ote Always use the Fragment Analysis Delete function to delete image files You cannot delete an Experiment Form file that is currently loaded Select the Close option from the Experiment Form s
31. inserted in the appropriate order either ascending or descending depending on the order you selected Changing a value To change a value in the list box 1 Inthe list box click the value to change 2 In the Value box type a new value 3 Click Change In the list box the old value is replaced with the new value Deleting a value To delete a value in the list box click the value you want to delete and then click Delete The value is removed 9 4 6 Saving changes to a standards file If you modified an existing file and want to save the changes click Save As to open the Save window and then e If you modified an existing file and want to save it as a separate file leaving the original file intact type a new name in the File Name box e If you want to save the changes to the existing file click OK A message asks if you want to replace the existing file Click Yes 9 4 7 Deleting a standards file To delete a standards file select the Delete command from the File menu section 6 6 When the Delete window appears search for siz or mas files select the file you want to delete and then click OK p9 10 Fragment Analysis User s Guide Chapter 9 Analysis menu 9 5 Band Finder The Band Finder command displays the Band Finder inspector figure 9 6 Use this inspector to initiate and control band finding Panes B and Finder Setup No of Bands in Std File i3 Scale Factor 0 1 Noise
32. lower the band finding threshold and then click Find Bands Bands that are slanted or nonuniform in intensity may also be overlooked In such cases try decreasing the Scale Factor parameter and then click Find Bands again For more information about the Scale Factor see the preceding paragraph Bands are difficult to select for deletion Be sure that you are clicking on the band marker the short line and not the band boundary the box Use either the M agnifier or Zoom Area tool for a closer look If the band marker is difficult to see open the View menu and select Display On the Display window turn off the Band Boundary display Also you can select a color for the band marker that is easier for you to see C 5 Retrieving a saved experiment When you attempt to retrieve a saved experiment the Open and Save options on the Experiment Analysis submenu are not available Be sure to first load the image on which the analysis was saved To recover open the File menu select O pen Image and then select the image file After the image appears load the experiment Fragment Analysis User s Guide pC 3 Index A Actual Size command 7 2 Add Band Manual command 8 4 Add Band Semiautomatic command 8 4 amount standards 3 6 9 9 amount in band statistics 3 24 9 15 Analysis menu 9 1 Arrange Icons command 10 3 Attach Toolbar command 10 2 auto in band statistics 9 15 Automatic Options window 4 14 automating the ana
33. on the image The pointer changes to a hand H old down the mouse button and move the hand to drag the image up down left or right Click Select from the menu or toolbar N to end the Pan mode 8 3 Pixel Locator The Pixel Locator command allows you to determine pixel locations and intensity values To determine pixel locations select Pixel Locator from the menu or toolbar The pointer turns into a cross Place the pointer over the image and click the mouse button A cross appears on the image and pixel information appears on the left side of the status bar When you release the mouse button the information shifts to the right side of the status bar The locator mark remains on the image until you return to the Select mode The information displayed includes the x and y location of the selected pixel P and the intensity value of the pixel To see the raw pixel value prior to conversion to the units used by the software hold down the CTRL key while clicking If you mark theimage with both the Pixel Locator and the Pixel Distance tools you can switch the information on the status bar from one type to the other by clicking the Pixel Locator or Pixel Distance toolbar button 8 4 Pixel Distance The Pixel Distance command allows you to measure the distance between pixels To measure distance select Pixel Distance from the menu or toolbar The pointer turns into a cross Drag the pointer to draw a line on the image The information
34. or the DELETE key The lane marker disappears p3 16 Fragment Analysis User s Guide Chapter 3 Fragment Analysis step by step FragmeNT Analysis Fi cAdatattutoritutortds ile View Tools Analysis Window c Adata tutor samplel fim Figure 3 8 Finding lanes To try it now 1 Place the pointer inside the rectangle in the Image window and drag the rectangle left or right to make sure that it uniformly surrounds the lane 2 Click Find Lanes The lanes are marked with rectangles and the number of found lanes is displayed figure 3 8 3 Review the placement of the rectangle markers and move or resize them to make sure that each lane is accurately defined 4 Click Done The Lane Setup inspector appears figure 3 9 5 Go to section 3 9 Fragment Analysis User s Guide p3 17 Partone Basics 3 9 Reviewing the lane setup information When the Lane Setup inspector appears figure 3 9 the lane identification information you entered in the Lane Setup window of the Experiment Form is shown in this inspector 3 9 1 Identifying the standards lanes The Lane Setup inspector is the counterpart of the Lane Setup window which you use to set up the experiment section 3 3 If you did not enter the lane setup information into the Lane Setup window you can use the same techniques to enter the information into the inspector N ote To select lan
35. points of the standards bands 4 1 1 Checking the isobar lines Isobar lines connect the standards bands that Fragment Analysis used to calculate the values of the unknown bands The lines should generally follow the smile of your gel caused by electrophoresis anomalies If they do not appear correct check to see that all the standards bands were found If they were not use an editing tool section 4 2 to identify the missing bands or to delete the spurious bands and then click Recalculate When checking the bands note that values for unknown bands that fall below the lowest or above the highest standards band are extrapolated 4 1 2 Magnifying portions of the display TheM agnifier tool is a magnifying glass for enlarging selected areas of the display figure 4 1 To use this tool click the M agnifier button da on the toolbar or select the M agnifier command from the Tools menu With the pointer now a cross shape on the Image window drag the M agnifier around the Image window to view different areas 4 1 3 Changing the line and label attributes You can hidethe isobar lines labels lane numbers and names and or band and lane marking objects You can also change the color of any of these objects as well as display intensity profiles of each lane To do this open the View menu and select Display The Display window appears Check boxes in the Display window allow you to select and deselect objects such as lane p
36. reenter the information and then click Set C 4 Band finding After you click Done in the Band Finder inspector a Found 0 bands message appears Be sure to click the Find Bands button before you click Done To recover on the Found 0 bands message window click Yes in answer to the Edit bands query and then click Find Bands Fragment Analysis found too many bands identified artifacts as bands TheN oise Factor parameter which controls the separation of relevant from irrelevant data is set too low Increase the N oise Factor value to raise the band finding threshold exclude more data from the analysis and then click Find Bands Fragment Analysis identified noise spikes as bands M ost spikes will be excluded as a normal part of the band finding process Fragment Analysis ignores spots that are less than half the width of a band To further exclude spikes increase the Scale Factor parameter The Scale Factor value indicates the degree to which the intensity of the left and right half of the band must match For example a value of 0 30 means that to be pC 2 Fragment Analysis User s Guide Appendix C Troubleshooting identified as a band the intensity value of the left half of the band must be at least 30 of the intensity value of the right half of the band Fragment Analysis overlooked bands The N oise Factor parameter is set too high To include more data in the analysis reduce the N oise Factor value to
37. standards file to the standards bands on the image Assignments are based on the order in which they are listed in the standards file Fragment Analysis estimates the volumes for all bands The volumes for the mass standards are then used in a linear regression analysis least squares straight line N ext the volumes of the unknowns are used to compute their quantities based on the linear regression figure B 3 pB 2 Fragment Analysis User s Guide Appendix B Calculating unknown band values y Volume Volume Oh p sse e unknown X Quantity Quantity of unknown Standards bands Figure B 3 Fragment Analysis performs regression analysis to determine the mass of unknown bands If only one mass standard value is listed in the mass standards file Fragment Analysis assumes a second standard with a volume of zero and a quantity of zero Fragment Analysis User s Guide pB 3 Appendix C Troubleshooting This appendix provides tips on how to avoid and resolve some of the problems you may experience when learning to use the Fragment Analysis software Problems in bold print are followed by causes and solutions C 1 Image setup After loading an image the region of interest and prototype band markers large red and small green rectangles appeared in their default mode instead of the way you set them You might have forgotten to save the changes you made to the rectangles the last time you
38. the mouse button A rectangular magnifying glass appears that enlarges the target area to the next magnification level section 7 5 R elease the mouse button to remove the M agnifier You can drag the M agnifier around the Image window to view different areas The status bar displays the magnification as well as the upper left pixel coordinates Pi and the lower right Pf pixel coordinates of the magnified area To increase and decrease the magnification press the plus or minus keys on the numeric keypad while holding down the mouse button Click Select from the menu or toolbar to end the M agnifier mode 8 9 Add Band Manual The Add Band M anual command allows you to manually identify a band that was not found using the current band finding settings To create a band click Add Band M anual from the menu or toolbar and then move the pointer to the area where you want to createa band Drag the pointer diagonally to create a rectangle that defines the band size When you release the mouse button Fragment Analysis inserts a band marker in the center of the rectangle you created Click Select from the menu or toolbar N to end the draw mode 8 10 Add Band Semiautomatic The Add Band Semiautomatic command instructs Fragment Analysis to insert a band marker in the area of maximum pixel intensity within an area you define To draw a band semiautomatically click Add Band Semiautomatic from the menu or toolbar and then move
39. the O pen option and then after the form appears click the Close button A basis for creating new forms For similar experiments you can retrieve an existing form select the O pen option modify it and then save it under a new name The form you use remains in effect until you select a new form close the application or unload a previously loaded form To unload a previously loaded form at either the beginning or end of the analysis session open the Experiment Form menu and select Close from the submenu p3 2 Fragment Analysis User s Guide Chapter 3 Fragment Analysis step by step 3 1 3 Working without a form You can perform an analysis without first creating a form In this case you load your image and then enter your specifications into the inspectors as they appear during the course of an analysis session At the end of the analysis the entries you made in the inspectors are automatically stored in a form which you can then choose to save Experiment Form Experiment Analysis Open Image F Export Results Generate Report Delete Rename Print Image Print Form F Print Setup Alt F4 Figure 3 1 Opening the Experiment Form window To try it now 1 On the main menu bar click File to display the File menu 2 Inthe File menu click Experiment Form A menu of options appears figure 3 1 3 From the list of options select New The Experime
40. wm 375 657 Fragment Analysis User s Guide Version 1 1 lt t Amersham FluorSep and ImageQuant are trademarks of Amersham Biosciences UK Limited Amersham and Amersham Biosciences are trademarks of Amersham plc Microsoft and Windows are trademarks of Microsoft Corporation Cover The image shown in this manual is courtesy of Data Genetics Hayward California The information in this document is funished for informational use only and is subject to change without notice Amersham Biosciences assumes no responsibility or liability for any errors or inaccuracies that might appear in this document All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them A copy of these terms and conditions is available on request Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Biosciences SV Corp 928 East Arques Avenue Sunnyvale CA 94085 4520 USA Amersham Biosciences Corp 2002 All rights reserved April 2002 Table of contents Preface About this qQuid s s 6 isus aaga a a r a ees 4 a era bres xi ASSUIMPUOMNS eaa aan a A a A A a Ta a E REA xi ASSAN CE danna a Soy ia
41. you just drew and then click the Delete Band button 34J to remove the band 5 In the Image window click any band Handles appear on the band and statistics for that band appear in the inspector 6 In the inspector click the Band Name box type your name and then click Set 7 Go to section 4 3 Fragment Analysis User s Guide p4 5 Partone Basics 4 3 Creating a report You can create a report that shows the results of the analysis Its contents can either be transferred to an Excel worksheet click the Report button or saved for export to a word processing or database application click the Export button See appendix A for examples of reports 4 3 1 Report versus Export Buttons on the Band Statistics inspector let you choose either R eport or Export Alternatively you can select Generate Report or Export Results from the File menu e Report Displays the Report window figure 4 3 for specifying report contents After you make your selections and click the Report button the Fragment Analysis Report window appears Double click in this window to transfer the results to Excel Data must be saved from Excel See section 6 5 for details e Export Displays the Export window for specifying report contents After you make your selections and click the Save As button the Save Export File window appears In the File Name box type a name for the report and then click OK Fragment Analysis adds either a txt or
42. you will use the magnification tool edit the bands create and print a report save the entries you made during this session automate the analysis and then exit Fragment Analysis If you want to quit now you can save the analysis and then retrieve it and finish the tutorial later See section 4 8 1 for instructions on saving the analysis see section 4 9 for instructions on quitting the analysis Fragment Analysis User s Guide p3 25 Chapter 4 Post analysis operations step by step This chapter explains the tasks you can perform after completing the analysis It also continues the tutorial that started in chapter 3 The tutorial portion of this chapter assumes that you have completed an analysis and that the analyzed image and Band Statistics inspector are displayed The topics in this chapter are e Inspecting the bands section 4 1 Editing the bands section 4 2 e Creating a report section 4 3 e Printing the results section 4 4 e Exiting from Excel section 4 5 Saving your specifications section 4 6 e Automating the analysis section 4 7 Saving the experiment analysis section 4 8 e Closing the Fragment Analysis software section 4 9 Fragment Analysis User s Guide p4 1 Partone Basics 4 1 Inspecting the bands W hen the Band Statistics inspector appears isobar lines are added to the image as well as labels on the outermost standards lanes showing the molecular weights or isoelectric
43. 0 14 3 0 234 502 004 Unassigned 7 447 Automatic HMN 1 Standard 5 900 18 2 0 297 699 848 Unassigned 10 382 Automatic HMN 1 Standard 6 800 21 8 0 356 790 388 Unassigned 11 725 Automatic HMN 1 Standard 7 700 27 4 0 448 1115 799 Unassigned 16 552 Automatic HMN 1 Standard 8 600 31 3 0 511 386 527 Unassigned 5 734 Automatic HMN 1 Standard 9 500 34 9 0 57 402 561 Unassigned 5 972 Automatic HMN 1 Standard 10 400 41 6 0 68 492 123 Unassigned 7 3 Automatic Lane Name Lane Number Lane Type BandName Band Number Band Size bp Distance BandRf BandVolume BandAmount Percent Band Source 2 Unknown 1 193 703 3 2 0 869 308 577 Unassigned 41 713 Automatic 2 Unknown 2 110 073 58 5 0 956 431 194 Unassigned 58 267 Automatic Lane Name Lane Number Lane Type BandName Band Number Band Size bp Distance BandRf BandVolume BandAmount Percent Band Source 3 Unknown 1 666 238 28 6 0 467 286 517 Unassigned 100 Automatic Lane Name LaneNumber Lane Type BandName BandNumber Band Size bp Distance BandRf Band Volume BandAmount Percent Band Source 4 Unknown 1 191 648 53 3 0 871 390 735 Unassigned 37 409 Automatic 4 Unknown 2 108 906 58 6 0 958 653 757 Unassigned 62 591 Automatic et Fragment Analysis User s Guide pA 3 Appendix B Calculating unknown band values This appendix explains how Fragment Analysis determines the values of unknown bands fragments The method of determining values for fragment size molecular weight and isoelectric focusing point is different from th
44. 110 073 108 906 1200 111 387 282 875 1200 1100 1100 1100 1000 1000 1000 300 900 300 800 800 800 700 700 700 600 600 600 500 500 500 400 400 400 300 300 300 200 200 200 100 100 100 Lane Report Report generated on 8 26 1994 8 12am Administrator c data tutoritutor1 ds Experiment Form Filename c data tutor sample1 frm Sample 1 8 25 1994 2 26pm c data tutoritutor1 DIR SEP1 GEL 1993 06 14 10 41 17 Lane Name Lane Number Lane Type Band Name Band Number Band Size bp Distance mm BandRf Band Volume BandAmount Percent Band Source 2 Unknown 1 193 703 53 2 0 862 470 00 Unassigned 46 33 Automatic 2 Unknown 2 110 073 58 5 0 948 544 53 Unassigned 53 67 Automatic See pA 2 Fragment Analysis User s Guide Appendix A Report examples Table Report Report generated on 8 26 1994 11 12am User Name Administrator Image Filename c data tutoritutor1 ds Experiment Form Filename c data tutor sample1 frm Experiment ID Sample 1 Experiment Date 8 25 1994 2 26pm Prepared On Image File c data tutor tutorl DIR SEP1 GEL Scanned on 1993 06 14 10 41 17 Lane Name Lane Number Lane Type BandName BandNumber Band Size bp Distance BandRf BandVolume BandAmount Percent Band Source HMN 1 Standard 1 1300 1 4 0 023 138 594 Unassigned 2 056 Automatic HMN 1 Standard 2 1200 5 0 082 507 144 Unassigned 7 523 Automatic HMN 1 Standard 3 1100 8 7 0 142 720 984 Unassigned 10 695 Automatic HMN 1 Standard 4 100
45. 4 Zoom Out The Zoom Out command halves the size of the image To reduce the image click the Zoom Out command 7 5 Magnification TheM agnification command displays a menu of magnification percentages To magnify or reduce the image click one of the percentages listed in the menu p7 2 Fragment Analysis User s Guide Chapter 7 View menu 7 6 Grey Color Adjust The Grey Color Adjust command displays the Grey Color Adjust window figure 7 1 To close the window click the Close button This window graphically reflects the intensity of the image displayed in the Image window High Display Level c data datblot ds Maximum Range Value Range Adjust Bar Log exp Contrast Intensity Profile Brightness Minimum Range Value Low Display Level Figure 7 1 The Grey Color Adjust window 7 6 1 What the Grey Color Adjust window does Your monitor can display 256 levels of grey but an image file can contain up to 46 700 levels of intensity The Grey C olor Adjust window allows you to view your data more clearly by excluding some of the unused intensity levels background and unused high values of your image In this way you evenly distribute the relevant image data over the 256 levels of grey You can also use the Grey Color Adjust window to change the contrast brightness and color of the image N ote Changing the image display using the Grey Color Adjust window affects only the display T
46. 5 p6 8 Fragment Analysis User s Guide Chapter 6 File menu Automation Options Automate the following steps L image CO Lane Finder jo Lane Setup E Standards Setup O Band Finder E Export Figure 6 5 Automation Options window no options selected How automation works To automate the analysis you click the boxes next to the steps you want to automate an X appears in the box You can select any or all steps Fragment Analysis uses the parameters in the currently loaded Experiment Form to automate the analysis If you are not certain that the region of interest indicated on the image by the large rectangle is appropriate for the image you have loaded it is best not to automate the Image Setup step About the automation options Checking the automation options for the inspectors produces the following results Image Setup Sets the region of interest and the prototype band size implements the Image Setup parameters rotates the image if applicable and displays the Lane Finder inspector Lane Finder Finds lanes and then displays the Lane Setup inspector Lane Setup Implements the lane setup parameters and then displays the Standards Setup inspector Standards Setup mplements the standards parameters and then displays the Band Finder inspector Band Finder Finds bands and then displays the Band Statistics inspector Fragment Analysis User s Guide p6 9 Part
47. Analysis window appears 3 In thelist of file names locate the experiment analysis file and then double click it or click the file name and then click OK The saved experiment appears The objects are displayed on both images and the Image Setup inspector is replaced by the Band Statistics inspector for whichever image is active To see the statistics for the other image double click the image p5 8 Fragment Analysis User s Guide Part two Reference w Chapter 6 File menu Experiment Form Experiment Analysis Open Image F7 Export Results Generate Report Delete Rename Print Image P Print Form F Print Setup Shift P Exit Alt F4 The File menu contains commands for accessing the Experiment Form managing and printing files generating reports and exiting Fragment Analysis The topics in this chapter are e Experiment Form section 6 1 e Experiment Analysis section 6 2 Open Image section 6 3 e Export Results section 6 4 e Generate Report section 6 5 Delete section 6 6 e Rename section 6 7 e Print Image section 6 8 Print Form section 6 9 e Print Setup section 6 10 e Exit section 6 11 Fragment Analysis User s Guide p6 1 Parttwo Reference 6 1 Experiment Form The Experiment Form command displays a submenu with the following options e New Opensa blank Experiment Form The form contains some default settings for running an an
48. Date _ ___ Number of Lanes o General Comments JE Channel Lane Setup Automation lie Experiment Type RAE T Standards Filenames ee SEE pK Amount east Squares Straight Line Figure 6 2 The Experiment Form set for an isoelectric focusing type experiment p6 4 Fragment Analysis User s Guide ww Distance log MW Distance Chapter 6 File menu 6 1 6 Selecting interpolation methods The Interpolation selection boxes allow you to select the curve fit method by which Fragment Analysis determines the size amount or isoelectric focusing point of unknown bands N ote that the curve fit choices can vary according to the type of experiment you select Before applying the curve fitting calculations Fragment Analysis mathematically superimposes the bands from the standards lanes onto the unknown lanes Isobar lines displayed on the image show the calculated location of the standards bands N ext Fragment Analysis calculates the curve through the standard points in the lane using the curve fit method you chose in the Interpolation area Finally the software determines the molecular weight values of the unknowns from the standard curve You can choose among the following curve fit methods point to point logarithmic is the preferred method Point to point linear Point to point linear uses the following formula to calculate the sizes or isoelectric
49. For detailed information about choices in the Experiment Form see section 6 1 For information about analyzing dual channel images see chapter 5 p3 4 Fragment Analysis User s Guide Chapter 3 Fragment Analysis step by step Form1 frm Experiment ID Experiment Date 7 20 1994 1 32pm Today Number of Lanes bB General Comments JE Channel Lane Setup Automation Experiment Type Standards Filenames Size Point to Point Logarithmic Amount east Squares Straight Line Figure 3 2 Setting up the experiment To try it now 1 Click Today Today s date appears in the Experiment Date box figure 3 2 2 Doubleclick the Number of Lanes box and then type 9 You will leave the other text boxes blank and accept the defaults in the selection boxes 3 Click Lane Setup The Lane Setup window appears 4 Go to section 3 3 Fragment Analysis User s Guide p3 5 Partone Basics 3 3 Entering the lane setup information After you click the Lane Setup button the Lane Setup window appears showing the number of lanes you typed in the Experiment Form Use the Lane Setup window to identify standards lanes and enter other identifying information about the lanes 3 3 1 Identifying the standards To identify the standards lanes display the Type list and then select the appropriate type Size standards define the molecular weight of each band Amount standards define the amount
50. Hated Wile oo a a a Peale a Pal wale an eh alee 10 2 10 6 Tile Horizontal 0 0 0 eee 10 2 10 7 Tile Vertical Srur sco contac tan dolce que ease A nae daa 10 3 10 8 Arrange ICONS wees eee Rew cee ee eee Pade oD ae 10 3 10 9 Close Alh Serieari a Ree A aad ena E AS 10 3 10 10 List of Open WINdoWS 00 0 ccc eee 10 3 Part three Appendixes Appendix A Report examples Appendix B Calculating unknown band values B 1 Determining the size or isoelectric point of unknown bands B 1 B 2 Determining the amount quantity of unknown bands B 2 Appendix C Troubleshooting GI mage SeU piire Reta Eee ere a eae cea Res C 1 C22 tane PINGING 324 6 iori Baie hese ee aa eae C 1 6 3 Lane SOU Dira eddie dence tea iaa DEAA wed an daod in a ead agate C 2 C4 Band finding sv iene sienna eee hae eee eae wanes C 2 C 5 Retrieving a saved experiment 0 ccc eee C 3 Fragment Analysis User s Guide ix Preface About this guide This guide explains how to use the Fragment Analysis software to analyze DNA and protein fragments separated by electrophoresis The manual is divided into three parts e Part one Basics Provides introductory information and step by step procedures for performing an analysis Part one also includes a tutorial chapters 3 and 4 that walks you through an analysis session from loading an image to creating a report The final chapter in part one explains how to analyze dual channel
51. ID ES Dat Interpolation Size Point to Point Logarithmic E Amount Least Squares Straight Line General Comments c data tutor tutor1 ds 4 095 i 2 429 Set Range Figure 3 6 Adjusting the image display To try it now 1 Click the Grey C olor Adjust toolbar button IFAI to open the Grey Color Adjust window figure 3 6 M ove the pointer to the title bar of the Grey Color Adjust window and drag the window so that it does not cover the image M ove the pointer to the top of the range adjust bar figure 3 6 drag the pointer down until the middle of the darkest bands begin to appear green and then release the mouse button The bands appear darker If the color appears purple instead of green click Reset and start again Click Set Range to save the changes the changes affect the display only and then click Close to close the Grey Color Adjust window Go to section 3 7 Fragment Analysis User s Guide p3 13 Partone Basics 3 7 Preparing the image for analysis When the image appears on the screen a large red rectangle and small green rectangle are overlaid on the image The rectangles define the region you want to process and the approximate size of the bands The Image Setup inspector includes a tool for rotating the image plus input areas for entering experiment setup specifications 3 7 1
52. ad the image you are analyzing Fragment Analysis then steps you through the analysis process by displaying inspectors a series of windows for entering additional specifications or for confirming the results figure 1 2 Image Setup File View Tools Analysis Window Help pame pun a _ m on 3 Date AEA T 10pm Taday mepa 2 Size Font to Fort Looarthme 3 as 5 flame Least Sauer Stig Line e ener Comment ny image Rotation _ zl LLLI ep 7 Ea ME Lane Finder l Image Setup File View Tools Analysis Window Help 5 e dataltutor tutorl ds z k Panes Lane Finder oj pa ped c hmi ja lam ial r A w a m a fm E _ lane eo lam a a a Jal fm
53. age to see if Fragment Analysis located the bands Then e If band finding was satisfactory click Done e If Fragment Analysis incorrectly identified artifacts as bands you can delete extraneous bands by selecting the bands and pressing the DELETE key You can also increase the N oise Factor and or Scale Factor values to exclude more data and click Find Bands again e If Fragment Analysis overlooked some bands you can mark them yourself using the editing tools section 4 2 You can also decrease the N oise Factor and or Scale Factor values to include more data and click Find Bands again For more information on the N oise Factor and Scale Factor parameters see section 9 5 2 p3 22 Fragment Analysis User s Guide Chapter 3 Fragment Analysis step by step FragmeNT Analysis File View Tools Analysis Window corr EE Setup No of Bands in Std File ha Scale Factor 0 1 Noise Factor 0 100 1 900 Scope EMIRI E T TT e data tutor samplel frm Figure 3 11 Finding bands To try it now 1 In the Band Finder inspector click Find Bands Found bands are marked with bars indicating the areas of maximum intensity and rectangles indicating the band region 2 Click Done The Band Statistics inspector appears figure 3 12 3 Go to section 3 12 Frag
54. alysis Select this option if you want to createa new Experiment Form This option is available only if no image is loaded or when the Image Setup inspector is displayed e Open Displays the Open Form File window from which you select an existing form Select this option to load an Experiment Form modify a form or create a new form based on an existing form To open an existing Experiment Form locate the form name on the File N ame list and double click the name or click once and then click OK This option is available only if no image is loaded or when the Image Setup inspector is displayed e Show Displays the Experiment Form that is currently in effect This option is available only at the beginning of the analysis session Image Setup at the end Band Statistics and whenever a form is loaded by itself no image If you did not load a form at the beginning of the analysis the Show command displays a form containing the entries you made during the session You can then save the form e Close Unloads the Experiment Form that is currently in effect After selecting this option you can run an analysis without using a form This option is available only at the beginning of the analysis session Image Setup at the end Band Statistics and whenever a form is loaded by itself no image 6 1 1 About the Experiment Form Use the Experiment Form figure 6 1 to set the parameters for running the analysis and to create a rec
55. and Press ENTER or click Add The value appears in the list box Repeat steps 4 and 5 for each remaining band value Click Save As A window for naming the file appears In the File N ame box typea name for the file and then click OK Fragment Analysis adds the extension either siz or mas 9 4 3 Loading an existing standards file To load an existing standards file 1 2 3 Click either the Size Std or pK Std or Amount Std button Click O pen The O pen Size A mount File window appears In the list of files double click the name of the file you want to use or click the name and then click OK The file name appears in the Standards File box and the values appear in the list box Fragment Analysis User s Guide p9 9 Parttwo Reference 9 4 4 Loading two sets of standards If you are using both size pK and amount standards either in the same or separate lanes first select one of the standards types such as Size Std and then load the file as explained in section 9 4 3 After you load one file select the second standards type and then load the second file To switch the band value display from one file type to another click the standards button in the Type area 9 4 5 Modifying the Standards Values You use the Add Change and Delete buttons to change the values in the list box Adding a value To add a value in the Value box type the number to add and then click Add or press ENTER The value is
56. and 5 3 show examples of two channel experiments Fragment Analysis User s Guide p5 3 Partone Basics standards first set of unknowns E S U U S U U S Uy Weide U Ue De U OEOI OOD D QD DDOD OOD OD 2 OO DODD D O22 Co Qo GD OO DED D DODODO D CDMODO OD ED ED Ca ZD D Gel Channel 1 image Channel 2 image second set of unknowns Legend S Standards U Unknowns Dyel C2 Dye2 amp Comigrating bands Figure 5 3 Placing two samples per lane labeled with different dyes to increase sample throughput Lanes 1 4 and 7 contain standards and unknowns lanes 2 3 5 and 6 contain only unknowns DOO Q2 D D OOO DD DOD D 2200222 D DD OD DOD QD QOD ZD CD D DPD ED ED GD GD ED ED ED CD ED ED GD GD ED OD Gel Channel 1 image Channel 2 image standards unknowns Legend S Standards U Unknowns Dyel Z Dye2 Figure 5 4 accuracy of analysis p5 4 Fragment Analysis User s Guide Placing standards and unknowns labeled with different dyes in the same lanes to ensure Chapter 5 Dual channel image analysis 5 4 Setting up for dual channel image analysis To prepare for dual channel image analysis access the Experiment Form and complete the form twice once for each channel N ote You can practice performing a two channel analysis using the tutor2 ds image file available in the tut
57. and is located above or below the highest or lowest standards band and that the calculations are based on extrapolation The Extr designation refers to only the size calculation 9 6 2 Naming bands To name a band click the band you want to name type a name in the Band Name box and click Set Fragment Analysis User s Guide p9 15 Parttwo Reference 9 6 3 Saving the analysis parameters Fragment Analysis keeps track of the entries you make during an analysis session You can save the entries to create a new Experiment Form if you had not previously created or loaded one or to update an existing form To save the parameters or view the current Experiment Form click the Show Form button The Experiment Form appears It contains the parameters you entered during the analysis session In the Experiment Form click Save to save the parameters to the current file name or click Save As to assign aname to a newly created form For more information see sections 6 1 10 and 6 1 11 9 6 4 Saving the experiment analysis To save the experiment analysis the objects such as band markers and the calculated results click the Save Analysis button The Save Analysis File window appears In the File N ame box type a name for the analysis and then click OK Fragment Analysis adds the exp extension To save a dual channel image experiment analysis use the Save Analysis command after completing the analysis of the channel 2 image To
58. ar at the bottom of the window displays messages about the function of current selections as well as the name of the Experiment Form you are using Fragment Analysis User s Guide p2 1 Partone Basics r Mb P Fragment Analysis FragmeNT nalysis File Window Ready Figure 2 1 Starting the Fragment Analysis software p2 2 Fragment Analysis User s Guide Chapter 2 Getting started 2 2 Understanding the toolbar After you load an image a toolbar appears at the edge of the Fragment Analysis window figure 2 2 The toolbar provides a handy way to access various functions The last three buttons on the toolbar do not appear until you reach the band finding step To usea tool click the button If you place the pointer on an image the pointer changes to a different shape depending on the tool you select For detailed information about the toolbar options see chapters 7 and 8 Select Return to select mode Pan Scroll image by dragging Pixel Locator Display pixel location and value Pixel Distance Determine the distance between two pixels Zoom Area Create Frame Enlarge the framed area Zoom Area Previous Reduce and view previous frames Map Show or change the portion of the total image currently displayed Magnifier Magnify selected area Channel 1 Display channel 1 image Channel 2 Display channel 2 image Side by Side Two Color Display images sid
59. ared directory on another workstation section 6 3 5 You can use the File N ame box to type the name of the file you want to display If the file you want is stored in a different directory than the one displayed you must include the full path For example c data dna ds Fragment Analysis User s Guide p6 13 Parttwo Reference 6 3 2 Selecting a file type The List Files of Type selection box displays the type of files currently listed in the File N ame box The default setting is for ds and gel file types To change file types click the List Files of Type box to display a menu of file types and then click one of the options A new list of files appears in the file list box 6 3 3 Changing the directory The Directories area displays the current directory path The general directory structure is as follows Double click the root directory c to display a list of directories After you locate the correct directory in most cases the data directory double click it Its contents appear in the files list box N ote The dir folders listed under the data directory identify the special directories that hold the image datasets and associated files Do not open a dir folder to access your dataset Your dataset is available to you by opening the data directory and selecting the correct ds file If you should accidentally delete the ds file you can obtain a copy of it in the dir folder that has the same name as the dataset
60. at for determining fragment amount quantity B 1 Determining the size or isoelectric point of unknown bands After band finding is complete Fragment Analysis assigns the values listed in the size standards file or pKI file to the standards bands on the image The assignments are based on the order in which the values are listed in the standards file N ext standards bands are mathematically superimposed onto lanes containing the unknown bands If the sample being analyzed contains more than one standards lane Fragment Analysis uses the information from the standards lanes located on both sides of the unknown Because bands in the standards lanes may not migrate exactly the same distances Fragment Analysis must determine where on an unknown lane to superimpose the standards values To do this Fragment Analysis performs a point to point linear interpolation between each band using the band values of the standards lanes that bracket the unknown lane figure B 1 e TA 4 425 gt L o Legend T je Standard band Unknown band ee gt Exported standard ee ee i i S Standards lane U Unknown lane Figure B 1 Fragment Analysis exports standards band values into unknown lanes using point to point linear interpolation to estimate where to locate the bands Fragment Analysis User s Guide pB 1 Part three Appendixes Linear interpolation cor
61. button E of the Image window A message asks if you want to save the experiment analysis Click No you already saved it Both the Image window and inspector close From the File menu click O pen Image The O pen Image window appears Double click tutorl ds The Image window and Image Setup inspector appear In the File menu click Experiment Analysis and then click Open The Open Analysis File window appears From the list of files in the O pen Analysis File window double click IM AGE1 The objects such as isobar lines and band markers appear on the image together with the Band Statistics inspector figure 4 9 Go to section 4 9 Fragment Analysis User s Guide p4 17 Partone Basics 4 9 Closing the Fragment Analysis software When you are finished using the Fragment Analysis software you should close all the open windows and then close the Fragment Analysis software 4 9 1 Closing the Image window To close the Image window double click the control menu button E on the Image window figure 4 9 If you did not previously save the experiment a message appears asking if you want to save the experiment If you select Yes the Save Analysis File window appears Type a name for the analysis file and then click OK 4 9 2 Closing the Report window If the Fragment Analysis Report window is open double click the control menu button E of this window to close it A message appears asking if you want to reopen Excel
62. csv extension depending on whether you select tab or comma as the field separator character delimiter See section 6 4 for details 4 3 2 Specifying the report contents The Report and Export windows allow you to choose among three report formats To select one of the formats click one of the buttons To select the contents for Band Reports band characteristics and Lane Reports lane numbers click the down arrow J next to the box to see a list of choices and then select from the list e Band Report Shows information about one band characteristic for all lanes The choice of characteristics is a subset of those listed in the Band Statistics inspector Lane Report Shows all band characteristics for one lane e Table Creates a table of all band characteristics for all lanes p4 6 Fragment Analysis User s Guide Chapter 4 Post analysis operations step by step Double click the window to open Excel Report Type bBandReport an O Lone Repent P ie Table Database Compatible FragmeNT Analysis Report 1 Band RF Report Report generated on 8 23 1994 1 07pm User Name Administrator Image Filename c data tutor tutor1 ds Experiment Form File c data tutor samplet frm Experiment Date 8 23 1994 1 04pm Prepared On Image File c data tutor tutor1 ds Scanned on 1993 06 14 10 41 17 Figure 4 3 Reporting results To try it now 1 In the
63. dards type and then click Open to display the O pen Size A mount File window Double click the desired standards file or click the file name and then click OK The band values appear in the Editor area of the inspector If you selected the wrong file you can select a new one If a single standard serves as both size and amount standards first load one standards file type and then the other Remember to specify the type before clicking O pen For more information about using the Standards Setup inspector see section 9 4 p3 20 Fragment Analysis User s Guide Chapter 3 Fragment Analysis step by step Loy ag Ana vja File View Tools Analysis Window Help a data 0 orl d vial Inspector M E T 7 8 8 Bee fstondoe seu Type pand Size Std Amount Std i pr E Standard File None Ch mad Editor __ Y 2 A eg GT A uit be C oane E am _ 1300 5 1 ml o LJ j f Order O Ascending Descending Manual m a a 7 aeronomia Ea 1 Hii HMN HMIN ia gt Ready _ c Adata tutor samplel fm X Figure 3 10 Creating a standards file To try it now 1 In the Standards Setup inspector make sure that the Size Std button is selected in the Type area and that Descending i
64. e Fragment Analysis automatically adds the csv or txt extension depending on the field delimiter you select Click OK Your file is saved p6 16 Fragment Analysis User s Guide Chapter 6 File menu 6 5 Generate Report The Generate Report command displays the Report window figure 6 11 Use this window to specify the contents and format of a report that you can transfer to an Excel worksheet Reports you create contain the results of an analysis session Report Type Band Rept aa Ormene Table Database Compatible Figure 6 11 The Report window 6 5 1 Selecting the format and content You can choose among three report formats To select one of the formats click the appropriate button Band Report Shows information about a selected band characteristic such as size amount or distance for all lanes To select a particular characteristic click the down arrow next to the Band Report box A list of characteristics appears Click the characteristic for which you want a report e Lane Report Shows all band characteristics for one lane To select a lane click the down arrow next to the Lane Report box A list of lanes appears Click the lane for which you want a report Table Creates a table of all band characteristics for all lanes After making your selection click on the Report button The Report window closes and the Fragment Analysis Report window appears showing your analysis data
65. e by side in two colors Overlay Display composite dual image Grey Color Adjust Adjust image display Add Band Manual Draw a band centered in the drawn box Add Band Semiauto Draw a band centered at area of max intensity Delete Band Delete a selected band Figure 2 2 The toolbar Fragment Analysis User s Guide p2 3 Partone Basics 2 3 Using inspectors To perform an analysis you use inspectors Inspectors are windows designed to set up and initiate a specific task or to display results figure 2 3 For example you use one inspector to find bands and another to set up standards As you go through the analysis Fragment Analysis presents each inspector in sequential order beginning with the Image Setup inspector After you click Done Fragment Analysis displays the next inspector You can also access inspectors from the Analysis menu which appears after you load an image or from within the currently displayed inspector Inspectors have the following features figure 2 3 e Panes Displays the current inspector name and contains a list of inspectors you have already used You can return to a previous step by selecting the inspector from the list N ote Because each step of Fragment Analysis builds on the preceding step the list of available inspectors depends on the stage of analysis you have reached e Help button Displays information about the inspector e Done button Finalize
66. e ee eee 3 8 3 4 1 How the form affects the analysis 00005 3 8 3 4 2 Unloadingaform 0 0 eee 3 8 3 4 3 Retrieving a form Open versus Show 0 005 3 8 Loading the image e ciacveise eae Pewee bee Bae ea ea 3 10 3 5 1 Selecting an image file 00 0 eee eee 3 10 3 5 2 After loading the file 0 cece eee ee 3 10 Adjusting the image display 0 0 cece eee ee 3 12 3 6 1 Moving and hiding the inspectors 0 000 3 12 3 6 2 Scrolling the image 0 6 eee ee 3 12 3 6 3 Optimizing the image display 00000 3 12 3 6 4 Enlarging and reducing the image 00 3 12 Preparing the image for analysis 000 cece eee eee 3 14 3 7 1 Understanding the rectangles 0 000 eee 3 14 3 7 2 Moving and resizing the rectangles 000 3 14 3 7 3 Rotating the image 0 eee ee 3 14 Finding thE lanes sas se ke seek iiias iae ia a a a ES 3 16 3 8 1 Adjusting the lane marker nnana aaa 3 16 3 8 2 Initiating lane finding aaa 3 16 3 8 3 Editing the lane markers auaa 3 16 Reviewing the lane setup information a s sanaaa eea 3 18 3 9 1 Identifying the standards lanes nsan anaana 3 18 3 9 2 Returning to a previous INSpector aaa aaa 3 18 Creating a standards file nananana 3 20 3 10 1 Creating a new standards file auaa 3 20 3 10 2 Saving the new standards file anaana aaa 3 20 3 10 3 Loading a
67. e toolbar 7 9 Display The Display command displays the Display window figure 7 4 Use this window to hide and show objects such as lane markers band position markers and the graph profile and to change their colors Display Bands Show Hide COLOR EJ Show Lane Names amp Band Position Position EI Show Lane Numbers Band Boundary Boundary L Isobar Grid Grid L Ip Isobar Labels Unknown Unused CO Lane Profile Profile Figure 7 4 The Display window To show or hide an object listed in the window click its check box an X in the box denotes show To select a color for the object click its associated color box to display a menu of colors and then click the color of your choice The buttons on the bottom of the screen work as follows Save Retains the choices and closes the window e Cancel Closes the window without applying the new selections e Apply Applies the changes without closing the window but does not save the changes e Close Closes the window Fragment Analysis User s Guide p7 9 Chapter 8 Tools menu Tools Select vx Pan Pixel Locator Pixel Distance Enlarge Reduce Zoom Area Magnifier Add Band Manual Add Band Semiautomatic Delete Band Del The Tools menu includes commands for careful examination of the image Tools menu selections change the function of the pointer from the select mode to a mode for performing a specific task For examp
68. eas but the bands are still unaffected release the mouse button e To change both levels together Place the pointer in the middle of the range adjust bar hold down the SHIFT key and drag the pointer up and down To save the setting click Set Range T he image redisplays with the new settings and the intensity profile graph in the Grey Color Adjust window changes to reflect the settings To restore the image to its original display limit values click the R eset button You can only reset the display limits if you have not clicked Set Range To expand the display limits after you click Set Range type new values in the Hi and Lo boxes N ote For instructions on changing the display values for dual channel images see section 7 6 6 p7 4 Fragment Analysis User s Guide Chapter 7 View menu c data dotblot ds Range Adjust Bar Figure 7 2 Adjusting the upper range Setting range values beyond those displayed To set the display limit values above or below those displayed in the Hi and or Lo boxes or to set display levels for dual channel images delete the current value in one or both of the boxes type the new value and then click Set Range If you are setting levels for dual channel images click the side of the dual channel Image window you want to change to activate it before changing the limit settings For additional information about working with dual channel images see section 7 6 6 7 6
69. ed in 15 gradations You can increase the number of gradations in one of the images by decreasing the number in the other image The minimum and maximum numbers are 2 and 118 respectively For example instead of viewing both images in 15 gradations you can view one image in 3 and the other in 78 gradations To do this move the pointer into the range adjust bar of the Grey Color Adjust window and click the right mouse button A pop up menu appears displaying the gradation options Click one of the options using the left mouse button For information on the choices of dual channel display modes and how they work see section 7 8 7 7 Map The M ap command displays a small version of the Image window including an outline that shows the portion of the image currently displayed figure 7 3 It also allows you to change the displayed area To reposition the image display place the pointer on the interior outline and drag the outline to a new location The main image display moves to reflect the new outline location To move the M ap window move the pointer into the M ap window and then SHIFT drag the window to a new location To return the M ap window to the upper left corner of the Image window position the pointer in the M ap window and click the right mouse button Select Reset from the pop up menu To close the M ap window click the Map command or the M ap toolbar button Fragment Analysis User s Guide p 7 Parttwo Ref
70. eee nee Ready e data ictertsarnplel im Lane Setup y File View Tool Help a zi l k Panes Lane Finder oj pa e i E 5 ma E lam a yr E w l m fel fo E len as a _ ot a le fm eee nee Ready T data ictertsarnplel im Window Help Ey Inspector BE ae eS anes Standards Setup z peni Amount Sed am bo lene ie fl Order pan Ascending Descending Manual Lew J ooe Leer Ji oone J H FN Ready e Band Finder y 5 FragmeNT Analysis aS File View lools Analysis Window Help 5 e dataltutoritutort ds h Sins aay E a 2 nes Band Finder al lal oP 1o of Bande in Std File aa nal actor 0 1 0 m pe tor 0100 5 4 E _ je kaal Sl oi hn Find Bands al j id cu a Oy ae Figure 1 2 The inspector sequence es Fragment Analysis User s Guide pl 3 Partone Basics 1 3 Creating a report You can select from a variety of report formats and then
71. elect a particular characteristic click the down arrow next to the Band Report box A list of characteristics appears Click the characteristic for which you want a report e Lane Report Shows all band characteristics for one lane To select a lane click the down arrow next to the Lane Report box A list of lanes appears Click the lane for which you want a report e Table Creates a table of all band characteristics for all lanes Fragment Analysis User s Guide p6 15 Parttwo Reference a 6 4 2 Selecting the field delimiter The Field Delimiter selection buttons allow you to choose either tabs or commas as the separator characters between fields in the report e Comma Inserts a comma between field entries in the report and creates a Comma Separated Values csv file type e Tab Inserts a tab between field entries in the report and creates a Tab Separated Values txt file type N ote Use Tab for export to Excel 6 4 3 Naming the report After you click the Save As button in the Export window the Save Export File window appears figure 6 10 with either txt or csv displayed in the File Name box depending on the delimiter you selected Save Export File File Name Directories A ee _Cancet_ data gt tutor C tutor1 dir tutor2 dir a Save File as Type Drives Tab Separated Values c B Figure 6 10 Naming the report In the File N ame box type a name for the report fil
72. en you save certain files asking if you want to replace the existing file with the new file Click Yes OK The topics in this chapter are e Accessing the Experiment Form section 3 1 Setting up the experiment section 3 2 e Entering the lane setup information section 3 3 e Saving the Experiment Form section 3 4 e Loading the image section 3 5 e Adjusting the image display section 3 6 e Preparing the image for analysis section 3 7 e Finding the lanes section 3 8 e Reviewing the lane setup information section 3 9 e Creating a standards file section 3 10 Finding the bands section 3 11 e Reviewing the band statistics section 3 12 Fragment Analysis User s Guide p3 1 Partone Basics 3 1 Accessing the Experiment Form You begin an analysis session by entering the information such as number of lanes and locations of size standards that Fragment Analysis uses to calculate data for the unknowns You enter this information into an Experiment Form 3 1 1 Creating a new form To access a new form open the File menu and select Experiment Form and then select N ew from the submenu figure 3 1 The Experiment Form window appears 3 1 2 Three ways to use a form You can use the Experiment Form as e A record of your experiments In this case you can create a new form for each analysis you perform e A template or method for running experiments that use the same parameters In this case select
73. erence FragmeNT Analysis c data tutor tutorl gel File View Tools Analysis Window piitel Figure 7 3 The Map window 7 8 Dual Channel The Dual Channel command opens a menu of display controls for dual channel images The Dual Channel menu provides the following commands Overlay Displays a composite image of two separate images Each filter is represented by a different color Areas where both filters produced a signal are shown in a third color For example when red and green overlap yellow is produced In this way you can differentiate between isolated sample signals and merged sample signals such as comigrating electrophoresis bands Side by Side Two Color Displays each channel Separated image ml in a separate side of the Image window The color representing each channel is displayed in its respective side Bl Channel 2 Displays in grey scale the image created by channel 2 ol Channel 1 Displays in grey scale the image created by channel 1 p7 8 Fragment Analysis User s Guide Chapter 7 View menu Side by Side Grey C olor Displays each channel separated image in a separate side of the Image window using the same color scheme for both images This command is not available from the toolbar Synchronize Links two images together so that you can scroll magnify enlarge and reduce both images simultaneously This command is not available from th
74. es you can click the lanes in the Image window as well as in the Lane List To select multiple lanes in the Image window hold down the SH IFT key while you click the lanes 3 9 2 Returning to a previous inspector To return to a previous inspector such as the Lane Finder click anywhere in the Panes box to reveal the inspector options and then click an option After returning to a previous inspector you must proceed through the normal sequence of inspectors by clicking Done in each one N ote Because each step of Fragment Analysis builds on the preceding step the list of available inspectors depends on the stage of analysis you have reached p3 18 Fragment Analysis User s Guide Chapter 3 Fragment Analysis step by step FragmeNT Analysis File View Tools Analysis Window cAdatattutoritutort ds 1 2 3 4 5 6 7 8 9 Lane Setup Image Setup EAE Unknown Size Std Unknown Unknown Unknown Size Std O O N N N MW I Figure 3 9 Identifying lanes To try it now 1 In the Lane Setup inspector examine the lane list Lanes 1 5 and 9 should be identified as size standards and HM N should appear on the image Click the Panes box A list of inspectors appears figure 3 9 Click Lane Finder The Lane Finder inspector appears Click Done The Lane Setup inspector appears Click Done The Standards Setup inspecto
75. f enlarged image frames that can be redisplayed in reverse order Create Frame M agnifies a selected image area To magnify a selected area click C reate Frame from the menu or toolbar Place the pointer on the image and drag the pointer diagonally to create a frame After you release the button the area you selected expands to fill the window Repeat the process to create a series of frames The series is saved until you redisplay the frames using the Previous Frame option or select the R eset option The series is not saved when you close the Image window Previous Frame Displays the previous frame If you created a series of frames using the Create Frame tool Previous Frame allows you to view the series stack To view the stack click Previous Frame from the menu or toolbar M ove the pointer into the image area and click to view the previously created frame Continue clicking to cycle through the remaining frames in the stack Reset R esets the image to its original view deleting any previously created frames This command is not available on the toolbar Click Select from the menu or toolbar N to end the Zoom Area modes Fragment Analysis User s Guide p8 3 Parttwo Reference 8 8 Magnifier The M agnifier command creates a magnifying glass for examining selected areas of the image To use the M agnifier select the M agnifier command from the menu or from the toolbar The pointer changes to a cross Hold down
76. forms the entire analysis automatically ending with creating a report file If you select the Export box additional options appear The options allow you to select the report format and contents the same as those discussed on section 4 3 To make your selections click the boxes to display menus beginning with Report Type e Report Type Choose among Table Report Band Report and Lane Report e Report O ptions T he options you see depend on whether you select Band Report or Lane Report N o options appear for the Table report type because all information for all lanes and bands is reported e De limiter Select either Tab Separated or Comma Separated to insert either a tab or comma between the data fields Fragment Analysis automatically names the report the same as your form file name but adds either a txt or csv extension depending on the delimiter option you choose The file is then available for export to a word processing or database application p4 14 Fragment Analysis User s Guide Chapter 4 Post analysis operations step by step Automation Options Automate the following steps q Image Setup Lane Finder J Lane Setup ig Standards Setup Figure 4 7 Selecting steps to automate To try it now 1 From the Experiment Form click the Automation button The Automation Options window appears 2 Click all the boxes in the Automation O ptions window except Export figure 4 7
77. he channel 1 left side of the Image window active perform a complete analysis as described in chapter 3 N ote To activate an image click in the image area not on the title bar The frame around a portion of the window darkens 2 Attheconclusion of the analysis after you have reached the Band Statistics inspector double click the channel 2 image Fragment Analysis returns to the Lane Setup step so that you can continue the analysis of the channel 2 data 3 Complete the analysis for channel 2 N otes If you did not specify a separate standards file for channel 2 Fragment Analysis uses the standards specified for channel 1 If after completing an analysis you save to the Experiment Form any new region of interest prototype band size noise factor or scale factor parameters that you entered during the analysis session only the parameters for channel 1 are saved not for channel 2 5 6 Dual channel reporting The procedure for creating a dual channel report is the same as that for creating a single channel report section 4 3 You create the report after analyzing each image First perform the analysis for the channel 1 side of the Image window and then create your report for that image N ext perform the analysis for the channel 2 side of the Image window and then create your report for that image The channel 2 report is appended to the channel 1 report Fragment Analysis User s Guide p5 7 Partone Basics 5
78. he original image and quantitation results are not altered Fragment Analysis User s Guide p 3 Parttwo Reference 7 6 2 Changing the display levels Because the range of intensity levels in the image may not extend over the complete range of possible values you set limits to the upper and lower display levels to eliminate unused values In this way the 256 possible grey levels are spread over a narrower range of intensity values The Grey Color Adjust window provides an interactive mechanism the range adjust bar for setting display limits As you use the bar to change the display limits color is added to the bar and the image display Green shows the pixels that will be assigned the intensity value shown in the H i box purple shows the pixels that will be assigned the intensity value shown in the Lo box background Using the range adjust bar single channel images You can use the range adjust bar to change the image display limits of a single channel image e To change the high display limit Place the pointer at the top of the range adjust bar and drag the pointer down figure 7 2 When the bands begin to turn green indicating high intensity areas move the pointer up slightly until the bands are again black and then release the mouse button To change the low display level Place the pointer at the bottom of the range adjust bar and drag the pointer up When the background is purple indicating low intensity ar
79. iciency and accuracy The topics in this chapter are e What is a dual channel image section 5 1 e How to create a dual channel image section 5 2 e Examples of two channel experiments section 5 3 e Setting up for dual channel image analysis section 5 4 Running a dual channel image analysis section 5 5 e Dual channel reporting section 5 6 e Automating dual channel image analysis section 5 7 e Saving a dual channel image experiment analysis section 5 8 e Loading a saved dual channel image experiment analysis section 5 9 5 1 Whatis a dual channel image A dual channel image results from the collection of two separate sets of data from one sample When you load a dual channel image the two sets of data are displayed in a side by side view figure 5 1 Fragment Analysis User s Guide p5 1 Partone Basics c Nl FragmeNT Analysis View Tools Analysis Window p c data 2clrfrag ds w fol tre ia fa Figure 5 1 A side by side dual channel image Fragment Analysis uses the side by side view for analysis H owever you can display the two channel image separately as an overlay or side by side in one Image window Buttons on the toolbar allow you to change the views Channel 1 Only the channel 1 data al Channel 2 Only the channel 2 data E Overlay A composite of the channel 1 and channel 2 data 1 2 Side by Side Channel 1 and channel 2 data in separate sides of the E Image wi
80. ick OK A second window appears in which you specify the pages to print and the number of copies To change from portrait to landscape orientation or to change the size of the paper click Setup The Print Setup window appears The choices in this window are printer dependent but at a minimum allow you to determine the page orientation See your printer documentation for more details l Center Print Options q Vertically Horizontally Printer System Printer TP_LPRINTER Print Range O Selection F Scaling A Scale to Fit Page Magnification H Pages O Collate Copies Figure 6 13 The Print Options and Print windows 6 9 Print Form The Print Form command allows you to print the Experiment Form To print the Experiment Form select the Print Form command The current form is printed 6 10 Print Setup The Print Setup command displays the Print Setup window You can also access this window by clicking the Setup button in the Print window Use this window to set preferences for print orientation and two sided printing The exact contents of this window are printer dependent See your printer documentation for more detail p6 20 Fragment Analysis User s Guide Chapter 6 File menu The Exit command closes Fragment Analysis You will be prompted to save any open files Fragment Analysis User s Guide p6 21 Chapter 7 View menu
81. ick in the Lane Name box and then type HMN CTRL 4click Lane1 Lane 5 and Lane 9 All three are highlighted Click Set The lane list is updated with the new information figure 3 3 Click OK The Lane Setup window closes Go to section 3 4 Fragment Analysis User s Guide p3 7 Partone Basics 3 4 Saving the Experiment Form To save the Experiment Form click Save The Save Form File window appears figure 3 4 Type a name for the form in the File N ame box and then click OK Fragment Analysis adds a frm file extension Alternatively you can accept the default name form1 frm in which case the next default name will be form2 frm and so on See section 6 1 10 for information on file name restrictions 3 4 1 How the form affects the analysis Fragment Analysis uses the information you entered into the form as input for the analysis session The current form remains in effect until you create a new form load another form or unload the form 3 4 2 Unloading a form If after loading a form you decide to analyze a sample without using the form you can close the form you loaded To do this from the File menu select Experiment Form and then select Close from the submenu N ote that you can close a form only at the beginning or end of an analysis session at the Image Setup inspector or at the Band Statistics inspector or before an image is loaded 3 4 3 Retrieving a form Open versus Show The O pen and Show commands
82. ick one of the color choices 7 6 5 Inverting the current values To invert the current display values click the Invert check box The intensity profile and the displayed pixel values invert Click the Invert check box again to turn Invert off 7 6 6 Working with dual channel images If you are viewing a dual channel image the Grey Color Adjust window operates as follows Active and nonactive sides of the Image window The display levels reflect the active side of the window To activate one side of the window click the image The title bar of the Grey Color Adjust window displays the number of the active window 1 or 2 The range adjust bar The range adjust bar does not operate in dual channel mode To change display levels type values in the Hi and Lo boxes Brightness and contrast controls The brightness and contrast controls work independently for each window For the channel 1 image use the right mouse button to operate the brightness and contrast controls For the channel 2 image use the left mouse button p7 6 Fragment Analysis User s Guide Chapter 7 View menu Side by Side Two Color mode In the Side by Side Two Color mode e Thecolor palette changes to two color schemes such as red green You select the combination you want from the list in the Palette area e Two lines appear on the intensity profile The line colors correspond to the channel colors Each color in a two color scheme is display
83. identified lanes click Set The Type designations in the Lane list box change to reflect your choice Fragment Analysis User s Guide p9 7 Parttwo Reference 9 3 2 Adding descriptive information You can type a lane name and description in the boxes provided for these items Before entering the information select the lane s to which the information applies Use the selection techniques described in step 2 of the preceding section If you add lane names they are displayed on the image To remove the names open the View menu select Display to open the Display window and then click the Lane Name check box to deselect it remove the X 9 4 Standards Setup The Standards Setup command displays the Standards Setup inspector figure 9 5 Use this inspector to load a standards file modify the file or create a new standards file The standards file provides the information needed to compute the values of the unknowns The inspector changes slightly depending on whether you select an isoelectric focusing experiment or a DN A protein experiment as shown in figure 9 5 Panes Standards Setup Type Size Std Amount Std Standard File None Editor vae OO C i Order Ascending Descending Manual Panes Standards Setup Type pKI Std Amount Std Standard File None Editor a Add Order Ascending Figure 9 5 Standards Setup inspector
84. images Part two Reference Provides information about all the items on the Fragment Analysis menus e Part three Appendixes Includes report examples technical information about how Fragment Analysis calculates band values and a troubleshooting guide Assumptions The instructions in this manual assume you have basic computer skills You should be familiar with a Windows based graphical user interface and know how to use a mouse If you do not have these skills refer to the M icrosoft Windows documentation Fragment Analysis User s Guide xi Preface xii Assistance If you have problems please contact Asia Pacific Te 852 2811 8693 Fax 852 2811 5251 Australasia Tel 61 2 9899 0999 Fax 61 2 9899 7511 Austria Tel 01576 0616 22 Fax 01 576 0616 27 Belgium Tel 0800 73 888 Fax 03 272 1637 Canada Tel 1 800 463 5800 Fax 1 800 567 1008 Central East and Southeast Europe Tel 43 1 982 3826 Fax 43 1 985 8327 Denmark Tel 45 16 2400 Fax 45 16 2424 Finland amp Baltics Te 358 0 9 512 39 40 Fax 358 0 9 512 17 10 France Tel 01 69 35 67 00 Fax 01 69 41 96 77 Germany Tel 0761 4903 291 Fax 0761 4903 405 Italy Tel 02 27322 1 Fax 02 27302 212 Japan Tel 81 3 5331 9336 Fax 81 3 5331 9370 Web site http w ww amershambiosciences com Fragment Analysis User s Guide La
85. ises Experiment ID Experiment Date 7 20 1994 1 32pm Number of Lanes b Channel O E General Comments Experiment Type DNA E Standards Filenames Cen Jd seveas Save As gt Automation Options Interpolation Automate the following steps Size Point to Point Logarithmic Image Setup Amount east Squares Straight Line Lane Finder J Lane Setup Standards Setup amp O Export Figure 1 4 Selecting automation options Fragment Analysis User s Guide pl 5 Chapter 2 Getting started This chapter provides the information you need to start using the Fragment Analysis software The topics in this chapter are e Starting the Fragment Analysis software section 2 1 e Understanding the toolbar section 2 2 Using inspectors section 2 3 Quitting the Fragment Analysis software section 2 4 2 1 Starting the Fragment Analysis software After you log on to the Windows desktop you double click the Fragment Analysis shortcut icon on the desktop If you do not see the icon you can start Fragment Analysis using the Start menu The Fragment Analysis main window appears figure 2 1 W hen Fragment Analysis first appears three menu items are displayed File Window and H elp figure 2 1 As you begin working on various tasks the menu items change according to the task you are performing The status b
86. known molecular weights are calculated based on migration distance Distance Volume For additional information about how Fragment Analysis calculates unknown band values see appendix B 9 1 5 Rotating the image To rotate an image that is not squarely aligned with the Image window click the image rotation button E A red line with black handles at both ends appears down the center of the image You move the line to indicate how the bands should be vertically aligned Drag the line itself to move the line Select the handles to rotate the line figure 9 2 Align the rotation line with a column of bands that form a lane For best results choose a column near the center of the image Click the Rotate button The image shifts to the degree indicated by the line p9 4 Fragment Analysis User s Guide Chapter 9 Analysis menu Figure 9 2 Moving the rotation line to the right shifts the image to the left 9 2 Lane Finder The Lane Finder command displays the Lane Finder inspector figure 9 3 together with a lane marker a new rectangle on the image that serves as a pattern for finding lanes Use the Lane Finder inspector to initiate automatic lane finding and to adjust the markers that identify lanes Inspector Panes Lane Finder Setup Number of Lanes Found s f Lane Editor Figure 9 3 The Lane Finder inspector Fragment Analysis User s Guide p9 5 Parttwo
87. le in the M agnifier mode the pointer becomes a magnifying glass The pointer remains in that mode until you select another function or the Select option N ote Tools options are also available from a pop up menu To access the pop up menu place the pointer in the Image window and click the right mouse button and then use the left mouse button to select a command In this chapter if a Tools option is available from the toolbar the appropriate toolbar icon is also shown The topics in this chapter are e Select section 8 1 Pan section 8 2 Pixel Locator section 8 3 e Pixel Distance section 8 4 e Enlarge section 8 5 e Reduce section 8 6 Zoom Area section 8 7 e Magnifier section 8 8 Add Band M anual section 8 9 Add Band Semiautomatic section 8 10 Delete Band section 8 11 Fragment Analysis User s Guide p8 1 Parttwo Reference 8 1 Select The Select command returns the pointer from an editing or special viewing mode to the select mode In Select mode you can select one or more objects on the image such as rectangles To select one object click the object To select a group of objects place the pointer in the image area next to one of the objects and drag the pointer diagonally to surround the group with a rectangle 8 2 Pan The Pan command enables scrolling in any direction To use the Pan tool select the Pan command from the menu or from the toolbar Place the pointer
88. lect from the Image window H owever parameters applied to individual lanes are not saved to the Experiment Form The Noise Factor parameter The default N oise Factor value of 1 0 represents the threshold setting established by Fragment Analysis The threshold indicates the point at which Fragment Analysis separated random variations noise on the image from relevant data N oise factors are computed for every lane in the image To instruct Fragment Analysis to find fewer bands exclude more data increase the N oise Factor value Increasing the value moves the threshold up to allow more data to be considered as noise and less as bands To instruct Fragment Analysis to find more bands include more data decrease the N oise Factor value Decreasing the value moves the threshold down to allow more data to be considered relevant and less as noise The Scale Factor parameter Fragment Analysis uses the default Scale Factor value of 0 25 to remove bands from analysis whose left and right sides are significantly different in intensity The 0 25 value means that the intensity value of the left half of the band must be at least 25 of the intensity value of the right half of the band for the band to be included The Scale Factor value is a measure of band uniformity Decreasing the scale factor includes more nonuniform bands in the analysis p9 12 Fragment Analysis User s Guide Chapter 9 Analysis menu 9 5 3 Limiting band finding to se
89. lected lanes The buttons in the Scope area allow you to limit band finding to a specific group of lanes the default selection is All Lanes To limit the band finding scope select one of the buttons in the Scope area such as Unknown and then click Find Bands You can also limit band finding by selecting individual lanes on the Image window as follows e To select one lane click the lane e To select multiple lanes one at a time SH IFT click each lane To select a group of lanes place the pointer outside the upper left corner of the first lane you want to select and drag the pointer down to the lower right corner of the last lane you want to select A rectangle encloses the lanes until you release the button H andles on the corners of the lanes indicate they are selected e To deselect individual lanes that you selected as a group see above SH IFT click each lane you want to deselect 9 5 4 After you click done standards bands verification After you click Done Fragment Analysis compares the number of bands it found in the standards lanes one lane at a time with the number of band values listed in the standards file If the number of bands in the lane does not match the number of bands in the standards file a message appears asking if you want to edit the bands If your response is Yes the Band Finder inspector remains displayed You can use the editing tools to add or delete band markers If your respon
90. lysis 4 14 6 8 Automation Options window 6 8 B Band Finder inspector 3 22 9 11 band name in band statistics 3 24 9 15 band number in band statistics 3 24 9 14 band report 4 6 6 10 6 15 6 17 Band Statistics inspector 3 24 4 2 9 14 bands adding 4 4 8 4 defining size 3 14 deleting 4 4 8 5 drawing 8 4 editing 4 4 finding 3 22 9 11 marking 4 4 naming 4 4 9 15 parameters for finding 9 12 selecting scope 9 13 C Cascade command 10 2 Channel 1 command 7 8 Channel 2 command 7 8 Channel in Experiment Form 6 4 Close All command 10 3 Close and Unload command 3 2 3 8 6 2 Fragment Analysis User s Guide Index 1 Index closing Fragment Analysis 6 21 colors adding to image 7 6 changing 7 9 saving 7 9 comma separated delimiter 6 11 6 16 Create Frame command 8 3 cubic spline interpolation 6 6 9 4 curved lanes C 1 D Date in Experiment Form 3 4 6 3 delete bands 4 4 8 5 files 6 18 lanes 3 16 9 6 Delete Band command 4 4 8 5 Delete command 6 18 delimiters in Export function 6 11 6 16 Description in Lane Setup window 3 6 directories 6 14 Display command 4 2 7 9 distance in band statistics 3 24 9 15 Draw Band Manual command 4 4 Draw Band S 4 1 Draw Band Semiautomatic command 4 4 drives 6 14 dual channel analysis 5 3 and Grey Color Adjust 7 6 commands 5 1 image 5 1 reporting 5 7 Dual Channel command 7 8 dual images and Grey Color Adjust 7 6 changing color gradations 7 7
91. m inspectors 2 4 hide band position markers 7 9 inspector 3 12 isobar line 7 9 lane markers 7 9 profiles 7 9 Index image display actual size 7 2 adding colors 7 6 adjusting brightness 7 5 adjusting contrast 7 5 adjusting grey scale range 3 12 7 3 enlarging 3 12 8 3 full size 7 2 inverting 7 6 magnifying 7 2 8 4 reducing 3 12 8 3 rotating 3 14 9 4 scrolling 3 12 8 2 zoom in 7 2 zoom out 7 2 zooming an area 8 3 image file loading 3 10 printing 4 8 Image Setup inspector 3 14 9 2 inspectors Band Finder 3 22 9 11 Band Statistics 3 24 4 2 9 14 hiding 3 12 Image Setup 3 14 9 2 Lane Finder 3 16 9 5 Lane Setup 3 18 9 6 moving 3 12 returning to previous 3 18 showing 3 12 Standards Setup 3 20 9 8 interpolation choices 6 5 9 3 invert image 7 6 isobar lines 4 2 L Lane Finder inspector 3 16 9 5 Lane Name in band statistics 3 24 9 14 Lane Name in Lane Setup window 3 6 lane number in band statistics 3 24 9 14 Fragment Analysis User s Guide Index 3 Index lane report 4 6 6 10 6 15 6 17 Lane Setup inspector 3 18 9 6 Lane Setup window 3 6 6 6 lane type in band statistics 3 24 9 14 lanes 3 16 adding descriptions 9 8 adjusting 3 16 curved C 1 deleting 3 16 9 6 displaying names 4 2 duplicating 3 16 9 6 finding 3 16 identifying 9 7 identifying standards 3 18 moving 9 6 naming 9 8 resizing 9 6 selecting 9 7 selecting in lane list 3 6 3 18 standards 3 18 least squares straight line in
92. me A text box in which you can type a name for a selected band section 9 6 2 e Size or pKI T he calculated size or the isoelectric focusing point in the units you specified e Rf The distance traveled by the band expressed as a percentage of the total migration range measured from the top to the bottom boundary of the lane e Distance The distance traveled in millimeters from the top of the lane to the band position region of greatest intensity e Amount The calculated amount quantity of the band in the units you specified e Volume The sum of all the pixel values in the band minus the background Source The method used to locate the band Auto Fragment Analysis found the band Semiauto Fragment Analysis found the band within an area you specified using the point of maximal intensity within the area Manual You specified the band location and dimension by drawing a rectangle Fragment Analysis inserted a marker in the center of the rectangle e Percent Percent of band volume relative to all bands in the lane N otes The designation Unassigned means either that no standards file exists for a particular type of calculation size or amount that no standard value is associated with the band amount type only or that the current standards file is inappropriate for the experiment The designation Extr following a source designation such as Auto Extr means that the b
93. ment Analysis User s Guide p3 23 Partone Basics 3 12 Reviewing the band statistics The Band Statistics inspector figure 3 12 displays the following information about any band you select To select a band click the band marker H andles appear on the marker to indicate the band is selected Lane Number T he number automatically assigned to the lane Lane Name T he name you assigned to the lane Lane Type T he lane designation such as Size or Unknown Band N umber T he band s position in the lane counting from the top Band N ame A box in which you can type a band name click Set Size pKI T he calculated size or isoelectric point in the units you specified Rf T he distance traveled expressed as a percentage of the migration distance measured from the top of the lane to the selected band Distance T he distance traveled in millimeters from the top of the lane to the band position region of greatest intensity Amount T he calculated amount in the units you specified Volume The sum of all the pixel intensity values in the band minus the background background is determined automatically Source T he method used to locate the band Auto Fragment Analysis found the band Semiauto Fragment Analysis found the band after you indicated its general location Manual Y ou indicated the location of the band Percent Percent of band volume relative to all bands in the lane The Ba
94. ment analysis To retrieve the saved experiment analysis first load the image from which the analysis was saved section 3 5 N ext open the Fragment Analysis File menu and select Experiment Analysis and then select O pen from the submenu The O pen Analysis File window appears In the list of file names locate the analysis file and then double click it or click the file name and then click OK The saved analysis appears The objects are displayed on the image and the Image Setup inspector is replaced by the Band Statistics inspector You can now reexamine the experiment results and generate additional reports using the procedure described in section 4 3 If you click Show Form a form named formn frm is displayed wheren isa unique number assigned by the computer It shows the experiment parameters associated with the saved analysis If you save this form it becomes the new active form p4 16 Fragment Analysis User s Guide Chapter 4 Post analysis operations step by step Save Analysis File File Name Directories fe data gt tutor tutor1 dir 5 tutor2 dir Save File as Type Experiment Files exp F igure 4 8 Saving the analysis To try it now 1 In the Band Statistics inspector click the Save Analysis button The Save Analysis File window appears figure 4 8 In the File N ame box type image1 and then click OK The window closes Double click the control menu
95. n existing standards file 0 3 20 Finding the bands 00 cece cee eee 3 22 3 11 1 Initiating the band finding 00 0000 3 22 3 11 2 Evaluating the band finding 0 0 000 3 22 Reviewing the band statistics 0 cee ees 3 24 iv Fragment Analysis User s Guide Table of contents Chapter 4 Post analysis operations step by step 4 1 Inspecting the bands 0 cece 4 2 4 1 1 Checking the isobar lines 0 0 0 0 c eee eee 4 2 4 1 2 Magnifying portions of the display 0005 4 2 4 1 3 Changing the line and label attributes 4 2 4 2 Editing the bands 2 3 200 0614 vee wees a eee es bane aes 4 4 4 2 1 Using the band editing buttons 0 0005 4 4 4 2 2 Recalculating the data eee eee 4 4 42 3 Naming bands s 2 3 ee ved eee Wi ee ae 4 4 4 3 Creating a report 0 tee 4 6 4 3 1 Report versus Export aaa 4 6 4 3 2 Specifying the report contents saasaa aaa 4 6 4 4 Printing the results uaaa 4 8 4 5 Exiting from Excel auauua 4 10 4 5 1 Saving yourreport 0 0 eee 4 10 4 5 2 Closing the worksheet 0 0 0 0 c eee eee 4 10 4 5 3 Exiting Excel resi ences aoe wasted a awe 4 10 4 6 Saving your specifications 00 0 eee 4 12 4 6 1 Specifications that are saved 1 es 4 12 4 6 2 The save procedure cece 4 12 4 6 3 Creating a new form based on the current form
96. n from the Experiment Form submenu N ote Do not use the following characters lt gt and space You can use the underline character Fragment Analysis User s Guide p6 11 Parttwo Reference 6 1 11 Saving the form to a new name The Save As button allows you to change an existing form file and save it to anew name After modifying an existing form click Save As The Save Form File window appears In the File Name box type a name for the form and then click OK The original form is retained with its original file name The modified version of the form is given the new name Fragment Analysis adds the FR M extension 6 1 12 Closing the window Select Close to close the Experiment Form window If you have changed the form Fragment Analysis will ask if you want to save the changes before closing the window 6 2 Experiment Analysis The Experiment Analysis command allows you to save and retrieve the analysis including the objects band markers lane markers and so forth and the calculated data Clicking Experiment Analysis displays a submenu with two options e Open Displays the Open Analysis File window To retrieve an analysis file locate the file in the list of files and then double click the file name or click the filename and then click OK The objects appear in the Image window in the same state as when you saved the experiment The Band Statistics inspector also appears e Save Displa
97. nce ax MW or distance ax pl where a is a constant determined by the slope of the line between two points on the standard curve MW is the molecular weight and pl is the isoelectric point Distance traveled is proportional to the molecular weight or isoelectric point Fragment Analysis User s Guide p9 3 Parttwo Reference SSS Point to point logarithmic Point to point logarithmic uses the following formula to calculate the sizes of log the bands based on their migration distances ae distance a x log MW Distance or distance a x log pl where a is a constant determined by the slope of the line between two points on the standard curve MW is the molecular weight and pl is the isoelectric point Distance traveled is proportional to the log of the molecular weight Cubic spline logarithmic Cubic spline logarithmic uses regression analysis to plot predicted values log we of the unknowns with the observed values of the standards The regression MW line creates a best fit sigmoidal curve through all the data points and then calculates the unknown molecular weights based on their migration distances and the log of the standard molecular weights Distance Least squares straight line Least squares straight line uses regression analysis to plot a best fit straight A line through all the data points and the origin Unknown amounts are MW x calculated based on total band volumes and un
98. nd Statistics inspector is the end of the analysis Go to chapter 4 for more information about the Band Statistics inspector as well as for instructions on creating a report saving your specifications saving the analysis automating the analysis and more See section 9 6 1 for additional details about band statistics p3 24 Fragment Analysis User s Guide Chapter 3 Fragment Analysis step by step FragmeNT Analysis File View Tools Analysis Window Inspector Panes Band Statistics Band Characteristics Lane Number Lane Name Lane Type Band Number Band Name Size Rf Distance Amount Volume Source Percent Unknown Help Recalculate Export Show Form Save Analysis i Sloleia lalclaicietsi c data tutor samplel fm Figure 3 12 Obtaining band statistics To try it now 1 Place the pointer on a band marker and click the mouse button H andles appear on the marker if they do not click again Statistics for that band appear in the inspector figure 3 12 2 Repeat step 1 for the other bands Compare the values on the standards labels with the values displayed in the Size box of the inspector 3 Go to chapter 4 to continue the tutorial In chapter 4
99. ndow i p5 2 Fragment Analysis User s Guide Chapter 5 Dual channel image analysis 5 2 How to create a dual channel image You create a dual channel image by labeling two sets of sample material such as two sets of unknowns with two different fluorescent dyes The emissions signals from the two dyes are of different wavelengths The instrument collects data for the two fluorochromes separately using different filters to collect the emissions of the two fluorochromes figure 5 2 Emission signals Emission Photomultiplier filter A tube detector S 99O O 9S S 9 O SO SG S9 Sample ooog O o O O ZED DDDDDODD gt g Overlay image Side by side image Emission filter B Legend D Dyel C Dye2 Figure 5 2 Diagram showing two channel data collection N ote Before analyzing a dual channel image you must first separate overlapping emission spectra using the FluorSep utility 5 3 Examples of two channel experiments Two channel experiments can be set up to serve a variety of purposes In particular two channel experiments allow you to load two samples in a single lane thereby increasing your efficiency You can also ensure accuracy by running standards and unknowns in the same lane thereby eliminating problems caused by smiling lanes or other gel artifacts Figures 5 3
100. nt Form window appears figure 3 2 4 Go to section 3 2 Fragment Analysis User s Guide p3 3 Partone Basics 3 2 Setting up the experiment To set up the experiment enter information into the form figure 3 2 3 2 1 Entering the experiment setup information Enter information in the Experiment Form as follows Optional Entries The Experiment ID Date and General Comments boxes are for your records only e Defaults The Channel Experiment Type and Interpolation boxes contain default values If the displayed defaults are not appropriate for your experiment choose another selection from the list Number of Lanes Enter the number of lanes that are in your sample e Standards Files The buttons in the Standards Filenames area provide access to windows from which you can select the standards files to use with your experiment If the file you need does not exist you can create the file at a later step 3 2 2 Entering the lane identification information To enter lane identification information first enter the number of lanes and then click Lane Setup The Lane Setup window appears see section 3 3 3 2 3 Automating the analysis After setting the experiment parameters you can automate the analysis by selecting options from the Automation O ptions window which you open by clicking the Automation button For information on using the Automation feature see section 4 7 3 2 4 For more information
101. nt type options appears Click the option of your choice DNA isoelectric focusing protein 9 1 2 Specifying the number of lanes To specify the number of lanes in your sample type the number in the N umber of Lanes box Fragment Analysis uses this information to find lanes 9 1 3 Entering information for your records optional You can use the ID Date and General Comments boxes to add information for your records The information you enter here is not used for the analysis but is included in the report These three text entry boxes duplicate those on the Experiment Form e ID UsetheID box to type identifying information for your experiment e Date You can type a date in the Date box or click the Today button to enter today s date in the box e General Comments Use the General Comments box to type additional notes about your experiment 9 1 4 Selecting an interpolation method You can choose among the various curve fit methods listed in the Size or pK and Amount selection boxes you should use the point to point logarithmic for size standards To see the options click the down arrow next to one of the boxes Click one of the options to change the curve fit method N ote that the choices vary depending on the experiment type you select Point to point linear Point to point linear uses the following formula to calculate the sizes or isoelectric points of the bands based on their migration distances dista
102. o not extend the full lane width You can also try dragging the left and right edges of the region of interest large red rectangle in or out to change the lane finding routine Click Done Fragment Analysis User s Guide pC 1 Part three Appendixes to return to the Lane Finder inspector In the Lane Finder inspector click Find Lanes If necessary adjust the lane markers so that they intersect all bands in the lane C 3 Lane setup The settings you specified in the Lane Setup window of the Experiment Form did not get transferred to the Lane Setup inspector When you enter information in the Lane Setup window of the Experiment Form be sure to click Set before clicking OK To recover enter the lane setup information in the Lane Setup inspector Remember to save the Experiment Form at the end of the analysis session select Show from the Band Statistics inspector and then select Save on the Experiment Form On the Lane Setup window or inspector when you changed the information in either the Type Lane Name or Description boxes the type lane name or description information that had previously appeared on the selected line either disappeared or changed You must be sure to set all three boxes Type Lane N ame and Description to the correct values for the selected lane before clicking Set Clicking a line does not automatically insert the type name and description information into their respective input boxes To recover
103. o try it now 1 On the Band Statistics inspector click the Show Form button The Experiment Form you created appears N ote that the standards file you created appears in the Standards Filenames area In the Experiment Form click the Lane Setup button The Lane Setup window appears figure 4 6 In the Lane Setup click the Use Lane Positions check box to save any adjustments you made to the lane positions and then click OK An X should appear in the box The Lane Setup window closes In the Experiment Form click Save Your new specifications are saved Go to section 4 7 Fragment Analysis User s Guide p4 13 Partone Basics 4 7 Automating the analysis You can automate all or part of the analysis process You do this by accessing the Automation Options window from the Experiment Form and then choosing the steps you want to automate 4 7 1 Accessing the Automation Options window To access the Automation O ptions window display the Experiment Form window and then click the Automation button The Automation Options window appears figure 4 7 After you make your selections in the Automation Options window click OK Then in the Experiment Form click Save to implement your selections 4 7 2 Selecting the steps to automate In the Automation O ptions window click the check boxes to indicate which steps you want to automate You can select any or all the steps If you select all the steps Fragment Analysis per
104. of DNA or protein in each band e pKI standards define the isoelectric point of each band Optionally you can also type Lane Name and Description information N ext in the lane list box click one or more of the lanes that represent standards To select more than one lane at a time hold down the CTRL key while you click the lanes After making your selections click Set The lane list is updated with the new information figure 3 3 Because all bands are initially labeled Unknown adding information about unknown lanes is not required To add identifying information such as Lane Name use the same technique as for identifying standards 3 3 2 Changing the information To change the information about the lanes click the lane to select it change the information in the selection and text boxes near the top of the window and then click Set To add a lane click a lane and then click Insert A new lane is added after the lane you selected To remove a lane click the lane and then click Remove p3 6 Fragment Analysis User s Guide Chapter 3 Fragment Analysis step by step Lane Setup Description l Description CONN ewn lz Size Std Unknown Unknown Unknown Size Std Unknown Unknown Unknown Size Std Figure 3 3 Identifying standards lanes To try it now 1 Oys SUT As SOI a Click the down arrow next to the Type box A list of lane types appears Click Size Std Cl
105. one The Lane Finder inspector appears with a rectangle over the first lane in the Image window Go to section 3 8 Fragment Analysis User s Guide p3 15 Partone Basics ee 3 8 Finding the lanes At the same time that the Lane Finder inspector appears a rectangle appears on the first lane in the image The Lane Finder inspector allows you to initiate automatic lane finding The rectangle serves as the prototype lane marker 3 8 1 Adjusting the lane marker The rectangle surrounding the first lane serves as a pattern for finding the remaining lanes If the rectangle does not correctly delineate the lane move it or resize it using the same techniques discussed in section 3 7 2 N ote that you cannot change the height of the rectangle only its width and position 3 8 2 Initiating lane finding To initiate lane finding click Find Lanes Fragment Analysis encloses the found lanes with rectangles and displays the number of lanes it found figure 3 8 The software finds the number of lanes you specified in the Experiment Form or Image Setup inspector 3 8 3 Editing the lane markers You can move and resize lane markers to fit the lanes You can also add and delete the markers To add a lane marker Click a lane marker and then click the duplicate button A rectangle encloses the lane to the right of the selected lane e To delete a lane marker Click the lane you want to delete and then click the delete button
106. or directory For both the channel 1 and channel 2 images use the same lane setup information as you did with the single channel tutorial You can load the standards file sizestd you created in the single channel tutorial To prepare the Experiment Form 1 Open the File menu and select Experiment Form and then select N ew from the submenu or select O pen if you want to create a new form based on an existing form The Experiment Form appears figure 5 5 Form1 frm Experiment ID Experiment Date Number of Lanes o General Comments JE Channel Lane Setup Automation Experiment Type Standards Filenames O Doo o i Size Point to Point Logarithmic Amount east Squares Straight Line Figure 5 5 Entering parameters for the channel 1 image 2 Complete the form for the channel 1 data make sure that a 1 appears in the Channel selection box N ote If you want to automate the analysis select the automation options as part of the set up See section 5 7 for more information Fragment Analysis User s Guide p5 5 Partone Basics E 3 Click Lane Setup The Lane Setup window appears with the lanes for channel 1 listed in the Lane List box figure 5 6 Lane Setup Type Unknowns Lane Name Channel 1 Description l No Type Name Description Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
107. ord of each experiment By creating a form you can partially or fully automate fragment analysis After you create a form it is automatically loaded and ready to use for the analysis The name of the form appears on the right side of the status bar The form you load remains in effect until you create a new form load another existing form or close the form using the Close command in the Experiment Form submenu You can create a new form for each analysis thereby keeping a separate record of each or you can create just a few forms to use repeatedly for experiments that are similar to one another p6 2 Fragment Analysis User s Guide Chapter 6 File menu You need not prepare a form to perform an analysis If you do not use a form you can enter parameters such as the number of lanes in the sample and the location of the standards lanes into each inspector At the end of an analysis session you can then choose to save the parameters to create an Experiment Form Form1 frm Experiment ID Experiment Date sd Today Number of Lanes fo General Comments Channel JE Lane Setup Automation Experiment Type Standards Filenames i a Size Point to Point Logarithmic Amount east Squares Straight Line Figure 6 1 The Experiment Form set for a DNA type experiment 6 1 2 Entering information for your records Use the following text boxes to create a record of your e
108. ort The topics in this chapter are Setting up the experiment section 1 1 e Performing the analysis section 1 2 e Creating a report section 1 3 e Automating the analysis section 1 4 Fragment Analysis User s Guide pl 1 Partone Basics ee 1 1 Setting up the experiment You begin a session by setting the experiment parameters such as the number of lanes in the sample figure 1 1 Form1 frm Experiment Form Experiment ID P Experiment Date 7 20 1994 1 32pm DE Eee Open Experiment Analysis Show Open Image F7 Export Results Generate Report Close Number of Lanes 3 Channel General Comments Delete Rename Print Image P Setup Print Form F Print Setup Shift P Automation Exit AlttF4 Experiment Type Standards Filenames Save Save As Interpolation Size Point to Point Logarithmic Amount Least Squares Straight Line lz Lane Setup Tape Lone Nane Cramme oomen C OO UOO OOOO OO O No Type Name Description 1 Size Std HMN 2 Unknown 3 Unknown 4 Unknown 5 Size Std HMN 6 Unknown 7 Unknown 93s Size Std HMN Figure 1 1 Accessing the forms for setting up the experiment format pl 2 Fragment Analysis User s Guide Chapter 1 The Fragment Analysis workflow 1 2 Performing the analysis N ext you lo
109. osoft Excel Workbook Figure 4 5 Saving the worksheet To try it now 1 Open the Excel File menu and select Save C opy As The Save As window appears figure 4 5 2 Inthe File N ame box type your name and then click OK Excel adds the xls extension A Summary Information window appears Click OK 3 Doubleclick the contro menu button E of the worksheet window not the Excel main window The worksheet closes 4 Onthe Excel title bar click the minimize button Excel is reduced to an icon below or behind the Fragment Analysis window 5 In the Fragment Analysis window double click the control menu button E of the Fragment Analysis Report window A message asks if you want to activate Excel Click No The Report window closes 6 Go to section 4 6 Fragment Analysis User s Guide p4 11 Partone Basics 4 6 Saving your specifications Before exiting the session you can update the Experiment Form with the entries you added during the analysis session 4 6 1 Specifications that are saved When you save the specifications you entered during the analysis you are updating the Experiment Form with the following e The degree of image rotation e Thesize of the largest band small rectangle e Theregion of interest large rectangle e Thetype of experiment e Theinterpolation methods e Thenumber of lanes in the sample e The spacing between lanes select Use Lane Positions The standards
110. ough the analysis 3 5 1 Selecting an image file To select an image file to load display the File menu and click O pen Image TheO pen Image window appears figure 3 5 In the list of files box locate the file you want to analyze and then double click the file name or click the file name and then click OK If you do not see the file name use the scroll bars to reveal hidden file names Also check the List Files of Type box to make sure that the correct file type is displayed usually M D Files ds gel and check the Drive and Directory areas Typically image files are stored in the data directory in drive c N ote For additional details on using the O pen Image window including information about the directory structure see section 6 3 3 5 2 After loading the file After you load the file the following events occur A progress bar appears on the screen indicating that the file is being loaded e Theimage appears sized to fit the available space on the screen e Two rectangles large and small are overlaid on the image e Thelmage Setup inspector appears figure 3 6 If you created an Experiment Form the settings you selected for N umber of Lanes Experiment Type Interpolation and other information are displayed in the inspector e Thename of the form appears on the right side of the status bar p3 10 Fragment Analysis User s Guide Chapter 3 Fragment Analysis step by step Open Image
111. overlay 7 8 viewing side by side in color 7 8 viewing side by side in grey 7 9 Index 2 Fragment Analysis User s Guide duplicating lane 3 16 E editing bands 4 4 lanes 3 16 Enlarge command 8 3 enlarging image 8 3 Excel closing 4 10 exiting from 4 10 opening 4 6 saving report from 4 10 transferring results to 6 18 Exit command 6 21 Experiment Analysis command 6 12 Experiment Form about 6 2 and dual channel analysis 6 4 command 3 2 editing 3 2 loading 3 2 3 8 opening 3 2 opening new 3 2 retrieving 3 8 saving 3 8 6 11 showing 4 12 submenu 3 2 6 2 updating 4 12 using 3 2 3 4 6 3 Experiment ID in Experiment Form 3 4 6 3 Experiment Type in Experiment Form 6 4 in Image Setup inspector 9 3 Export button in Band Statistics inspector 9 16 in Automation Options 4 14 6 10 report options 6 10 window 6 14 Export Results command 4 6 6 14 9 16 extensions in file names 6 18 extrapolation in band statistics 9 15 F File menu 6 1 file name extensions 6 18 file types 3 10 files renaming 6 19 standards 3 20 finding bands 9 11 lanes 3 16 fluorochrome separation 5 3 Fragment Analysis closing 4 18 6 21 main menu 2 1 quitting 2 5 starting software 2 1 frames creating 8 3 viewing 8 3 Full Size command 3 12 7 2 G General Comments in Experiment Form 3 4 6 3 in Image Setup inspector 9 3 Generate Report command 4 6 9 16 grey levels 7 3 Grey Color Adjust command 3 12 7 3 H Help fro
112. pen the Analysis menu and click the inspector name 3 6 2 Scrolling the image For images that are larger than the Image window several scrolling tools are available the window scroll bars the Pan tool c and the M ap window tool The Pan tool available from the Tools menu or toolbar allows you to move the image in any direction The M ap window available from the View menu and toolbar displays a small replica of the entire image with an outline showing the currently displayed area You move the outline to shift the image display 3 6 3 Optimizing the image display If the image is difficult to see you can adjust the grey scale range or invert the grey tones of the image using the G rey C olor Adjust window figure 3 6 available from the View menu or toolbar fF Section 7 6 explains how to use the Grey Color Adjust window 3 6 4 Enlarging and reducing the image Both the View and Tools menus offer options for changing the image display For example to scale the image to fit the window select Full Size from the View menu To magnify a particular region of the image select M agnifier from the Tools menu or toolbar dap p3 12 Fragment Analysis User s Guide Chapter 3 Fragment Analysis step by step Top of range adjust bar FragmeNT Analysis File View Tools Analysis Window edatattutoritutornds v Inspector Panes Image Setup Experiment Type DNA Number of Lanes 9
113. points of the bands based on their migration distances distance ax MW or distance ax pl wherea isa constant determined by the slope of the line between two points on the standard curve MW is the molecular weight and pl is the isoelectric point Distance traveled is proportional to the molecular weight or isoelectric point Point to point logarithmic Point to point logarithmic uses the following formula to calculate the sizes of the bands based on their migration distances distance a x log MW or distance a x log pl wherea is a constant determined by the slope of the line between two points on the standard curve MW is the molecular weight and pl is the isoelectric point Distance traveled is proportional to the log of the molecular weight Fragment Analysis User s Guide p6 5 Parttwo Reference Distance MW Distance Volume Cubic spline logarithmic Cubic spline logarithmic uses regression analysis to plot predicted values of the unknowns with the observed values of the standards T he regression line creates a best fit sigmoidal curve through all the data points and then calculates the unknown molecular weights based on their migration distances and the log of the standard molecular weights Least squares straight line Least squares straight line uses regression analysis to plot a best fit straight line through all the data points and the origin Unknown amounts are calc
114. r records p6 6 Fragment Analysis User s Guide Chapter 6 File menu Type Unknowns Lane Name Channel 1 Description l No Type Name Description 1 Unknown 2 Unknown 3 Unknown 4 Unknown 5 Unknown 6 T 8 9 1 Unknown Unknown Unknown Unknown 0 Unknown Figure 6 3 The Lane Setup window available from the Experiment Form When the window appears the lanes are listed in the lane list box transferred from your entry in the N umber of Lanes box on the Experiment Form Initially all lanes are typed as Unknowns Specifying the location of standards lanes To change the lane types from unknown to one of the standards selections 1 Click the down arrow button next to the Type box and then select among the choices on the list figure 6 4 Type Unknown sd Lane Name Channel 1 Description Fia Sud No T Size Amt Std Unk nknown Unknown Unused Description Unknown Unknown Unknown Unknown Unknown Unknown 0 Unknown O0NDNAUN Figure 6 4 Selecting the standards type 2 Inthelanelist box click the laneto which you want to assign the standards type designation To select multiple lanes CTRL click each lane 3 Click Set The type designations change from Unknown to the type of standards you selected Fragment Analysis User s Guide p6 7 Parttwo Reference Eas Assigning names and
115. r appears Oh SON o Ww Go to section 3 10 Fragment Analysis User s Guide p3 19 Partone Basics ee 3 10 Creating a standards file The Standards Setup inspector figure 3 10 allows you to create load or modify a standards file The standards file provides the information the software uses to compute the unknowns 3 10 1 Creating a new standards file To create a new standards file first select the type of file you will be creating by clicking one of the buttons in the Type area N ext in the U nit box type the appropriate unit for the value such as bp kb or ng In the Order box select the order in which you want the values listed Finally in the Value box type the first value and then click Add or press ENTER Thesystem adds the value to the list inserting it in an appropriately sorted order Repeat to add more values To remove a value from the list click the value and then click Delete To change a value click the value type a new value in the Value box and click Change To insert a value type a new value in the Value box and then click Add The value is inserted in the appropriate ascending or descending order 3 10 2 Saving the new standards file To save the new file click the Save As button The Save Size A mount Standard window appears In the File Name box type a name for the file and then click OK 3 10 3 Loading an existing standards file To load an existing standards file select the stan
116. rects for smiling bands in which bands of the same size migrate at different rates during electrophoresis If an unknown lane is not bracketed by standards lanes no standard lane on either its left or right size values are exported from only one standards lane its nearest neighbor to the left or right using horizontal extrapolation figure B 2 If an unknown band is located above the uppermost standards band or below the lowest standards band its size is estimated using extrapolation Fragment Analysis indicates this condition by adding Extr to the Source field in the Band Statistics inspector For example the Source designation for a band that Fragment Analysis found and calculated using extrapolation would be designated as Auto Extr Standard bands exported in a Straight line Figure B 2 For unknown lanes that are not bracketed by standards lanes molecular weight values are exported from only one standards lane using horizontal extrapolation Finally Fragment Analysis computes the size or isoelectric focusing point of each band using the interpolation method you select point to point linear point to point logarithmic cubic spline logarithmic or least squares straight line See section 6 1 6 for an explanation of the interpolation methods B 2 Determining the amount quantity of unknown bands After band finding is complete Fragment Analysis assigns the values listed in the amount
117. retrieve the experiment first open the image from which the experiment was saved N ext open the File menu select Experiment Analysis and then select O pen from the submenu A window appears for selecting the file to load 9 6 5 Creating a report To create a spreadsheet report of the data click the Report button to display the Report window The Report window allows you to specify the report format and then transfer the results to Excel You can also access the Report function by selecting Generate Report from the File menu For more information on the Report function see section 6 5 9 6 6 Exporting results To save the data in a file that you can export to a word processing or database application click the Export button The Export window appears The Export window allows you to specify the report format and open a window for naming the file You can also access the Export function by selecting Export Results from the File menu For more information on the Export function see section 6 4 p9 16 Fragment Analysis User s Guide Chapter 10 Window menu Show ToolBar v Attach ToolBar v Show Status 1 c data tutor tutorl gel Cascade Shift F5 Tile Shift F6 Tile Horizontal Tile Vertical Arrange Icons Close All The Window menu includes commands for rearranging windows and their components The topics in this chapter are Show Toolbar section 10 1 Attach Toolbar section 10 2 Show Status section
118. rofiles that you want to show or hide A color menu allows you to change object colors Click Apply to apply your changes to the selected objects or Save to save and apply the changes to all subsequent analyses For more information on using the Display window see section 7 9 p4 2 Fragment Analysis User s Guide Chapter 4 Post analysis operations step by step FragmeNT Analysis File View Tools Analysis Window l inspector ua Band Characteristics Lane Number Lane Name Lane Type Unknown Band Number Band Name Size Rf Distance Amount Volume Source Percent elp Recalculate Show Form Save Analysis Pi 102 90 Pf 270 258 Mag x2 _ Mode Magnifier Figure 4 1 Using the Magnifier to inspect bands To try it now 1 On the toolbar click the M agnifier button dal and then place the pointer anywhere in the image and hold down the mouse button A magnifying glass appears figure 4 1 2 Drag the glass around the image to examine the bands and then release the mouse button 3 Click Select 3 to end the M agnifier mode 4 Open the View menu and select Display The Display window appears 5 Inthe Display window click the Lane Profile box to select it
119. s 00 00 e eee 9 9 9 4 2 Creating a standards file 2 2c eee 9 9 9 4 3 Loading an existing standards file 0 9 9 9 4 4 Loading two sets of standards 0 c eee eee eee 9 10 9 4 5 Modifying the Standards Values 0 cece eeuee 9 10 9 4 6 Saving changes to a standards file 0 ceca 9 10 9 4 7 Deleting a standards flle 0 0 eee eee 9 10 Ba ndFindei se risrg gakuen non EEE Ae Gea RNAS 9 11 9 5 1 Finding bands i c2 ace pees Sak ead dai ma aiei 9 11 9 5 2 Adjusting the band finding parameters 9 12 9 5 3 Limiting band finding to selected lanes 9 13 9 5 4 After you click done standards bands verification 9 13 viii Fragment Analysis User s Guide Table of contents 9 6 Band Statistics 0 ck eee 9 14 9 6 1 Displaying band statistics eee 9 14 9 6 2 Naming bands 225 05 020s54 50404 be ee dees be edie 9 15 9 6 3 Saving the analysis parameters 00 0 eae 9 16 9 6 4 Saving the experiment analysis 000 ee eae 9 16 9 6 5 Creatingareport 0 cee 9 16 9 6 6 Exporting results aoaaa 9 16 Chapter 10 Window menu 10 1 Show Tool ba sassi ara ie ce te te eine Pane aoe va Soe whe 10 2 10 2 Attach Toolbar seta sierran ea reie bay eee eid 2 ten a 10 2 10 3 SNOW StAtUS 2 secret eee eee ae Mead Serna AS 10 2 10 4 Cascaden ciora ahiek AE Resin HEE PAS WNL ae 10 2 TOSA Tle sc vg aie bea doe
120. s selected in the Order area 2 Click the Value box type 1300 and then click Add or press ENTER The value 1300 appears in the list of values box figure 3 10 3 Next type 1200 in the Value box and click Add Continue entering values in decreasing 100 increments until you reach 100 You should have 13 entries Use the scroll bar to check 4 Click Save As The Save Size File window appears 5 Inthe File N ame box type SIZESTD and then click OK Fragment Analysis adds the siz file extension to the file name 6 Click Done The Band Finder inspector appears figure 3 11 7 Go to section 3 11 Fragment Analysis User s Guide p3 21 Partone Basics 3 11 Finding the bands The Band Finder inspector figure 3 11 initiates and controls the automatic band finding process When the Band Finder inspector appears the Setup area displays the number of bands in your standards file as well as the default band finding parameters In addition three band editing buttons are added to the toolbar see section 4 2 1 for an explanation of the buttons 3 11 1 Initiating the band finding The Scope area allows you to specify the lanes for which you want Fragment Analysis to find bands First click one of the selections in the Scope area the default is All Lanes and then click Find Bands Fragment Analysis locates the bands and marks the band positions and boundaries 3 11 2 Evaluating the band finding Check the im
121. s the step and displays the next inspector in the series Banes Lane Setup H Lane Identification Tape Description Panes Lane Setup Image Setup Lane lde ane Finder TEET Size Std D1580 Unknown Unknown Unknown Size Std Unknown Unknown Unknown Size Std D1580 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 Size Std D1580 Figure 2 3 Example of an inspector p2 4 Fragment Analysis User s Guide Chapter 2 Getting started Se 2 4 Quitting the Fragment Analysis software To quit the Fragment Analysis software save the results of your analysis Then from the File menu click Exit The Fragment Analysis main window closes Fragment Analysis User s Guide p2 5 Chapter 3 Fragment Analysis step by step This chapter takes you through a complete fragment analysis session Before you begin analyzing your own data use this chapter as a tutorial to learn how to analyze the provided image The tutorial image file is named tutor1 ds The tutor2 ds image fileis a dual channel image that can be used in chapter 5 To use this chapter as a tutorial look for the words To try it now on each odd numbered page To begin the tutorial display the Fragment Analysis main window as described in section 2 1 N ote During the course of the tutorial you will be creating files and giving them names specified in the tutorial If someone has used the tutorial before you a message will appear wh
122. s to the degree indicated by the line For complete instructions on rotating see section 9 1 p3 14 Fragment Analysis User s Guide Chapter 3 Fragment Analysis step by step Large rectangle Small rectangle re ag vja File View Tools Analysis Window Help data tutor tutor1 d vial Inspector w Panes image se H 1 Experiment ak Te DNA dS _ a Number of Lanes 9 4 1p my Date 87187153 11pm Tay I Interpolation Eod Size Point to Point Logarithmic E p _ Amount Leart Squares Suan Line x General Comments 3 T 2 Image Rotation ef gt Ready c data tutor samplel fim Figure 3 7 Adjusting the region of interest and band prototype rectangles To try it now 1 Place the pointer inside the small rectangle and drag the rectangle to enclose the largest band figure 3 7 Resize the small rectangle to fit the band Place the pointer on one of the black handles and drag the handle right or left up or down Click the large rectangle H andles appear at the corners Use the handles to adjust the size of the large rectangle Pull the top and outside edges inward so that they are close to but not touching the bands Pull the lower edge up to exclude the bottom row of bands from the region of interest figure 3 7 Click D
123. scade command To bring a hidden window to the top of the stack click its title bar 10 5 Tile The Tile command arranges multiple mage windows in a tiled fashion both horizontally and vertically if more than three are displayed The image size is reduced to accommodate a tiled arrangement To arrange windows in a tiled fashion select the Tile command 10 6 Tile Horizontal The Tile H orizontal command arranges multiple Image windows side by side horizontally The image size is reduced to accommodate the tiled arrangement To arrange windows horizontally select the Tile H orizontal command p10 2 Fragment Analysis User s Guide Chapter 10 Window menu 10 7 Tile Vertical The Tile Vertical command arranges multiple mage windows side by side vertically The image size is reduced to accommodate the tiled arrangement To arrange windows vertically select the Tile Vertical command 10 8 Arrange Icons The Arrange Icons command realigns icons displayed in a window To arrange icons select the Arrange Icons command 10 9 Close All The Close All command closes all open windows You will be prompted to save files if appropriate To close all windows select the Close All command 10 10 List of Open Windows The bottom of the Window menu displays the names of the open windows If the paths to the files are different from the currently displayed document the path names are also displayed You can select a file name to displa
124. se is No the Band Statistics inspector appears You can also edit bands when you reach the Band Statistics inspector and then click the Recalculate button Fragment Analysis User s Guide p9 13 Parttwo Reference Sz 9 6 Band Statistics The Band Statistics command displays the Band Statistics inspector figure 9 7 This inspector allows you to see a variety of statistics for any band you select In addition from the Band Statistics inspector you can access the Experiment Form as well as report the analysis results Inspector Panes Se Band Characteristics Lane Number 7 oo O O Lane Name Lane Type Unknown Band Number 77bp Band Name Size Rf Distance Amount Volume Source 7 6 Percent Bere poe Figure 9 7 The Band Statistics inspector 9 6 1 Displaying band statistics To see the statistics click any band found by the system The Band Characteristics area displays the following information Lane Number The lane number assigned by Fragment Analysis e LaneName Thename you assigned to the lane using the Experiment Form or the Lane Setup inspector e Lane Type The lane designation Size Std or pK Std Amount Std Size A mt Std pK I Amt Std Unknown or Unused e Band Number The band s position in the lane counting from the top p9 14 Fragment Analysis User s Guide Chapter 9 Analysis menu e Band Na
125. stics inspector Lane Report Shows all band characteristics for one lane e Table Creates a table of all band characteristics for all lanes No Report O ption is available for Table reports because all data is reported p6 10 Fragment Analysis User s Guide Chapter 6 File menu 2 Click the Report O ption box and then choose the report contents Fora Band Report type select the band characteristic such as Size or Rf for which you want a report e Fora Lane Report type select the lane for which you want a report 3 Click the Delimiter box and then select one of two options Tab Separated Inserts a tab between fields and creates a file with a txt extension Comma Separated Inserts a comma between fields and creates a file with a csv extension 6 1 10 Saving the Experiment Form To save to the specifications that you entered directly or that were automatically entered during an analysis session click the Save button The Save Form File window appears figure 6 7 Save Form File File Name Directories Form1 frm c data tutor A ioe Cancer data gt tutor tutorl dir tutor2 dir E Save File as Type Drives fom Fes E Figure 6 7 Saving an Experiment Form Type a name for the form in the File N ame box and then click OK Fragment Analysis adds the frm extension You can retrieve the form by selecting Experiment Form from the File menu and then clicking Ope
126. terpolation 6 6 9 4 List Files of Type 3 10 loading Experiment Form 3 8 image 3 10 M Magnification command 7 2 Magnifier command 3 12 4 2 8 4 manual in band statistics 9 15 Map command 3 12 7 7 markers lane 3 16 prototypes 3 14 Index 4 Fragment Analysis User s Guide N network 6 14 New command 6 2 noise factor parameter 9 12 Number of Lanes in Experiment Form 3 4 6 3 in Image Setup inspector 9 3 0 Open command Experiment Form 3 8 Open Image command 6 13 Open Edit command 3 2 3 8 6 2 Overlay command 7 8 P P pixel distance 8 2 Pan command 3 12 8 2 Panes in inspectors 3 18 parameters saving 9 16 percent in band statistics 3 24 9 15 Pf pixel distance 8 2 Pixel Distance command 8 2 pixel intensity 4 4 8 4 Pixel Locator command 8 2 pKI standards 3 6 9 9 pKI in band statistics 3 24 9 15 Po pixel distance 8 2 point to point linear interpolation 6 5 9 3 point to point logarithmic interpolation 6 5 9 4 pop up menu 8 1 Previous Frame command 8 3 Print Form command 6 20 Print Image command 6 20 Print Setup command 6 20 printing Experiment Form 4 8 6 20 image 4 8 6 20 report from Excel 4 8 R recalculating data 4 4 rectangles band size 3 14 lane 3 16 moving 3 14 region of interest 3 14 resizing 3 14 selecting 3 14 Reduce command 8 3 reducing image 8 3 region of interest 3 14 Rename command 6 19 report band 4 6 6 10 6 15 6 17 creating 4 6 9 16 exporting 9 16 lane 4 6 6 10
127. the format and content 0 cece eee 6 17 6 5 2 Transferring the results to Excel cece eee ane 6 18 6 6 Deletes vanor ORAA es eae Sie pew wae a 6 18 6 7 REAME ccc teehee ee ge See eee Pee ee wae 6 19 6 8 Printimage 0 cece 6 20 6 9 Print FORM en asa ais ada aida PG AN eee 6 20 6 10 Print Sep panida eee gdb Wee eee Peed eet etre 6 20 Gr EX sets ee ARS A Me A E BR A AT A A Re 6 21 Chapter 7 View menu Ti Actal Size Fasc aor dna Sie hele a A a a OA 7 2 TQ FOWSIZEs vied oes dd atiad ove ae beat ele Sapo 7 2 Tas ZOOUMIN sees howe eae aye Red RAE ee eRe ere 7 2 TA ZOOM QUE iach cease OR eRe Rtn hte a AN NA REAA 7 2 1 5 Magnification 2 0 8 e eek aces da boernes a piner paete nates 7 2 7 6 Grey ColorAdjust 0 0 ccc ects 7 3 7 6 1 What the Grey Color Adjust window does 7 3 7 6 2 Changing the display levels 00 0 0 0 eee eae 7 4 7 6 3 Adjusting brightness and contrast 0 cece eee 7 5 7 6 4 Adding color to the displayed image 0 05 7 6 7 6 5 Inverting the current values 0 00 0 c eee eee ee 7 6 7 6 6 Working with dual channel images 0005 7 6 Bel NGG Diet Taane ethene dette Garces satiate els creeds bens gen Ber Ay AAA hatte SSE 7 7 7 8 Dual Channels os nes asipite boca bag ee Dele Sack a Dales 7 8 79 lt DiSplayste scotia ea eeines Mea aes AeA eee 7 9 Chapter 8 Tools menu Gi SC ISE Ea EE SRNR adel cae E BRE a OE
128. the pointer to the area in which you want the system to locate a band D rag the pointer diagonally to create a rectangle that defines the band size W hen you release the mouse button Fragment Analysis inserts a band marker in the area of maximum pixel intensity Click Select from the menu or toolbar N to end the draw mode p8 4 Fragment Analysis User s Guide Chapter 8 Tools menu 8 11 Delete Band The Delete Band command deletes a selected band To delete a band first click the band you want to delete and then click Delete Band from the menu or toolbar The band marker is deleted You can also delete selected band markers using the DELETE key To select multiple bands for deletion SH IFT click the bands Fragment Analysis User s Guide p8 5 Chapter 9 Analysis menu Image Setup Lane Finder Lane Setup Standard Setup Band Finder Band Statistics The commands on the Analysis menu enable you to access the inspectors you use to perform an analysis The availability of each inspector depends on the stage of analysis you have reached Each inspector contains a H elp button All but the Band Statistics inspector contain a Done button The H elp button accesses information about the inspector The Done button displays the next inspector N ote To use the keyboard to switch between an inspector and the Image window press ALT F6 The topics in this chapter are e Image Setup sec
129. tin America Tel 55 11 3667 5700 Fax 55 11 3667 87 99 M iddle East and Africa Tel 30 1 96 00 687 Fax 30 1 96 00 693 N etherlands Tel 0165 580 410 Fax 0165 580 401 Norway Tel 2318 5800 Fax 2318 6800 Portugal Tel 21 417 70 35 Fax 21 417 31 84 Russia amp other C 1 S amp N I S Tel 7 095 232 0250 956 1137 Fax 7 095 230 6377 Southeast Asia Tel 60 3 8024 2080 Fax 60 3 8024 2090 Spain Te 93 594 49 50 Fax 93 594 49 55 Sweden Tel 018 612 1900 Fax 018 612 1910 Switzerland Tel 01 802 81 50 Fax 01 802 8151 UK Tel 0800 616928 Fax 0800 616927 USA Te 1 800 526 3593 Fax 1 877 295 8102 Part one Basics Chapter 1 The Fragment Analysis workflow The Fragment Analysis software provides the tools and processing power to e Set up the layout of your experiment for DNA fragment analysis protein band analysis and isoelectric point determination e Find lanes and bands automatically e Edit lanes and bands manually e Calculate sizes amounts and isoelectric points of bands e Examine the image with special viewing tools e Obtain reports of size amount and migration statistics about any band in the sample e Perform two color analysis e Transfer data to a M icrosoft Excel spreadsheet Automate the entire analysis process The software steps you through the analysis process beginning with setting up the experiment and ending with obtaining a rep
130. tion 9 1 Lane Finder section 9 2 e Lane Setup section 9 3 Standards Setup section 9 4 Band Finder section 9 5 Band Statistics section 9 6 Fragment Analysis User s Guide p9 1 Parttwo Reference a 9 1 Image Setup The Image Setup command displays the Image Setup inspector figure 9 1 together with two rectangular objects on the image You can move and resize the rectangles to define the area of interest and the largest band size The Image Setup inspector allows you to rotate the image to select the experiment type and interpolation method and to add experiment specific information for your records Inspector Panes EE Experiment Type Number of Lanes ID Date 8 18 1994 1 10pm Size Point to Point Logarithmic Amount Least Squares Straight Line General Comments O Figure 9 1 The Image Setup inspector If you load an existing Experiment Form at the Image Setup step the settings for the area of interest and band size the two rectangles are not in effect Fragment Analysis recognizes the default settings as changes to the form You will need to readjust the rectangles p9 2 Fragment Analysis User s Guide wo Distance Chapter 9 Analysis menu 9 1 1 Selecting the experiment type To select the experiment type click the down arrow button next to the Experiment Type box A list of experime
131. transfer the results to an Excel spreadsheet figure 1 3 or export the results to a file for later retrieval in a word processing or database application Show Form Save Analysis View Tools Analysis Window Help c data tutor tutort gt i Inspector Report Type ae 4 CET J Panes Band Statistics g ee nm saimi A A ee RE Lane Number 7 i Ar Lane Name Lane Type Unknown arts aE O kemener al Dae alal Band Name et oc m Size 677p Gel y Table Database Compatible a ae Amount Unossianed oy Jie Volume Source uomaic o T mij Somwa iuam Bag rot e styl _ Report Export Help Bepon_ Cancel Ready e dataNtutorisarplelfim t Fragment nalysis Report 1 Band RF Report Report generated on 8 23 1994 1 07pm User Name Administrator Image Filename c data tutor tutor1 ds Experiment Form File c data tutor sample1 frm Experiment Date Prepared On Image File Scanned on 8 23 1994 1 04pm c data tutor tutor1 ds 1993 06 14 10 41 17 Microsoft Excel Worksheet in FragmeNT Analysis Insert Format Tools Data Window Help S File Edit View
132. two Reference E Exporting the results automatically Selecting the Export option instructs Fragment Analysis to automatically create a report file containing the results of the analysis The file which you can export into a word processing or database application is automatically assigned the same name as the Experiment Form but with a different extension For example if your form name is samplel frm your report file will be samplel txt or samplel csv If you have previously used an Experiment Form to export a report automatically a message will appear asking whether you want to overwrite the previously created report When you click the Export box new items appear in the Automation Options window giving you the opportunity to select the report type report contents and field delimiter type from selection boxes figure 6 6 Automation Options Automate the following steps g Image Setup J Lane Finder ig Lane Setup ig Standards Setup J Band Finder Export Options Report Type Table Report Option l l Tab Separated Delimiter Figure 6 6 The Automation Options window all options selected To automate exporting 1 Click the Report Type box A list of report options appears Band Report Shows information about one band characteristic for all lanes The characteristics options available from the R eport O ption menu are a subset of those listed in the Band Stati
133. ubmenu p6 18 Fragment Analysis User s Guide Chapter 6 File menu 6 7 Rename The Rename command displays the Rename window figure 6 12 Rename Figure 6 12 The Rename window To rename a file 1 Type the name of the file in the From box Include the path if the file is located in a directory or drive that is different from the current directory or drive Alternatively you can click the Select File button to display the Select Source File window and then select a file just as you do when selecting a file from the O pen Image window section 6 3 The file name including the path is automatically inserted in the From box 2 In theTo box type the new name for the file Include the path if you want to store it in a different drive or directory 3 Click OK To use an existing file name as the base for a new name click the To box and then click Select File The Select N ew N ame window appears Click an existing file name and then change the name in the File N ame box An error message appears if you try to save the file using the same name N ote You cannot rename an Experiment Form file that is currently loaded Select the Close option from the Experiment Form submenu Fragment Analysis User s Guide p6 19 Parttwo Reference 6 8 Print Image The Print Image command displays a Print O ptions window for printing the image figure 6 13 Select centering and scaling options and then cl
134. ulated based on total band volumes and unknown molecular weights are calculated based on migration distances For additional information about how Fragment Analysis calculates unknown band values see appendix B 6 1 7 Loading the standards file Before loading a standards file to use for your experiment you must first indicate the type of file to load Size standards files Contain sizes molecular weights of each standard band Size standards file names include a siz extension e pKI standards files Contain the isoelectric point values PI of each standard band pK standards file names include a siz extension Amount standards files Contain the amount of material in units such as nanograms in each standard band Amount standards file names include a mas extension If you load both amount and size standards in a single lane load both types of standards files To select a standards file click the Size or pKI or Amount button The O pen Size Amount window appears Double click the file you want to use or click the file name and then click OK The file name appears in the text box Alternatively you can type the name of the file in the text box 6 1 8 Entering the lane setup information Click the Lane Setup button to display the Lane Setup window figure 6 3 Use the Lane Setup window to inform Fragment Analysis of the location of standards lanes and to add names and descriptive information for you
135. used this Experiment Form After making changes you must display the form and select Save in order to retain new parameters Or you might have loaded an Experiment Form after loading the image in which case the default rectangle parameters take precedence To recover from either situation readjust the rectangles Then open the File menu and select Experiment Form From the submenu select Show to display the current form N ext select Save and then click the Close button You can also save the changes at the end of the analysis at the Band Statistics inspector C 2 Lane finding The lane markers on the outer lane s of your image are shifted toward the center of the image instead of aligned properly over the lane The bands in the outer lane s do not have high enough signals for accurate correlation To recover return to the Image Setup inspector and increase slightly the width of the region of interest marker large red rectangle and then click Done In the Lane Finder inspector click Find Lanes again Curved lanes on the image make it difficult for Fragment Analysis to define lanes accurately To improve lane finding on images with curved lanes return to the Image Setup inspector and resize the prototype band the small green rectangle to make it narrower about half a lane width Note Do not use this method if you are looking for amount information Also note that band quantitation is not accurate for lane markers that d
136. xperiment optional e Experiment ID Type any text to use for identifying your experiment e Date Typea date in the box or click the Today button to enter the current date e General Comments Type any special notations in this box 6 1 3 Entering the number of lanes In the Number of Lanes box type the total number of lanes you will be analyzing The number you enter here will be transferred to the Lane Setup window Fragment Analysis User s Guide p6 3 Parttwo Reference 6 1 4 Selecting 1 or 2 channels Use the Channel box to set up the experiment for a one or two channel image click the down arrow to see the second selection For single channel images leave the Channel setting at 1 For dual channel image analysis first set the Channel selection to 1 and fill out the form including Lane Setup for the channel 1 image and then set the Channel selection to 2 and fill out the form including Lane Setup for the channel 2 image For more information about dual channel image analysis see chapter 5 6 1 5 Selecting the experiment type Select the type of experiment you are running from the Experiment Type box The list displays the following experiment types e DNA fragment analysis e Protein fragment analysis e Isoelectric focusing Selecting Isoelectric focusing modifies choices on the Experiment Form For example the Size button changes to pKI figure 6 2 Form1 frm Experiment ID Experiment
137. y To end the drawing mode click the Select button 4 2 2 Recalculating the data After adding or deleting band markers click the Recalculate button to instruct Fragment Analysis to recalculate the data If you add or delete standards bands and recalculate the isobar lines are redrawn 4 2 3 Naming bands To label a band with a unique name click the Band N ame box and then type a name and click Set The band name appears in the report p4 4 Fragment Analysis User s Guide Chapter 4 Post analysis operations step by step FragmeNT Analysis File View Tools Analysis Window l Inspector Eas Band Characteristics Lane Number Lane Name Lane Type Unknown Band Number Band Name Size Rf Distance Amount Volume Source Percent Help Show Form Save Analysis c data tutor samplel fm i Sloleia lalclaicietsi Figure 4 2 Editing bands To try it now 1 Click the Draw Band M anual button Ef 2 Movethe pointer to an empty spot in any lane of the Image window and then drag the pointer to draw a rectangle figure 4 2 W hen you release the mouse button a band marker appears 3 Click Select I to exit the draw mode 4 Select the band marker within the box
138. y a hidden file Fragment Analysis User s Guide p10 3 Part three Appendixes Appendix A Report examples The following pages show examples of the three types of reports you can create Band Report Shows the values of one user selected band attribute such as size for all bands in all lanes e Lane Report Shows all the statistics for all bands in one user selected lane Table Report Shows all information for both bands and lanes Reports are divided into two sections The upper section shows general information about the report as well as information collected as part of the scanning process The lower section shows detailed information about lanes and bands The column headings are explained in section 9 6 1 The examples on the following pages were created using Excel formatting features Fragment Analysis User s Guide pA 1 Part three Appendixes User Name Image Filename Experiment ID Experiment Date Prepared On Image File Scanned on Units Band Size Report Report generated on 6 25 1994 10 51am Administrator c data tutor tutor1 ds Sample 1 8 25 1994 10 41am c data tutoritutor1 DIR SEP1 GEL 1993 06 14 10 41 17 bp Lane1 Lane 2 1300 193 703 666 238 191 648 1300 198 116 676 961 369 394 1300 Lane3 Lane 4 Lane5 LanG Lane Lane 8 Lane 9 User Name Image Filename Experiment ID Experiment Date Prepared On Image File Scanned on 1200
139. ys the Save Analysis File window To save the analysis type a name in the File N ame box and then click OK Fragment Analysis adds the exp extension You save an analysis when the session is complete the Band Statistics inspector is displayed You can initiate the Save command from either the Experiment Analysis submenu or by selecting Save Analysis from the Band Statistics inspector You retrieve open a saved experiment after loading the image from which the experiment was saved p6 12 Fragment Analysis User s Guide Chapter 6 File menu 6 3 Open Image The O pen Image command displays the O pen Image window figure 6 8 Open Image File Name Directories c data last gel testto ds testscan gel western ds wheat gel List Files of Type MD Files ds gel Figure 6 8 The Open Image window To open an image file locate the file name among the list of files and double click the name or click the file name and then click OK 6 3 1 Locating the file you want When you open the window the File N ame box initially displays ds gel indicating that all the files listed are image datasets or files Below the File N ame box is a list of existing ds and gel files If the file name is not listed check that e The list represents the correct file type section 6 3 2 e Thedirectory is correct section 6 3 3 e The drive is correct section 6 3 4 e The file is located in a sh

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