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1. Figure 2 6 Casebook dialogue File Information tab 2 7 2 Making your first run 5 9 2 8 Enter a name for the result file by typing M yresult in to the Name field Check that the file path is correct If necessary click on the Home button to recall the location of your user folder Click on the Conditions tab There you can see the experimental conditions that will be used for this run N o changes are necessary Click on the Sample Information tab and ensurethat the Clones tab in the lower left part of the dialogue is selected In the Name column enter M 13 for all ten clone positions Add any comments in the Comment column Larmbmst FS eqeence che odes doo td Figure 2 7 Casebook Clones tab in Sample Information Click on the Notes tab Enter relevant notes in the Pre run notes field Making your first run 2 Fig irierabon Cargo Sapin indoresioe Feine arem Hater Penna FP flare pres N a aali Erakon ner aj Figure 2 8 Casebook dialogue Notes tab 10 Click on OK in the Casebook dialogue T he dialogue closes and the Save Casebook dialogue is displayed Click on Save As and the Save Casebook As dialogue is opened Give a new file name to the casebook file so that your colleagues can use the Sequencelst cbk file for example Run1 cbk Click on Save 2 2 7 Preset
2. hi Fean Ardd nr Edit Weer x Lear ream a Homa tokier E AAP D eect AL Pearni Hora Broeene Backup kidar F AP Beh AL Fain Hore Binders Oenutrent addres a Phone sumba gB Figure 2 2 ALFwin Add or Edit User dialogue Type your name in the User name field Y ou can use up to a maximum of 256 characters Y our user and backup folders are created automatically when you enter a user name Y our home folder is automatically shown when you work with other ALFwin modules Click on OK to save the information and to return to the ALFwin Administrator dialogue For further information on adding new users see Section 3 2 2 2 3 Linking ALFwin to the instrument Define the connection of the instrument to ALFwin 1 From the ALFwin Administrator dialogue highlight a User name and select an instrument in the Available applications field Click on System Setup The ALFwin Edit System Settings dialogue opens 2 3 2 Making your first run A a Feis Edi Syok Gettin laanurani D Caine iritunaent Tracing F inclids dran iraco I fretunent iare erir Figure 2 3 ALFwin Edit System Settings dialogue The name of the instrument is shown in the top field of this dialogue Y ou can name your instrument here the name is shown in the ALFwin Control Title bar during the run 3 Typeany comments in the Comment field 4 Typethe number of the COM port to which your instrument is connect
3. 4 6 6 Viewing curve amplitude information To examine the amplitude of the curves 1 Click on the Curve window to activate it 2 Select View Signal Levels or press the lt Ctrl Shift S gt keys The maximum and minimum amplitudes and the difference in amplitude for each curve is shown in a table Signal Levels Clone 1 Eg 1 28 2 82 1 54 1 39 2 86 1 48 G 1 35 2 53 1 18 T 1 32 2 08 0 76 Help Figure 4 26 Signal levels dialogue The table relates to the parts of the curves and the current curve group shown in the curve window and the amplitude values are updated if you scroll in the curve window or change curve group The difference column shows the difference between the minimum and maximum signal level of the curve Differences in the order of 1 arenormal The unit refers to the percentage of the maximum possible amplitude 4 6 7 Writing run notes in the casebook You can write Run notes in the casebook Select Edit Casebook and select the Notes tab Changes and additions can not be made in this field after the run is finished ALFwin Control 4 4 6 8 Starting a run on another instrument Y ou can start a run on another instrument with ALFwin Control This requires that you open a second copy of ALFwin Control see Section 4 1 3 4 7 After the run is finished Therun stops automatically when the run time is out The selected post run actions are then performed The lights in the title bar an
4. These can be generated for each of the aligned sequences and you can generate amino acid sequences for the collective data in any or all of the three possible reading frames Selected sample sequences and all aligned sequences can be exported in M SF or PHY LIP format The consensus or the amino acid sequences can be exported in ASCII format The statistical function provides a quantitative measurement of the quality of the alignment Alignment of sequences from different sources Interactive editing features Sequence Alignment 6 Sequences can be included for alignment either as a clone sequence from ALFwin Sequence Analyser as imported sequences in ASCII format and as imported sequences in SCF format The alignment table display allows you to quickly jump to selected parts of the sequences where you can examine the bases in each sequence and view the related parts of the raw or processed data curves Full editing facilities allow you to make changes on the basis of this inspection Any changes you make are documented automatically in the alignment log and there is a note pad where you can write your own notes a MSF is the multiple sequence alignment format of the GCG sequence analysis package Information can be found at http www embl heidelberg de predictprotein new D exa optin_msfDes html b PHY LIP the PH Y Logeny Inference Package is a package of programs for inferring phylogenies evolutionary t
5. before the name To delete a group 1 2 6 Click on the Curve window to make sure that it is activated Select View Properties The Curve Properties dialogue is shown Select the Group to be deleted from list of Group names Click on Delete group A dialogue asks if you are sure you want to delete the group Click on OK The selected group is deleted from the list of Group names Click on OK to close the dialogue 4 8 11 Changing the Y axis scaling Y ou can use menu commands to select the type of scaling for the currently selected clone Curve window 4 53 7 win Control Command Effect View Scale Separately This automatically scales each of the curves separately so that the displayed amplitude T scale for each curve is adjusted to best fit the window displayed This may be useful in discerning the peaks in a curve that has a relatively low detection signal during sequencing N ote that selection of separate scales can be misleading when for example trying to establish if a peak is a true peak or background noise View Scale Together This automatically maximizes the size of the largest peak visible in the current view E of the Curves window The amplitude scale for the curve containing this maximized peak in the current view is used as the basis to scale the remaining curves Thus all peaks are correctly proportioned in relation to the largest visible peak This allows you to look at the relative
6. Explanation The algorithm usually needs a clear primer peak to be able to start its operation This problem may appear in solid phase sequencing since it shows just a minor primer peak Too extensive sampling i e too many data points per peak may interfere with downstream processing since the processing is optimised for a certain number of data points per peak Solution Start processing manually Use correct Sampling interval i e sampling every 2 sec for the standard cassette and 0 5 1 sec for shorter gels that run very fast The base calling algorithm has stopped Problem Y ou have got shorter reading length than expected Explanation M any different reasons are possiblewhen the base calling algorithm has stopped early Smiling effects chemistry problem template quality etc Solution Use manual start time Apply shift Edit Apply Shift perform shift and process again Check further reasons in the main trouble shooting guide for ALFexpress The base calling seems to be totally wrong Problem Y ou realise that the evaluation is totally wrong Explanation The selected lane order is wrong you have forgotten to exclude unused lanes or excluded lanes with samples Solution None Perform the run again Troubleshooting Strange fonts when you use copy clone or export to metafile Problem Strange fonts when you use copy clone or export to metafile format Explanation The export uses an expor
7. Figure 6 17 Sequence Alignment Export dialogue 3 Select from the Export includes options the items to be exported Entire alignment To export all the alignment data Selected sequences To export the base sequences selected in step 1 Consensus To export only the consensus sequence Amino acid sequences To save the amino acid sequences selected in step 1 If you select this option open the Reading frame drop down list and select the desired reading frame 4 Open the Format drop down list and select the appropriate format from the options available 5 Click on OK The Save As dialogue opens 6 33 6 Sequence Alignment 6 Use the dialogue to select the folder where the file is to be saved 7 Enter a name for the file 8 Click on Save 6 14 Opening a previously saved sequence alignment item To open a sequence alignment item that you have previously saved 1 Select File Open or click on the Open button The Open dialogueis displayed 2 Select from the Files of type drop down list alignment files sqa 3 Open the folder containing your sequence alignment files Select the appropriate file and click on Open The sequence alignment window is opened Note If another sequence alignment item was already open you are allowed to confirm a save of the item before it is closed 6 15 Configuring the sequence alignment module 6 34 The configuration of the sequence alignment module can be adjusted to
8. Foa guier Damsbori Eader cba EEE E A EE Figure 4 8 Casebook dialogue File Information tab Use this dialogue to enter the name and location of the result file 2 Select the Name of the result file You can name the file according to Casebook name This is the default setting and uses the casebook name Date Use the current date Name Use the name typed into the Name field 3 By default each file name includes the casebook name and a counter number The counter number increases with each successive result filethat you produce T his ensures that previously created files are not over written If the number passes 999 it will begin again at 1 and you may have to move rename or delete files with low serial numbers The counter is user specific and independent of what name you select on your result file The counter will only increase when it is on To disable this auto numbering function deselect the Add counter no to filename button ALFwin will warn you if you then select a filename that will overwrite an existing file ALFwin Control 4 4 Edit the File path field if you want to change the folder where the result file is stored If you want to bein your home folder click on Home and the path to your home folder will be automatically given Alternatively use the Browse button to open the Select Directory dialogue and useit to locate the destination folder for the result file Remember that you can create a
9. If you want to modify the casebook refer to Sections 4 4 When you have performed all of the modifications of the casebook click on OK This will close the Casebook dialogue 5 The Save Casebook dialogue opens see Section 4 3 4 for details 4 3 3 Creating anew casebook ALFwin Control allows you to create a new casebook by supplying an unnamed casebook file with default parameters set for the instrument To create a new casebook file Casebook button The Unnamed Casebook dialogue with default Cj 1 Select File New Casebook lt Ctrl N gt or click on the New values for the appropriate instrument is shown 7 win Control Note If you have both ALFwin Sequence Analyser 2 00 and ALFwin Fragment Analyser 1 00 software applications installed on your computer the Select Application dialogue will be displayed Click on Sequence Analyser and click on OK Sealer App anhun 3 Awsilaile spplcetions Fragment nahra Soquence Anak Cancel Heip Figure 4 5 Select Application dialogue 2 Edit the new casebook according to the procedures described in Section 4 4 4 3 4 Saving the casebook Y ou must close the Casebook dialogue before you start a run If you have created a new casebook file or opened and modified an existing casebook file it must be saved when closing the Casebook dialogue To close and save the Casebook dialogue 1 Click on OK in the Casebook dialogue The Save Casebook dialogue is shown Sa
10. RO 1 Select either View Zoom with Mouse the F 10 gt key or click on the EH Zoom with Mouse toolbar button 2 Position the mouse pointer in the top left corner of the area you want to magnify in the Gel View window Press and hold the left mouse button and drag out a box to encompass the area that you want to magnify Release the left mouse button 5 27 5 ALFwin Sequence Analyser Resetting the magnification Q To reset the magnification factor to the default level select either View Reset Zoom the F 9 gt key or click on the Reset Zoom toolbar button 5 6 4 Adjusting the Gel View window properties Adjusting the Gel View contrast Y ou can adjust the contrast of the Gel View window to improve visualisation of the bands in the lanes 1 Select View Gel Properties The Gel Properties dialogue is displayed Click on the Data Set tab Gel Properties x Appearance Data Set Processed data Cancel ERPI Figure 5 16 Data Set tab of the Gel Properties dialogue 2 If necessary you can select the data set in which the contrast changes are to be made Select the appropriate data set either Raw data or Processed data The data displayed in the Gel View window are correspondingly displayed 5 28 ALFwin Sequence Analyser 5 3 Select the Appearance tab Gel Properties x Appearance Data Set m Scale region Visible Axis vv Wz l M Time Whole run I Clone number m S
11. The curve view for a clone consists of four colour coded overlapping curves that represent the sequencing data for thefour basesA C T G The curve represents the signals detected by the laser system in the ALF family instrument and each peak shows the presence of nucleotide Im FEI ALFwin Sequence Analyser 5 material in the sample If the data have been processed for the viewed clone the calculated sequence is displayed directly beneath the curves The order of the bases A C G T in the sequence corresponds to the order of the peaks in the curves and are similarly colour coded to the curves they represent It is also possible to view ambiguity codes which are the IUPAC codes denoting the presence of more than one base component at a single base position Ambiguity codes are discussed in Section 5 7 1 and Appendix C Thebases of the sequence will consist of capital letters or small letters and can be either upright or italic Viewing curve legends To view the legends showing which of the four bases the curves represent select View Show Legends The legends are displayed on the right side of the curve view Mouse pointer position in Curves view Click in the Curves view and the mouse pointer is accompanied by an information window which gives the position value for time W hen the pointer touches a curve the typeof curveand its amplitude at the point of contact is displayed If you zoom with the mouse see Section 5 5 6 you c
12. 4 26 store table information in template clones 4 25 system requirements A 1 system setup 2 3 3 5 3 6 T Table Settings button 4 25 table settings for lanes 4 25 temperature 4 18 automatic raising of gel temperature 4 5 indicator light 2 9 pre heating the gel cassette 4 39 presetting 2 9 three letter amino acid codes 6 27 tick mark blue 6 12 investigating imperfections 6 12 red 6 12 yalow 6 12 TIFF from alx alf 5 61 tile all Curve windows 5 16 tile all windows 5 16 time using manual start or stop times 5 38 Tip of the Day 1 3 4 4 5 4 Title bar 4 6 title bar in ALFwin Control 4 6 in Sequence Analyser 5 6 toolbar hiding and showing 4 48 in ALFwin Control 4 7 in Sequence Analyser 5 7 moving around screen 5 37 5 40 toolbar button raw or processed data 5 12 tools 5 58 adding 5 59 arguments 4 37 automating selection 4 36 delete 5 60 edit settings 5 59 supplied with ALFwin 5 58 Index using tools 5 58 view settings 5 59 total number of called bases 5 33 trace functions 3 5 transmittance 4 40 two instruments on one computer 4 4 typographical conventions in this manual 1 3 U undo 6 21 uninstalling ALFwin i updates on the Web 1 2 4 8 4 9 upright bases 5 34 upright bases ambiguities 5 33 Upright bases A mbiguities column 2 15 upright status assigning 5 36 user adding 3 2 adding user details 2 3 choosing for Sequence Analyser 5 3 creating user name and folder 3 2 default 3 2 deleting 3 4 editing 3 4
13. 5 2 Introducing the ALFwin Sequence Analyser interface 5 2 1 Windows 5 2 2 Bars 5 3 Opening a result file 5 3 1 Contents of a result file 5 3 2 To open a result file 5 4 Presenting results in the Result window 5 4 1 Clone icons 5 4 2 Viewing the lane information 5 5 Presenting curves in the Curve window 5 5 1 O pening Curve windows 5 5 2 Viewing raw or processed data curves 5 5 3 Curve view 5 5 4 Orientation view 5 5 5 Navigating the Curve window 5 5 6 Using the zoom function in the Curve window 5 5 7 Stacking the curves 5 5 8 Selecting individual base curves for viewing 5 5 9 Viewing complementary or reverse curves 5 5 10 Arranging Curve windows 5 5 11 Linking Curve windows for comparison of clones 5 5 12 Changing the axes 5 5 13 Changing the presentation of the curves within the curve windows 4 57 4 58 4 58 4 59 4 60 5 3 5 3 5 5 5 5 5 6 5 7 5 7 5 8 5 9 5 9 5 1 5 10 5 11 5 12 5 12 5 13 5 14 5 14 5 15 5 15 5 15 5 15 5 16 5 17 5 19 5 5 14 Showing the signal levels for the currently displayed curve 5 19 5 5 15 Changing the default setting for the base curves shown in curve view 5 5 16 Changing the default setting for the type of data curve displayed in curve view 5 20 5 20 5 5 17 Changing modes in orientation view 5 5 18 Including instrument data as curves 5 5 19 Changing the font and colours in the Curve window 5 5 20 Additional information in the curv
14. Zoomin Zoom out Reset zoom Show complementary and reverse curves Hide or show lanes A C G orT Show signal levels The Levels dialogue is shown Activate or inactivate Edit mode The Edit toolbar is shown or hidden N ote that Replace mode is activated first Activate or inactivate Apply Shift mode The Shift toolbar is shown or hidden Find ambiguity or sequence The Find dialogue box is shown Ctrl C Ctrl L CtritHome Ctri End Page Up Page Down Left or right arrow key Keyboard shortcuts D Copy clone sequence or gel depending on the active part of the screen Link open Curve windows for similar presentation of curve views M ove to the first base in the run When the sequence is reversed move to the last base in the run M ove to the last base in the run When the sequence is reversed move to the first base in the run M ove zoomed area one step to the left M ove zoomed area one step to the right M ove to base one step to the left or right D 5 5 In the Curve view in the Curve window Home End M ove to the first base in the sequence shown in the curve view Always counts from the left M ove to the last base in the sequence shown in the curve view Always counts from the left D 5 6 In the Orientation view in the Curve window Curve and bar mode Home End Left or right arrow key M ove to the beginning of the run When curves are reversed moveto the end
15. casebook file any automated post run actions Preset the instrument and You can preset the temperature of the start the run instrument prior to starting the run 4 1 7 win Control 4 2 Perform any run activities You can monitor the run on the screen as it proceeds and check the amplitude of the curves You can perform other activities such as enter sample information standard settings and other analysis settings in the casebook include run notes in the casebook manipulate the screen layout for presentation purposes pause the run start another run on a second instrument analyse pre result data End therun The run ends automatically or you can end the run manually This chapter is divided into the following sections 4 1 Starting ALFwin Control 4 2 Introducing the ALFwin Control interface 4 3 Working with casebooks 4 4 M odifying casebook parameters 4 5 Starting a run 4 6 Activities that can be performed during a run 4 7 After the run is finished 4 8 Changing the screen layout 4 9 Exiting ALFwin Control ALFwin Control 4 4 1 Starting ALFwin Control ALFwin Control is normally started from ALFwin Administrator Y ou can also move into Control from ALFwin Sequence Analyser 4 1 1 Starting ALFwin Control from ALFwin 1 Administrator From the Windows 95 Start button menu options select Programs ALFwin software Use the ALFwin Administrator dialogue to select your user name fro
16. documents option from Windows 95 start button B 3 dust particles 2 6 E editing alignment table bases by using the Curve window 6 22 Index casebook 4 13 changing the order of the sequences in the sequence alignment list 6 7 column header 4 24 column settings 4 23 deleting bases in the alignment table 6 21 disabling the sequence alignment editing functions 6 25 edit again 4 13 enabling disabling sequence editing 5 24 experimental conditions in the casebook 4 17 group 4 52 inserting bases in the alignment table 6 21 lane information table 4 25 moving the edit toolbar 5 40 moving the shift toolbar 5 37 remove sequences from an alignment 6 7 replacing bases in the alignment table 6 20 sample information columns 4 23 sequence bases 5 39 sequence bases in sequence alignment 6 20 showing or hiding edited bases 5 44 toolbar 5 40 tools settings 5 59 user 3 4 user and backup folders 3 4 electrodes fitting 2 11 EMBL 4 31 E 2 export format 4 31 5 51 ending a run manually 4 43 entire alignment export 6 33 evaluating results 2 12 excluding lanes 4 26 exit from Control 4 60 experimental conditions in the casebook detail 4 17 in the casebook overview 4 8 experimental notes 4 9 export alignment facilities 6 2 alignment sequences 6 32 amino acid sequences 6 33 ASCII format 6 32 automating data export 4 30 consensus sequence 6 33 curves in M etafile format 5 49 entire alignment 6 33 export file formats available 4 31 filename
17. editing user and backup folder 3 4 user name and folder creating 3 2 A4 vertical cursor line 5 13 view signal levels 4 46 5 19 tools settings 5 59 viewing alignment statistics 6 18 alignment table bases by using the Curve window 6 22 cascade windows 5 16 casebook information 5 45 complementary or reverse curves 5 15 lane information 5 10 legends in curve view 5 13 raw data or processed data in the Gel View window 5 26 raw or processed data in the Curve window 5 12 run conditions 4 40 selected curves 5 15 5 20 XXV Index xxvi stacked curves 4 49 status bar 4 48 tile all windows 5 16 tiling all curve windows 5 16 time and curve amplitude at mouse pointer 5 13 toolbar 4 48 voltage electrophoresis 4 18 W water level in instrument 2 5 Web Amersham Biosciences address 4 8 4 9 Pharmacia address 1 2 white cell in the alignment table 6 13 window cascade 4 47 link 5 16 Result 5 8 5 10 tile as default 4 47 Windows earlier versions of M icrosoft Windows B 6 windows arranging 4 47 in Sequence Analyser 5 5 saving window positions on exit 4 47 Windows 95 check option boxes B 4 closing a window B 5 dialogue boxes B 4 introduction B 1 moving windows B 5 my computer B 2 network neighborhood B 2 on line help B 6 option buttons B 5 radio buttons B 4 recycle bin B 2 screen display B 1 scrolling windows B 5 sizing windows B 5 start button B 2 starting and logging on B 1 taskbar B 2 text fields B 5 toolbars B
18. view moretips click on the Next Tip button If you do not want to see the dialogue at each startup uncheck the Show Tips on StartUp box To view the Tip of the day dialogue again select Help Tip of the Day Note This dialogue must be closed before you proceed further with Control Click on Close 4 1 2 Starting ALFwin Control from Sequence Analyser 1 Select File Start Instrument 2 Use the ALFwin Select System dialogue to select the instrument 3 Click on Start to enter ALFwin Control 4 1 3 Using two instruments ALFwin Control can simultaneously run two instruments from one computer The instruments are distinguished by the computer s COM port to which they are connected Connection with the instruments is ALFwin Control 4 established automatically when you start ALFwin Control and the software identifies the type of instrument connected so that screen contents and control procedures can be adjusted accordingly If a computer is being used to control two instruments ALFwin Control must be started twice Both instances reside on the taskbar One instance controls Instrument 1 the other Instrument 2 To connect a second instrument repeat the steps described in Section 4 1 3 selecting the second instrument at Step 3 4 1 4 Automatic settings for ALFexpress family instruments If you start ALFwin Control when using an ALFexpress II or ALFexpress analyser with a gel cassette in place the following parameters are set
19. 1 Oncetheset temperature has been reached indicated by the steady illumination of the yellow temperature indicator lamp 2 Carefully remove the comb from the gel Lift the comb straight out avoiding bending in any direction Attention Use gloves to avoid skin contact with the gel 3 Rinse out the wells very thoroughly with running buffer to remove any smear of non polymerised gel Use a 50 ml syringe fitted with a 0 6 or 0 8 mm needle 4 Prepare the Eppendorf Gelloader tips by manually shortening them by about 1 5 cm Fit a tip to a micropipette 0 20 ul and load 5 ul of the A base sample to the first lane for each clone i e lanes 1 5 9 etc 5 Wash thetip in the upper buffer reservoir before taking each sample Load the samples accordingly for each clone in the order of basesA C G T 6 Load gel loading buffer or dummy samples in the two outer lanes 0 and 41 7 9 Making your first run 2 Fit the electrodes to each of the buffer reservoirs The upper electrode has a black male lead that is plugged into the black female socket to the right of the mounted cassette and the lower electrode has a red male lead that is plugged into the red female socket to the right of the mounted cassette Realign the laser by clicking on the Realign Laser button in the Manual window of ALFwin Control Close the lid of the instrument 2 2 10 Starting the run Y ou will now start the run 1 Check that the laser
20. 1 Select the relevant Curve window 2 Select View Properties and click on the Style tab in the Curve Properties Layout dialogue 3 Tochangecolour select the appropriate item under the Colour list and click on the Set Colour button T he Colour dialogue is shown 4 Select a colour 5 Click on OK Note Only solid colour will be printed or copied to the clipboard 6 Select a curve width Double click in the Curve width field and enter the line thickness required in the range of 1 to 50 pixels 7 To effect the changes without closing the dialogue click on Apply To close the dialogue and effect the changes click on OK The colour is changed in all Curve windows To change the font 1 In the Style page of the Curve Properties Layout dialogue select the Set Font button The Font dialogue is shown 5 23 5 ALFwin Sequence Analyser 5 24 2 Select a Font Font style and Size 3 Click on OK 4 To effect the changes without closing the dialogue click on Apply To close the dialogue and effect the changes click on OK If you want to restore the default settings click on the Default Settings button 5 5 20 Additional information in the curve window Y ou can configure the Curve window to show legends that labels each curve with their base type a vertical cursor line that assists positioning within a Curve window an information window that displays curve information alongside the mouse pointer in the curve vi
21. 4 To effect the change without closing the dialogue click on Apply To close the dialogue and effect the change click on OK 5 5 14 Showing the signal levels for the currently displayed curve 1 Select any open Curve window 2 Select View Properties and click on the Settings tab in the Curve Properties Layout dialogue 3 To display the Signal Level table that shows the minimum and maximum amplitudes and the differences in amplitude for the part of the curves currently displayed select check the Show signal levels option 4 To effect the change without closing the dialogue click on Apply To close the dialogue and effect the change click on OK 5 19 5 ALFwin Sequence Analyser 5 20 5 5 15 Changing the default setting for the base curves shown in curve view 1 Select any open Curve window 2 Select View Properties and click on the View tab in the Curve Properties Layout dialogue 3 By default all four base curves are shown in a curve view To change this default setting select the base curves you require by highlighting them in the Base curves field 4 To effect the change without closing the dialogue click on Apply To close the dialogue and effect the change click on OK 5 5 16 Changing the default setting for the type of data curve displayed in curve view 1 Select any open Curve window 2 Select View Properties and click on the View tab in the Curve Properties Layout dialogue 3 Select the
22. 4 4 Modifying casebook parameters The Casebook dialogue is divided into five tabs each containing parameters that may be modified before a run and some modified during a run M odifications that are made before a run is started are saved in the casebook file see Sections 4 3 4 and 4 3 5 and changes made during a run are stored in the result file The five tabs are File information see Section 4 4 1 Conditions see Section 4 4 2 Sample information see Section 4 4 3 Post run Actions see Section 4 4 4 Notes see Section 4 4 5 4 4 1 File Information tab Theinformation in this tab specifies thenameand location of the result file extension alx so that saving can proceed automatically during the run This section also stores operator and date information Y ou have the option of changing the default result file name before the run is started A result file contains the following raw data a copy of the casebook file used the logbook e the analysis log empty until an analysis is performed in Sequence Analyser e data from post run processing 4 15 7 win Control 1 Select the File Information tab in the Casebook dialogue Ceatbeet Segoe oih File latoa oadama Sarake levcamentows Pesii dioas antes Maree Ceb rae E Cte p i E i cauna ne to ia raa File posi CAPRE Fike AP Banke Fer ceeey Durval tows lees inkaa Daunia I Rur dab I Lent acct bee Hodisani
23. 5 using the mouse B 3 Index Windows 95 start button options documents B 3 find B 3 help B 3 programs B 3 shut down B 3 Windows M etaFile 1 2 X X axis changing the X axis 5 17 X axis units 5 17 Y Y axis changing the scaling 4 53 changing the Y axis 5 18 fixed scale 5 18 scale fixed 4 54 scale separately 4 54 5 18 scale together 4 54 5 18 yellow cell in the alignment table 6 13 Z zoom channels in or out 5 27 curve view 5 14 Gel View 5 27 time in or out 5 27 using with curves 4 50 zoom in 5 14 zoom out 5 14 xxvii Index xxviii El m tE S5 8e HE T oe a Short instruction ALFwin Sequence Analyser 2 00 Perform a run 1 Turn on the instrument and computer 2 Place the gel cassette into the instrument 3 Start ALFwin Control 4 Define a sequence analysis casebook 5 Preset the instrument conditions 6 Load samples 7 Realign the laser 8 Start the run Analyse the result file Ambiguity codes 1 Start ALFwin Sequence Analyser Code Unresolved bases M AC 2 Open a result file j R AG 3 Check the gel picture Ww AT l S CG 4 Check the curve view Y CT 5 Process the raw data for selected clones K GT V ACG 6 Check the results after processing H ACT D AGT If necessary adjust the sequence result by B oa Applying shift and process again Setting manual start time and process again N ACGT Editing bases manually Ali
24. Alignment Figure 6 12 Alignment Item Properties tab of the Alignment Properties dialogue When you close ALFwin the setting is retained and becomes the default setting for thenext session W hen saving an alignment item the start label is also saved 6 8 Amino acid sequences 6 26 Amino acid sequences can be generated and displayed below the sample sequences in the alignment table These can be generated for any or all of the three reading frames Note Amino acid sequences can not be edited 6 8 1 Generating amino acid sequences The following procedure is used to configure the amino acid display These settings will hold until the application is closed If you want to alter the default startup configuration see Section 6 15 2 To configure the display of amino acid sequences 1 Select the sequence s for which amino acid sequences will be generated Y ou can select them by clicking their names in the Label Sequence Alignment 6 list or in the alignment table Use the lt Ctrl gt and lt Shift gt keys with the mouse button to select separate and grouped sequences respectively 2 Select View Amino Acid Sequences and select check one of the three reading frames R epeat this process to generate amino acid sequences for additional reading frames E t WHR we we oo a rs SBR SRSRR ESS aaa agada n i ee ee Figure 6 13 Reading frame 1 three letter code amino acid sequences displayed for the sequence
25. Alt Shift 1 9 or 0 F7 F8 Shift F 7 Shift F 8 F9 Ctrl Page Down or Ctrl End Clone group 1 10 is displayed Clone group 11 20 is displayed if it exists Clone group 21 30 is displayed if it exists Clone group 31 40 is displayed if it exists Zoom in by large steps Zoom out by large steps Zoom in by small steps Zoom out by small steps Reset zoom M ove zoomed area to the end of the run D Keyboard shortcuts CtritPage Up or Ctrit H ome Page Up Page Down Left or right arrow key Ctrl Shift S M ove zoomed area to the beginning of the run M ove zoomed area one screen to the left M ove zoomed area one screen to the right M ove zoomed area onetenth of a screen to the left or right Show signal levels The Signal Level dialogue is shown Curve mode D 4 3 In the Manual window Tab or Shift T ab D 5 ALFwin Evaluation D 2 D 5 1 General Fl Ctrl 0 Ctrl S Ctrl P Ctrl Shift P Ctri F4 Alt Tab Alt F4 Alt underlined letter M ove between experimental conditions and buttons Help Open result The Open dialogue box is shown Save result under the existing name Print the selected Gel view or Curve window The Print dialogue box is shown Print all open Curve windows The Print dialogue box is shown Close the active result Switch between opened programs Exit ALFwin Evaluation Select commands in menus or in dialogue boxes Keyboard shortcuts D C
26. Preface Appendix A Appendix B Appendix C Appendix D Appendix E Index This details the computer hardware system requirements This is a short guide to using Windows 95 This describes the base calling algorithm used to process raw data This lists the keyboard shortcuts associated with ALFwin Sequence Analyser Reference list Contents Installing the software 1 Introduction To install the software To uninstall the software 1 1 Key features of ALFwin Sequence Analyser 1 2 Using ALFwin Sequence Analyser 1 2 1 Windows 95 operating system 1 2 2 Menu commands 1 2 3 Tip of the day 1 2 4 On line H elp 1 3 Using this manual 1 3 1 Typographical conventions 2 Making your first run 2 1 Before starting a run 2 1 1 Safety 2 1 2 Pre run activities 2 2 Making a run 2 2 1 Turning on the instrument and computer 2 2 2 Adding a user 2 2 3 Linking ALFwin to the instrument 2 2 4 Starting ALFwin Control 2 2 5 Mounting the gel cassette into the instrument 2 2 6 Entering information into a casebook 2 2 7 Presetting the instrument 2 2 8 Denaturing the samples 2 2 9 Loading the samples 2 2 10 Starting the run Contents N 0 N NN rot 1 rot FooOowWwo an Ul BW WN N Pr ay NNN 1 1 1 Contents 2 3 Analysing the results of your first run 2 3 1 Starting AL Fwin Sequence Analyser 2 3 2 O pening the result file 2 3 3 Processing the data 2 3 4 Analysing the processed data 2 3
27. Sequence Analyser 5 vy Did au boner Tha dsn bar diopdaye riman abot a Cures ca ie mirii F Gatan Bet Ti Figure 5 3 Tip of the Day dialogue Y ou can read tips about using the program To view more tips click on the Next Tip button If you do not want to see the dialogue at each startup uncheck the Show Tips on StartUp box Y ou can open the Tip of the Day dialogue from the program by selecting Help Tip of the Day Click on Close to leave this dialogue 5 2 Introducing the ALFwin Sequence Analyser interface 5 2 1 Windows Three main windows are used to analyse results contained in a result file The Result window Clone and lane information is shown ina Result window for each opened result file The Curve View window This provides a graphical view of run data for a selected clone for raw data or processed data The Gel window This shows the run data for the whole run as a fluorogram 5 5 5 ALFwin Sequence Analyser Title bar Menu bar Tool bar Result window Gel view window Curve window MA AN hl PheGkt Cue e Redes qe ene retpennr s Smale eke renee gee per T ELT e wae TE Status bar EE TThiitiit te we secemeaereeeay Figure 5 4 ALFwin Sequence Analyser screen with the Curve window and Gel View window open displaying results for the result file open in the Result window Note that you can maximize the display of any window or change the size of the windows by dragging the wi
28. Three Letter Codes 5 Open the Interpretation Table list in the Alignment item properties tab and select the table required 6 Click on OK to close the dialogue 6 35 6 Sequence Alignment 6 36 The format selected is applied to all the sequences Y ou can edit the display of amino acids sequences on one or more of the base sequences but only one of the letter codes can be used throughout see Section 6 8 1 6 15 3 Configuring the operation of the alignment algorithm Y ou can adjust e thedirection of the sequence data which may be considered for alignment e thealgorithm used for alignment To configure the alignment algorithm 1 Select View Properties to open the ALFwin Alignment Properties dialogue 2 Select the Alignment Item Properties tab 3 Usethe buttons in Algorithm Options to select the sequence orientation permitted during alignment Check Both Directions This is the default setting When the alignment calculation is performed the sequences are checked for best alignment in both the forward and complement reverse directions Check Current Direction When the alignment calculation is performed the sequences are checked for best alignment in the direction in which the sequences were imported 4 Click on the Algorithm button The ALFwin Sequence Alignment Algorithms dialogue opens Available types of algorithms can be set here The general sequence alignment algorithm considers only two s
29. Windows 95 operating system to make your use of ALFwin Sequence Analyser as effective and intuitive as possible It is assumed that you have a working knowledge of M icrosoft Windows 95 and you arerecommended to read appropriate operating system literature for further information A basic summary of useful Windows 95 operations are presented in Appendix B Introduction 1 1 2 2 Menu commands M any of the menu commands in ALFwin Sequence Analyser can be activated using the toolbar buttons keyboard shortcuts and the right mouse button menu The availability of these command options is dependent on the active field or window in which you are currently working Thefunction of a toolbar button is displayed when you place the mouse pointer over a button Keyboard shortcuts are summarised in Appendix D Right mouse button menu commands are quickly found through use of the program 1 2 3 Tip of the day The Control and Sequence Analyser modules always start up with a Tip of the Day dialogue to give you useful tips on using the program If you do not want to view this dialogue at start up deselect the Show Tips at StartUp option To view tips of the day you can select the Tip of the Day command in the Help menu 1 2 4 On line Help ALFwin SequenceA nalyser is supplied with a context sensitive O n line help If you want an explanation about a specific dialogue in which you are working either click on the Help button in the dialogue or press
30. an ALFwin ALX ALF data file a powerful feature of the sequence alignment module allows you to select a sample sequence base position in the alignment table and view the corresponding curves in the Curve window of ALFwin Sequence Analyser Note Edit operations that you perform in Sequence Alignment do not affect the original sequence data If you want to edit the original data you must do this in the Curve window of ALFwin Sequence Analyser 6 7 1 Editing a base position The outcome of editing a base position depends on whether the change is made to any of the included sample sequences or to the consensus sequence M oreover the displayed result for the consensus sequence resulting from an edit operation is dependent on the selected consensus mode see Section 6 5 1 W hen you change a base in a sample sequence and the consensus sequence is recalculated the change is reflected in the consensus sequence The edited base is underlined When a base is changed in a consensus sequence the bases at that position in all the sequences are changed to validate the consensus All the sample sequence bases at that position become underlined letters but the consensus base you edited is not underlined To replace a base To replace a base first activate the Replace mode selected by default by m selecting Edit Replace or clicking on the Replace Mode button Click on the letter of the base that you wish to replace and type the alternative l
31. and locate the appropriate filein the displayed dialogue Click on Open to return to the previous dialogue The name of the file is displayed in the Compressed file field The destination path and filename should be automatically displayed if the Tools Add file command is checked If required alter the destination path and folder for the file after decompression Use the Browse option to define the path using the standard Windows 95 dialogues Click on OK to decompress and open the file 5 14 3 Converting ALX ALF files to TIFF Y ou can convert ALF or ALX files into a Tagged Image File Format TIFF that can be imported as a graphic into other software applications This will be viewed as a fluorogram gel 1 2 4 Select Tools Convert ALX ALF to TIFF T he Convert ALX ALF to TIFF dialogue is displayed To open an ALX or ALF file for conversion click on Open and locate the appropriate file Click on Open to return to the Convert ALX ALF to TIFF dialogue for the opened file Select the Lanes that will be included in the TIFF file By default all are selected Specific lanes can be selected by holding down the lt Ctrl gt key Alter any other information as required Start time Stop time sec These fields allow you to change the default range of the sequencing data included in the TIFF file based on the start and stop times that you enter 5 61 5 ALFwin Sequence Analyser Resample Data Type Gutter Lane wid
32. close the dialogue To change set values in the Manual window 1 Type or select the new values in the lower box black characters of the conditions to be changed If you want to cancel these new settings click on Undo This command can only be used before you click on Set Click on Set N ew values are sent to the instrument and can be seen in the Logbook window Actual values green characters will change after a while ALFwin Control 4 Figure 4 25 Manual window in ALFexpress II Note that if you are using ALFexpress the Manual window does not have the Realign Laser button and the Transmittance is expressed as the Laser value 4 6 5 Analysing running samples Y ou can analyse the present run before it is finished Before you open ALFwin Sequence Analyser save the part of the run that is ready 1 Select Run Pre result The Enter name for pre result dialogue is shown 2 Typeaname for the preresult file Choose a different name from that of the present result file 3 Click on Save Note No post run actions will be performed at this stage 4 Start ALFwin Sequence Analyser to check and analyse the pre result 4 45 7 win Control 4 46 Locate and select ALFwin Software from the Windows 95 Start menu options In the ALFwin Administrator dialogue select your user name and then Sequence Analyser 2 00 from the Available applications list Click on Start and ALFwin Sequence Analyser starts see Chapter 5
33. condition changes select it in the Commands field and click on Remove 4 4 3 Sample Information tab This tab allows you to enter information about the samples clones and the lanes in the gel Information is entered in a tabular format which allows standard editing procedures to be implemented so that you can quickly enter and edit information in the clone and lane sample information tables Normally sample information is only retained in the result file but you can use the Store table information in casebook file option to retain sample information as part of the casebook file ALFwin Control 4 Adding sample information for the clones 1 From the Casebook dialogue select the Sample Information tab In Samples Information select the Clones tab Casebook Seqdem1 cbk x File Information Conditions Sample Information Post un Actions Notes Excl Lanes Apply to All Store table I information in casebook file Figure 4 12 Casebook dialogue Sample Information Clones tab 2 Edit theinformation in thethree default columns for sample Name and Comment Several basic functions are available to add information select the cells and copy and paste information between cells Adding information To fill in information Select a cell by clicking on it and about your lanes type in the information To edit the Lane Order Select the lane order cell Click on the drop down
34. default Dataset required Raw or Processed 4 To effect the change without closing the dialogue click on Apply To close the dialogue and effect the change click on OK 5 5 17 Changing modes in orientation view If the raw data for a selected clone have been processed it is possible to view sequence information in three different modes within the orientation view of the Curves window To change mode for one clone select the Curves window for that mode and click anywhere in the orientation view Select one of the following ALFwin Sequence Analyser 5 Command Effect View Curves m This is the default mode Figure 5 11 Orientation view Curves View Sequence Bar In this view ambiguities are shown with higher bars than other bases Usethis to help find ambiguity positions Figure 5 12 Orientation view Sequence Bar View Sequence Text Bi Damai aha Clonal MF fira Group 3 TE AB T ET CME ME GAT COS HA IM OTA ou R E eg es Group 10 i i ph ena kiti as erati H13 fir THOR ECR ACTCT AMAER OBECDC OETA CrbAt CTOHA TURE CEPE ARETO TECHE ACh EP PEDIG Ek TOA TORET TEEET ZECES Theor GEZOS Ce CHITE Sequence text can be viewed in groups of three five or ten bases depending on the Figure 5 13 Orientation view Sequence Text selected option grouped 3 5 10 from top to bottom 5 21 5 ALFwin Sequence Analyser 5 22 To change mode for all open Curve windows do the follo
35. factor to the default level select View Reset Zoom or the lt 9 gt key 5 5 7 Stacking the curves To stack the curves in the curve view select check the View Stack Curves command or click on the Stack button Select either of these commands again to toggle back to overlapping curves 5 5 8 Selecting individual base curves for viewing By default all the base components are shown in the curve view Section 5 5 8 describes how to change this default display To view selected curves only in the curve view deselect uncheck unwanted base components using View Include Curves 5 5 9 Viewing complementary or reverse curves Y ou can display the complementary and or reverse curves for the run data Select check View Complement to view the complementary data curve and or select check View Reverse to view the curve for reverse data Alternatively to simultaneously view complement and reverse data curves View Comp and Rev selects both To return to the default view select uncheck the option s again 5 5 10 Arranging Curve windows If you have opened several Curve windows select the one that you want to view in the Window menu Alternatively there are several alternative ways to arrange the display of all the windows 5 15 5 ALFwin Sequence Analyser 5 16 Command Effect Window Cascade This places all open windows on the ALFwin Sequence Analyser workspaceon top of oneanother The last selected window is display
36. first sequencing run analyse the data and print a report This will introduce you to the three ALFwin Sequence Analyser modules Administrator Control and Sequence Analyser Y ou will gain practical experience of using ALFwin Sequence Analyser with an instrument to obtain sequencing data For the purposes of the first run you should use the R eady T o Go ALFexpress M 13 Ladder order no 27 4560 80 which you can obtain from your local Amersham Biosciences representative Read through this chapter carefully before making a run Consult the appropriate instrument user manual for information about the instrument you are using 2 1 Before starting a run 2 1 1 Safety Before starting a run it is essential that e you know how to prepare the instrument for a sequencing run You should follow the relevant user manual for the ALF family analyser with which you are working e you are familiar with the safety information concerned with using the instrument and the use and disposal of hazardous chemicals Attention If you have any doubts or questions concerning aspects of safety contact your local Amersham Biosciences representative 2 1 2 Pre run activities Before you start your first run the following must be ready e the gel cassette for the instrument must be assembled and the gel freshly cast and polymerised in the cassette e sufficient running buffer 2 litres must be prepared for the instrument e thelower buffer rese
37. info window 5 24 show legends 5 24 show line 5 24 tiling all curve windows 5 16 viewing raw or processed data 5 12 working in the Curve window 5 10 curves amplitude information 4 46 arranging curve windows 5 15 changing the look of the curves 4 48 colour selection 4 55 curve view 4 48 curve width 5 23 cutting clone curves 5 44 displaying stacked or overlapped 4 49 exporting in M etafile format 5 49 font selection 4 55 magnification in curve view 4 50 presentation of the curves within the curve windows 5 19 printing a curve 5 57 selecting curves for viewing 5 15 5 20 stack curves 5 19 stacking 5 15 viii Index types of curves 5 31 viewing complementary or reverse curves 5 15 width 5 23 zooming 4 50 cut clone 5 44 cutting clone curves 5 44 D data automating data export 4 30 processing options in Analyser overview 5 1 save in alf format 5 60 data processing overview 5 30 reviewing the results 5 33 dataset 5 20 decompress ALF File 5 58 5 61 default folder 3 2 user 3 2 define custom colour 4 56 delete bases 5 40 5 43 column header 4 24 group 4 53 tools 5 60 user 3 4 demo instrument 3 7 demonstration instrument 3 6 connecting 3 6 denaturing the samples 2 10 department address 3 3 detector view 4 48 dialogue Add or Edit User 2 3 3 2 3 3 3 4 ALFwin Administrator 2 2 ALFwin Edit System Settings 3 5 3 6 ALFwin Select System 4 4 ALFwin Administrator 3 1 4 3 5 4 Appearance tab of Gel Pr
38. insertion To insert bases to the end of a sequence Y ou can usethe mouse method described aboveto add bases to the end of a sequence Alternatively 1 Select the last base in a sequence 2 Insert the same base 3 Select the last base using the cursor key and moveit to the right by using lt Ctrl right cursor key gt 4 Insert the first additional base 5 Select the last base again and shift it to the right as in 3 above 8 ALFwin Sequence Analyser 5 Insert the next additional base Repeat steps 5 and 6 until all the additional bases have been inserted Delete the last base 5 8 3 Moving a base To adjust the position of a base to fit the curve better 1 2 Click on the Curve window to make sure that it is activated Select Edit Edit Sequence or press the lt Ctrl E gt keys to activate the edit mode The Edit Toolbar is shown Select the base to be moved M ove the selected base by dragging it with the mouse or by clicking the move base right left buttons on the Edit Toolbar or by using the lt Ctrl cursor gt keys right left Y ou can move the last moved base back to its original position by clicking the Undo button on the Edit Toolbar or by selecting Edit Undo Move or press the lt Ctrl Z gt keys To exit edit mode deactivate Edit Edit Sequence or click on the Close button in the Edit toolbar 5 8 4 Deleting a base To delete a base 1 2 Click on the Curve window to make
39. lane near the assumed position and no peaks in the other lanes the symbol corresponding to the lane is chosen If instead a significant peak is found near the position in more than one channel an ambiguity symbol is chosen If there are peaks in all four channels near the position it is considered a full stop and the symbol N is chosen Update parameters Upright end point Stop point The base calling algorithm C If there are no significant peaks near the position or if neighbouring peaks are too close the existence of a base is questioned and a lower case version of the symbol is chosen This is called a frame ambiguity A peak is considered to be near a position if its height at the position is high enough or if it has its maximum close enough to the position After each base has been assigned the parameters are re tuned so that they are adapted to local changes in the signals The algorithm estimates the point in the sequence after which the error frequency might become bigger than 1 This point is called the upright end point At this point at least one of the following criteria is satisfied The peaks in the raw data are too poorly resolved There are too many ambiguities in a subsequence of a certain interval Therearetoo many ambiguitiesin a limited interval after a frame ambiguity The peaks are not well resolved and thereis a long interval without any well shaped and resolved peaks Af
40. menu button and select the lane order required 4 21 7 win Control To move between cells Selecting cells To select a single cell To select a number of separate cells To select a block of cells To select a column of cells To select a row of cells To select the entire table Use the left and right lt cursor gt keys to move around in the cell Use the lt T ab gt key to jump to the cell in the next column or lt Shift Tab gt to jump back to the adjacent cell in the previous column Also use the up and down lt cursor gt keys to move within a column Click on it or move to it by using the lt tab gt keys or up and down lt cursor gt keys H old down the lt Ctrl gt key and click the cells Click on a cell at one corner of the block Hold down the lt Shift gt key and click the cell located in the opposite corner of the block Alternatively click on one cell and drag select the remainder M ove the mouse pointer into the header at the top of the column The pointer turns into a black arrow Click on the left mouse button M ove the mouse pointer into the margin at the left of the row The pointer turns into a black arrow Click the left mouse button Click in the upper left corner of the table ALFwin Control 4 M ovingand copying cell contents To move the contents of Select the cell s to be moved and one or more cells press the lt Ctrl X gt keys Select the cell located at the
41. name Demo 001 alx Files of type ALF files alf alx hd Cancel Figure 5 5 Open dialogue for selecting result files The Result window see Figure 5 6 is displayed in the ALFwin Sequence Analyser workspace El SeqDemo alx Result of x SeqDemo alx et CloneQ1 Smt DNA RT Clone02 Smt DNA AT Clone03 Smt DNA RT Clonel4 Smt DNA RT CloneO5 Smt DNA RT CloneQ6 Smt DNA AT Clone0 Smt DNA RT Clone08 Smt DNA RT CloneO9 Smt DNA RT Clone10 Smt DNA Open Open All Help Figure 5 6 Result window ALFwin Sequence Analyser 5 3 Repeat step 1 if you want to open more result files Note All opened result files are visibleon the ALFwin Sequence Analyser desktop This means that you can open other windows for example Curve windows see Section 5 4 1 for any of the clones in the result files 4 Switch between open windows in the Window menu Note In ALFwin Sequence Analyser you can open an flx file but you can not save results in flx format 5 4 Presenting results in the Result window The Result window contains a tree structure of all the clones contained in the opened result file From this window it is possible to open the curves for each clone see Section 5 4 1 and to process the data see Section 5 7 5 4 1 Clone icons In the Result window each clone is represented by an icon to indicate w
42. new folder in this dialogue using the standard Windows 95 functions Click on Open to complete and close this dialogue 4 4 2 Conditions tab Theinformation in this tab specifies the run settings for the ALF family instrument controlled by the computer when the run starts and the settings able to be set are dependent on the instrument connected These settings specify the run time temperature the voltage current and power limits and the sampling interval T hese settings can be programmed to change during the course of a run You must complete any editing of run conditions in the casebook before you start a run Editing run conditions 1 From the Casebook dialogue click on the Conditions tab Fie iriomaton Condition faepie inkaras Postar Acer Hoten Fir ira ron mi pH ST Tawiri z b Winge mo 6 Ry Cae Fe To Pa a Teepe F THA I Larve haan bie na fulorgic ondon com 5 wp aed Aare rac Commeandt Figure 4 9 Casebook dialogue Conditions tab 2 Edit the run conditions as required Y ou can use the lt Tab gt key to move between fields 7 win Control N ote The Voltage Current and Power settings specify the maximum values that can not be exceeded during the run It is usual to select a limiting value for one of the three settings for example Power and to specify high non limiting values for the other two settings Voltage Current Power Run time Temperature Sample int Leav
43. of consensus 6 16 display of consensus line 6 11 display of consensus sequence 6 13 exporting the consensus sequence 6 33 initiating a recalculation manually 6 16 introduction to consensus sequence 6 14 least common denominator mode 6 14 majority mode 6 15 modes 6 14 restoring automatic recalculation 6 16 contrast in Gel View adjustment 5 28 scale all lanes together 5 29 scale each lane separately 5 29 Control automatic condition control during run 4 18 4 19 connecting an instrument 3 5 controlling voltage current and power 4 18 exit from Control 4 60 launching from Administrator 3 7 run controlled by computer 4 38 running two instruments 4 5 starting ALFwin Control 4 3 starting ALFwin control 2 4 vii Index starting from ALFwin Administrator 4 3 starting from Sequence Analyser 4 4 convert ALX ALF to TIFF 4 37 5 58 5 61 copy Clone 5 52 clone or Gel View 5 52 current electrophoresis 4 18 cursor rectangular cursor in sequence alignment overview 6 11 Curve View window contents of Curve window 4 6 Curve window arranging Curve windows 5 15 base curves 5 12 changing the colour 5 23 changing the font 5 23 changing the scale 5 18 configuring the sequences 5 24 displaying signal levels 5 39 linking curves 5 16 navigating the Curve window 5 14 opening curve windows 5 11 opening layout 5 25 orientation view selection 5 22 presentation of the curves 5 19 saving layout 5 25 scrolling the curve 5 14 show
44. on its Curve window to make sure that it is activated 2 Select Edit Cut Clone The Cut Clone dialogue is shown Cut Clone Demo 001 alx Clone EG Start Xi E z End 800 09 E I Cut on all clones in the result file Cancel Help Figure 5 23 Cut Clone dialogue ALFwin Sequence Analyser 5 3 Usethis dialogue to set the time range of the part of the clone that will be preserved Data outside the limits set in the Start and End fields will be deleted Click in the Start field and enter the start time in minutes and seconds Y ou can enter the time by typing or you can click on the start point in the Curve window Positioning in the Curve window can be aided by first selecting View Show Line to display a vertical cursor line at the mouse pointer 4 Click in the End field and enter the time as above 5 Select Cut on all clones in the result file if you want to apply the same cut to all the clones in the result file 6 Click on OK The Verify Cut Clone warning is shown 7 Click on OK to cut the clone If you wish to abandon a cut before confirming OK click on Cancel in the Cut Clone or Verify Cut Clone dialogues If you wish to reverse a cut that you have made close thefile without saving it and then open it again Any unsaved changes you have made to the file will be lost 5 9 Viewing casebook information and adding evaluation notes A copy of the casebook information is included in the result f
45. or combination of A C G T buttons to set the type of the selected base Insert a base to the left of the currently selected base and select the new base I E Delete the currently selected base and select the next base to the right Es M ove the currently selected base to the left M ove the currently selected base to the right Undo the last action Bee a celt ALFwin Sequence Analyser 5 Edited clones areidentified by a Ae pen symbol in their ACGT icons in the Result window Edited bases are underlined in the Curve window if Show Edited Bases is selected in the View menu or in the Sequence tab of the Curve Properties Layout dialogue 5 8 1 Replacing a base To replace a base 1 2 Click on the Curve window to make sure that it is activated Select Edit Edit Sequence or press the lt Ctrl E gt keys Replace Base is selected as default and the Edit Toolbar is shown M ove the mouse pointer over the sequence base in the Curve window The pointer turns into a text caret Click on the base component letter to select it Replace the base component by clicking on the appropriate base component button in the toolbar or type the base letter Selecting more than one base component will automatically give the appropriate ambiguity code see Appendix C The base component buttons toggle between on and off for the respective bases at least one base must be on Y ou can undo the most rece
46. pointer in the centre of the rectangular cursor press and hold the left mouse button and drag the rectangular cursor back and forth to select the appropriate area of the curve 2 Release the mouse button Alternatively click in the orientation view outside of the rectangular cursor or use the page up page down keys to scroll quickly Use the left and right scroll arrows to scroll only a few bases at a time 5 5 6 Using the zoom function in the Curve window Y ou can alter the magnification of the Curves view in the Curves window Changing magnification with the zoom commands Command E ffect View Zoom In Successively increases the magni lt 7 gt key fication View Zoom Out Successively decreases the magni lt 8 gt key fication Changing the magnification in orientation view Y ou can change the magnification of the curves by altering the size of the rectangular cursor in the orientation view 1 Position the mouse pointer over the left or right edge of the rectangular cursor The pointer becomes a bi directional arrow to the left and right ALFwin Sequence Analyser 5 2 Press and hold the left mouse button and drag the border inwards or outwards A larger rectangular cursor will reduce the magnification of the curves displayed in curve view and conversely a smaller rectangular cursor will increase the magnification of the curves 3 Release the mouse button Resetting the magnification To reset the magnification
47. shifted to another window click on the pin button icon in the top left corner of the dialogue The pin button icon then appears depressed To remove the effect of pinning click on the pin button icon again so that it appears to pop out or elseclick on the dialogue close button in the top right corner Y ou can switch the auto display function on and off by selecting View User Preferences and selecting or deselecting Auto Display of Statistics Popup Table To again display the alignment statistics select View Alignment Statistics The ALFwin Sequence Alignment Statistics dialogue opens 6 6 2 Explaining the alignment statistics The Sequence Alignment Statistics dialogue can be used to view statistics for the whole alignment or for a specifically selected sequence T hisis determined in the Sequence drop down list which by default shows Total to indicate that the displayed statistics are for all sample sequences To view the statistics for a specific sequence click on the drop down list and select a specific sequence Alignment quality An overall measure of the quality of the alignment is also provided through the three parameters Mean of Pairwise Alignment Ratios Maximum Pairwise Alignment Ratio and Minimum Pairwise Alignment Ratio A pairwise alignment ratio is calculated in the following manner The maximum total score value from the forward and complement reverse alignments is divided by the smallest length of the two sequenc
48. shown 4 Click on Save 4 13 7 win Control Note If you edit a casebook during the run you are warned that the changes are only saved in the result file You can switch this warning off by selecting Don t show this warning in the future and clicking on OK 4 3 6 Printing a casebook A casebook can be printed before starting a run 5 1 Complete the editing of the casebook prior to starting a run Select File Print Casebook or press the lt Ctrl P gt keys or click on the Print Casebook button The Print Casebook dialogue is shown m Select info to print M Conditions I File info IV Sample info clone M Sample info lane V Casebook notes Cancel Help Figure 4 7 Print Casebook dialogue 2 Select the parts of the casebook to be printed 3 Click on Print The Print dialogue is shown Note You can also print out the casebook once the run has ended and the result file has been automatically saved by selecting File Report in Sequence Analyser see Section 5 12 4 3 7 Print Setup Printing is made according to the print setup parameters that you have defined for the current printer in Windows 95 Y ou can alter the print setup by select Settings Printers from the W indows 95 Start button In the Printers dialogue select the appropriate printer icon and select File Properties M ake the appropriate selections Note The same print setup is used to print the post run report ALFwin Control 4
49. signal detection sensitivity of the curves for the area of the result that you are viewing in the Curve view View Scale Fixed This displays the portion of the curve that corresponds to minimum and maximum ait amplitude limits that you have defined in the Curve Properties dialogue see below To change the scale for the Curve window 1 Click on the Curve window 2 Select View Properties and click on the Settings tab in the Curve Properties dialogue Note If you change the properties the changes will affect all curve groups ALFwin Control 4 3 Select the relevant Y axis Scale either Separately or Together see above or Fixed If you select Fixed you must enter a minimum and maximum amplitude value e g an entered range of 0 to 50 means that the portion of the curves with amplitude between 0 and 50 of the possible range is shown 4 Ifyou want the scale to befixed according to the current scale area of the automatically calculated scales i e as calculated for Separately or Together check the Use current scale area box Whenever you select Fixed you can scroll along the result and view the peaks at their real height relative to the maximum amplitude for the whole run 5 To implement the changes without closing the dialogue click on Apply To close the dialogue and effect the changes click on OK Vers Baigi 5p Fie Curas recie Daciu mad Seas Seas Arae C joar Pim igrae fF E Eoria fomi
50. sure that it is activated Select Edit Edit Sequence or press the lt Ctrl E gt keys to activatethe edit mode The Edit Toolbar is shown Select the base to be deleted D elete the base by clicking on the Delete Base button on the Edit Toolbar or by selecting Edit Delete Base or by pressing the D a gt key Y ou can undo the most recent delete by clicking the Undo button on the Edit Toolbar or by selecting Edit Undo Delete or by pressing 5 43 5 ALFwin Sequence Analyser 5 44 the lt Ctrl Z gt keys 6 To exit edit mode select Edit Edit Sequence to deactivate it or click on the Close button in the Edit toolbar 5 8 5 Showing or hiding edited bases Edited bases are underlined by default Section 5 8 5 describes how you can change this default setting To hide this underlining 1 Click on the Curve window to make sure that it is activated 2 Select View Show Edited Bases The underlining is hidden R epeat the above to show the underlining again 5 8 6 Cutting clone curves The first and last parts of a run do not always give meaningful information To reduce the size of the result file you can cut selected parts of the clone Warning W hen you cut a clone and save the file the deleted data is lost Take care when making a cut It is prudent to save the file before applying a cut so that you can if necessary later return to the saved uncut version To cut a clone 1 Select the clone to be cut and click
51. that can be performed during a run 4 6 1 Pausing therun 4 6 2 Manually ending the run 4 6 3 Reloading the gel 4 6 4 Changing the run conditions 4 6 5 Analysing running samples 4 6 6 Viewing curve amplitude information 4 6 7 Writing run notes in the casebook 4 6 8 Starting a run on another instrument 4 7 After the run is finished 4 8 Changing the screen layout 4 8 1 Arranging open windows 4 8 2 Saving window positions on exit 4 8 3 Hiding and showing the toolbar and status bar 4 8 4 Changing the look of the curves 4 8 5 Showing stacked or overlapping curves 4 8 6 Using the Zoom function with curves 4 8 7 Selecting the group to be shown 4 8 8 Creating your own lane grouping 4 8 9 Editing a group 4 8 10 Deleting a group 4 8 11 Changing the Y axis scaling 4 8 12 Changing fonts and the colours in the Curves window Contents 4 8 4 8 4 9 4 11 4 12 4 13 4 14 4 14 4 15 4 15 4 17 4 20 4 28 4 37 4 38 4 39 4 41 4 42 4 43 4 43 4 43 4 44 4 45 4 46 4 46 4 47 4 47 4 47 4 47 4 47 4 48 4 48 4 49 4 50 4 51 4 51 4 52 4 53 4 53 4 55 Contents 4 8 13 Saving or opening a layout for the Curve window 4 8 14 Changing the appearance of the M anual window 4 8 15 Changing the font in the M anual window 4 8 16 Changing the font in the Logbook window 4 9 Exiting ALFwin Control 5 ALFwin Sequence Analyser 5 1 Starting ALFwin Sequence Analyser 5 1 1 Starting AL Fwin Sequence Analyser
52. that no residual daylight signal is saved from the detectors 4 6 2 Manually ending the run To manually end the run before the run time is completed 1 Select Run End or click on the End button A dialogue asks you to confirm you want to end the run 2 Click on Yes The run ends and a dialogue asks if you want to perform post run actions 3 Select Yes or No 4 6 3 Reloading the gel Y ou usethe Reload command to end a current run and start anew run using the same gel Reload allows access to the Casebook dialogue so that you can edit the casebook before restarting the run A reload is automatically documented in the logbook 4 43 7 win Control 4 44 Select Run Reload A warning dialogue is displayed asking you to confirm your action Click on OK to proceed Post run actions are executed and the Samples Standards tab of the Casebook dialogue is shown Use the Casebook dialogue to edit the prerun notes and samples standards as appropriate Click on OK to close the casebook The Start Run dialogue is shown Check the result file name and click on OK Reloading is noted in the logbook in the form Reload x where x is incremented for each reload 4 6 4 Changing the run conditions Warning Do not exit ALFwin Control or shut off the computer during the run otherwise the data from the run will not be saved If you try to exit ALFwin Control you are warned that you cannot do so during a run Press OK to
53. the displayed texts may also be reduced to display more information 4 2 1 Windows The following windows are visible The Curve View window This provides a real time graphical view of the lanes from the run The Manual window This shows the experimental setup conditions in black and the present measured values in green The Logbook window This logs all information pertaining to the current run 4 2 2 Bars The following bars are available Title bar This shows information about the instrument to which ALFwin control is 4 6 ALFwin Control 4 connected the name of the open casebook or result file and the status of the run M enu bar Click on any menu option to view and select the specific commands Toolbar The toolbar buttons allow you to quickly implement the most commonly used commands The range of active buttons is dependent upon the currently active window in the Control workspace Unavailable buttons are greyed out U sethe mouse pointer to get a description of each button Y ou can hide the toolbar by deselecting unchecking the active View Toolbar command DSA Preset Start Pause Continue End Group 1 Al Ee ze Rim amp oO 1 ALFwin Control Instru Status bar This gives you detailed information about for example the position of the mouse pointer and current mode selected on the screen Y ou can hide the status bar by deselecting unchecking the active View Status Bar comma
54. time shifts will be used correctly See also Section C 2 2 Setting the stop point ensures that no processed data or sequence will be generated after that point This saves disk space when storing the results Situations when it can be useful to set the stop point manually include e when it is known that there is no data of interest after a certain time for example when the samples are PCR fragments with a limited maximum length when the data are not interpretable after a certain time The base calling algorithm C C 2 2 When to manually adjust the time shift before processing If there are differences in the mobilities of the DNA fragments in different lanes belonging to the same clone the base calling program may have problems in automatically compensating for the resulting time shifts between the A C G and T signals This problem is most common in the outermost clones of the gel In such cases it is appropriate to manually adjust the shift before processing the clone It is often a good idea to manually set the wanted start point for the processing to ensure that the base calling starts in the interval where the shift has been adjusted To make the base calling program use the manually adjusted shifts Use applied shift must be selected in the Process Setup window before the processing is carried out You can also select Use these shifts and enter the required shifts from the keyboard This function can be used to reproduce an ea
55. top left hand corner of the target position and press the lt trl V gt keys To copy the contents of Select the cells to be copied and one or more cells press the lt Ctrl C gt keys Select the cell located at the top left hand corner of the target position and press the lt CtrI V gt keys To deletethe contents of Select the cell s to be cleared and one or more cells press the lt D el gt key or press the lt Ctrl X gt keys To copy the contents of Select the cell to be copied Click on one cell to all the cells in Apply to All the column W here appropriate you can add up to another four columns to the table To add further columns click on the Table Settings button The Clones dialogue opens Column labels Add Comment Lane Order Edit Deaete Move Down Figure 4 13 Edit Columns dialogue 4 23 7 win Control To add a column To edit a column header To delete a column To change the order of added columns Click on Add Typethenameof the column header in the dialogue and click on OK Select the header and click on Edit Type the changes in the dialogue and click on OK N ote that you cannot edit the column headers for the three default columns Name Comment and Lane Order Select the column by clicking its name in the Column labels field Click on Delete then click on OK to confirm the deletion N ote that you cannot delete the three default columns Name Com
56. you have selected another window click on the pin button icon Theicon is depressed To cancel the dialogue click on the pin button icon again so that it pops out Select another window and the dialogue automatically disappears Alternatively click on the dialogue close button in the top left corner e The automatic display of IUPAC codes when an ambiguity is selected in the alignment table can be suppressed by selecting unchecking View User Preferences Auto Display of Ambiguity Popup Table e If the dialogue is hidden from view select View IUPAC Codes 6 6 Sequence alignment statistics The sequence alignment module provides a statistical overview which you use to assess the quality of the alignment The statistics allow a quantitative assessment of the initial alignment and can be used to measure the effect of any adjustment you make to the data 6 6 1 Displaying the alignment statistics The statistics overview is displayed automatically when alignment processing is completed a Mean of Pairwise Alignment Ratios o7ast Maximum Pairwise Alignment Ratio 3836 Minimum Pairwise Alignment Ratio 506 m Sequence Specific Statistics Sequence Total Number of Mismatches Number of Ambiguities Number of Gaps WT Number of Edit Operations Figure 6 8 Sequence Alignment Statistics dialogue 6 17 6 Sequence Alignment To make the statistics window stay on screen as the focus is
57. you have used an earlier version of Windows B 1 Starting Windows 95 and logging on 1 Turn on the computer and Windows 95 will be automatically started 2 If your computer is connected to the network you will be prompted to enter your user name and password Enter these and click on OK Alternatively if you do not want to be connected to the network click on the Cancel button Y ou will now only be able to work locally using the computer s hard disk 3 You may also beasked to supply your user name and password for Windows 95 if you have supplied these before H owever if you areusing the computer for thefirst time you must enter a user name and password in the User name dialogue If you do not want enter the password each time you start the computer leave the Password field blank and click on OK When prompted to confirm the password leave all fields blank and click on OK again B 2 What is on your screen Depending on how your computer is set up various items appear on your screen when you start Windows 95 H ere area few of the important items B 1 B Short guide to Windows 95 El My Computer 27 My Computer Eh Network Neighbourhood RE zj Network Neighborhood Start button Windows 95 taskbar Doubleclick this icon to view the hardware and software content of your computer If your computer is connected to the network double click this icon to see available network resources Y ou can click t
58. your requirements Temporary configuration using View Consensus see Section 6 5 and View Amino Acid Sequences see Section 6 8 has already been described When ALFwin Sequence Analyser is closed the current settings become the default settings for the next session The following operations use the View Properties options to change the default configurations 6 15 1 Configuring the consensus mode The two consensus modes are described in Section 6 5 1 To change the default consensus mode 1 Select View Properties to open the ALFwin Alignment Properties dialogue and select the Alignment Item Properties tab 2 Usethe Consensus Mode buttons to select Least Common Denominator or Majority Sequence Alignment 6 6 15 2 Configuring the amino acid display The amino acid display options are described in Section 6 8 To change the default display of amino acid sequences 1 From the ALFwin Sequence Alignment dialogue select View Properties T he ALFwin Alignment Properties dialogue is shown 2 Select the Display Properties tab Alignment item propemes Dinis propis Ghee Fons Sample Raut Amno iod Sequences ahea ts Replace kierbos Charter F Pagesdinegy Frere 1 CONE eLiglaglt r l Pemra l Fapesding Frer T dira Latter Cadi Thea Leie Codes Figure 6 18 Alignment Properties dialogue Display Properties tab 3 Usethe dialogue to select the reading frames required 4 Select One Letter Codes or
59. z m a r he cani mak ama PF Eimi gurs elat Sear Biaiy GET PTT TTT TT Figure 4 31 Curve Properties dialogue Settings tab 4 8 12 Changing fonts and the colours in the Curves window Y ou can change the colour of the curves and the background Y ou can also change the font for the X axis and legends To change colours 1 Select the Curve window 2 Select View Properties and click on the Style tab in the Curve Properties dialogue 4 55 7 win Control Vern Seminge Sto Fie Figure 4 32 Curve Properties dialogue Style tab To change colour select the appropriate item under the Colour list and click on the Set Colour button The Colour dialogue is shown Select a colour To make your own colour first define the colour and then click on Add Custom Colour Click on OK To implement the changes without closing the dialogue click on Apply To close the dialogue and effect the changes click on OK Note Only the solid colours can be used for curves printed and exported to clipboard To change the scale and legends font 1 2 Click on the Set Font button The Font dialogue is shown Select a Font Font style and Size Click on OK To effect the changes without closing the dialogue click on Apply To close the dialogue and effect the changes click on OK ALFwin Control 4 If you want to restore the default font and colour settings click on the Default Settings button 4
60. 3 Contents 5 13 Printing Curve and Gel Views and sequences 5 13 1 Printing the contents of a single view 5 13 2 Printing all views 5 14 Using tools 5 14 1 The alx and alf formats 5 14 2 D ecompressing alf files 5 14 3 Converting AL X ALF files to TIFF 5 14 4 Add file 5 15 Exiting ALFwin Sequence Analyser 6 Sequence Alignment vi 6 1 Creating a sequence alignment item 6 1 1 Creating a new sequence alignment item 6 1 2 Adding further sequences before alignment 6 1 3 Changing the order of sequences 6 1 4 Removing sequences 6 2 Aligning the sequences 6 2 1 Aligning all sample sequences 6 2 2 Aligning additional sequences to the previous alignment 6 3 The alignment overview 6 3 1 Sample sequence lines and alignment direction 6 3 2 Consensus line 6 3 3 Rectangular cursor 6 3 4 Alignment imperfections 6 4 The alignment table 6 4 1 Sample sequences 6 4 2 Consensus sequence 6 4 3 Alignment direction 6 4 4 Alignment imperfections 6 4 5 Adjusting the alignment table 6 5 The consensus sequence 6 5 1 The consensus modes 6 5 2 Recalculating the consensus sequence 6 5 3 Viewing the IUPAC codes 5 57 5 57 5 57 5 58 5 60 5 61 5 61 5 62 5 63 Pa P AMA P Oo oo NNN A 1 h o 6 10 6 11 6 11 6 11 6 12 6 13 6 13 6 13 6 13 6 14 6 14 6 14 6 16 6 16 Contents 6 6 Sequence alignmeni statistics 6 17 6 6 1 Displaying the alignment statistics 6 17 6 6 2 Explaining the alignm
61. 38 settings for report 2 18 sharpen peaks C 2 shift auto shift 5 32 recalling previously applied shifts 5 38 shifting curves manually 5 37 Shift A C G T column 2 15 Index shortcut keys 1 4 to other programs 5 58 show edited bases 5 24 info window 5 13 italics 5 36 legends 5 13 line 5 13 tips at startup 1 3 4 4 vertical cursor line 5 13 showing or hiding axes and bases in Gel View 5 30 edited bases 5 44 shut down option from Windows 95 start button B 3 Signal Level table 5 39 signal levels 4 46 displaying in Curve window 5 39 displaying in curve window 5 19 small letter bases assigning upright status 5 36 why they are shown 5 35 split file 5 47 stacking curves 4 49 5 15 5 19 STADEN SCF export format 4 31 import 6 38 STADEN SCF export format 4 31 5 50 E 1 standard routines location 3 2 start button in Windows 95 B 2 start instrument 4 4 start label for bases 6 25 start point C 2 Start Time and Stop Time columns 2 14 starting arun 4 38 ALFwin 3 1 ALFwin Control 2 4 ALFwin Sequence Analyser 5 3 Windows 95 and logging on B 1 statistics configuring auto display 6 17 displaying the statistics 6 18 in sequence alignment 6 17 status bar hiding and showing 4 48 in ALFwin Control 4 7 in Sequence Analyser 5 6 Status column 2 14 xxiii Index XXiV step change at 4 20 Step change at button 4 19 step conditions 4 18 4 19 stop point C 3 Store 4 26 store table information in casebook file 4 20 4 25
62. 4 32 formats 1 2 formats for sequence export 4 31 5 50 E 1 xi Index xii M SF format 6 32 PHY LIP Interleave format 6 32 selected sequences 6 33 selecting the alignment export format 6 33 selecting the filenames 5 51 selecting the files for export 5 49 selecting the sequence modeto be exported 5 51 selecting the types of bases for export 5 51 sequences 5 49 Windows Metafile 1 2 F Fl key 1 3 features of ALFwin 1 1 file backup automating 4 36 information 4 15 information tab in casebook detail 4 15 information tab in casebook overview 4 8 layout 2 18 selecting the clones for a split file 5 48 split file 5 47 file type calf 6 38 alx 6 38 cbk 4 10 dyt 4 57 5 25 spt 4 33 5 53 wmf 5 49 find next 5 36 find option from Windows 95 start button B 3 find previous 5 36 finding ambiguity positions 5 35 special sequences 5 34 5 35 first run 2 1 folder Backup Folder 3 2 Create N ew Folder button 3 3 default 3 2 home 3 2 standard routines 3 2 font changing font in Curve window 5 23 in curve window 4 55 logbook window 4 59 manual window 4 58 Index format for alignment export 6 33 forward sequence direction 6 10 forward sequencing direction symbol 6 13 G gaps in alignment sequences 6 12 6 13 GCG export format 4 31 5 50 E 1 gel checking for dust particles 2 6 preparing loader tips 2 10 printing a gel 5 57 gel cassette mounting 2 5 Gel View adjusting magnification 5 27 adjusting magnifi
63. 5 O ther options 2 3 6 Viewing casebook information and adding analysis notes 2 3 7 Printing a report 2 3 8 Saving the whole result file 3 ALFwin Administrator 3 1 Starting ALFwin Sequence Analyser 3 2 Adding a user 3 2 1 Adding a new user 3 2 2 Editing a user 3 2 3 Deleting a user 3 3 Connecting an instrument to ALFwin Control 3 3 1 Connecting an ALF family instrument 3 3 2 Connecting to the demonstration instrument 3 4 Launching the ALFwin applications 3 4 1 Launching AL Fwin Control 3 4 2 Launching AL Fwin Sequence Analysis 4 ALFwin Control 4 1 Starting ALFwin Control 4 1 1 Starting AL Fwin Control from AL Fwin Administrator 4 1 2 Starting ALFwin Control from Sequence Analyser 4 1 3 Using two instruments 4 1 4 Automatic settings for AL Fexpress family instruments 4 2 Introducing the ALFwin Control interface 4 2 1 Windows 4 2 2 Bars 4 2 3 The light indicator 2 12 2 12 2 13 2 14 2 15 2 16 2 16 2 18 2 18 4 3 Working with casebooks 4 3 1 Introducing casebooks 4 3 2 O pening an existing casebook 4 3 3 Creating a new casebook 4 3 4 Saving the casebook 4 3 5 Editing a casebook beforea run 4 3 6 Printing a casebook 4 3 7 Print Setup 4 4 Modifying casebook parameters 4 4 1 File Information tab 4 4 2 Conditions tab 4 4 3 Sample Information tab 4 4 4 Post run Actions tab 4 4 5 Notes tab 4 5 Performing a run 4 5 1 Preset and prepare the instrument 4 5 2 Starting the run 4 6 Activities
64. 8 13 Saving or opening a layout for the Curve window Y ou can savea Curve window layout for later use All properties of the Curve window defined in the properties tabs are saved If you do not save these the default layout will be applied when you next use ALFwin To save a layout or open a saved layout 1 Select the Curve window 2 Select View Properties and click on the File tab in the Curve Properties dialogue Coat re Prip ei airat tld Ed item Betre Ske A Soe Led te tweet Hey Figure 4 33 Curve Properties Layout dialogue File tab 3 Select a button Open Layout The Open dialogue is shown Use this dialogue to open a saved layout and apply it Save Layout As O pens the Save As dialogue which allows you to save the layout as a lyt file which you can use again later The last used layout file will be applied by default the next time ALFwin Control is started by the same user Use the dialogue to locate and name the layout file 5 Click on OK 7 win Control 4 8 14 Changing the appearance of the Manual window Y ou can select the parameters that are displayed in the Manual window and you can choose the fonts that are used The settings are saved for the current user and are used the next time that ALFwin Control is started Changing the contents of the Manual window Select the parameters to be displayed in the Manual window All parameters are selected as default To select parameters 1
65. ALFwin Sequence Analyser 2 00 18 1125 90 Amersham Edition AA e KY Biosciences Important user information Reading this entire manual is recommended for full understanding of the use of this product Should you have any comments on this manual we will be pleased to receive them at Amersham Biosciences SE 751 84 Uppsala Sweden Warranty and Liability All goods and services are sold subject to the terms and condi tions of sale of the company within the Amersham Biosciences group which supplies them A copy of these terms and conditions of sale is available on request Trademarks ALFwin ALFexpress ALF and ALFred are trademarks of Amersham Biosciences Limited or its subsidiaries In view of the risk of trademark degeneration it is respectfully suggested that authors wishing to use these designations refer to their trademark status at least once in each article The following designations are trademarks owned by other companies GCG the Wisconsin Package TM GCG Genetics Computer Group a subsidiary of Oxford M olecular Group Inc DNASISTM by Hitachi Software Genetics Systems Group PC GeneTM by Oxford M olecular Group Windows 95 is registered Trademark of M icrosoft Corporation Copyright Amersham Biosciences UK Limited 1998 All rights reserved N o part of this publication may be repro duced stored in a retrieval system or transmitted in any form by any means without permi
66. ALFwin Software M enu command cannot be found Problem M enu command inactive greyed out Explanation The wrong window or part of the window is activated Solution Click the part of the window you want to work with by clicking on it The computer is working slowly amp Error messages concerning low memory resources Problem The computer is working slowly Error messages like Out of memory Low memory resources etc may appear Explanation N ot sufficient RAM memory or disk space left You may be running to many applications at the same time Solution Try to close down programs files you are not using If you can not do this then shut down and restart the computer If your computer has a small hard disk lt 300 M B perform theshut down and restart operation on a regular basis or between runs Error messages concerning file handling corrupt program files Problem Error messages like R equired filexx xxx was not found or Error occurred while initialising the algorithm settings appears Solution Re install the ALFwin Software Printing problems Problem Last curve window on each page not properly printed Explanation Insufficient memory installed in printer Solution Increase the memory of the printer or use a recommended printer see Section A 2 For more information on printing problems see the appropriate web site for the computer dealer for example www hp com 7 1 Troublesh
67. B 3 3 Using a dialogue box B 3 4 Sizing windows B 3 5 Closing a window B 3 6 M oving windows B 3 7 Using toolbars B 3 8 Scrolling windows B 3 9 On line Help B 3 10 Creating a shortcut B 4 If you have used an earlier version of Windows w N ius vvu 1 1 o4 1 4 1 1 O dbninn C The base calling algorithm C 1 How the base calling algorithm works C 2 How to use the base calling algorithm C 2 1 When to set the start and or stop point manually C 2 2 When to manually adjust the time shift before processing C 2 3 The different types of processing O 0O00 Oa OO O unk A C 3 Base ambiguity codes D Keyboard shortcuts D 4 ALFwin Control D 4 1 General D 4 2 In the Curve window D 4 3 In the M anual window ovu g Veb 4 viii Contents D 5 ALFwin Evaluation O Aann AWUN N 5 1 G eneral 5 2 In the Clone view in the Result window 5 3 In the Gel view in the Result window 5 4 In the Curve window 5 5 In the Curve view in the Curve window 5 6 In the O rientation view in the Curve window 5 7 In the Edit mode after Ctrl E in the Curve widow 5 8 In the Process Status window 5 9 In the Casebook D 6 Sequence Alignment ooog o JoJo g E Reference list E 1 References for export formats E 1 E 2 Other references E 3 Contents Installing the software Installing the software Y ou should check that your computer complies with the minimum hardware specification requirements detailed i
68. Casebook dialogue Sample Information Lanes tab Lanes that have been excluded are represented by an asterisk and the lanes belonging to each clone have been adjusted accordingly Warning The excluded lanes settings are lost if you close the casebook File Close casebook or open another casebook The excluded lanes settings are only stored in the result file 4 27 7 win Control 4 4 4 Post run Actions tab This tab allows you to specify activities that will proceed automatically when the run is completed T hese include data processing export printing launching tools and file backup The following tasks can be automated Process data Process the raw data and producea processed data file Export a sequence result Convert the data into standard format and save it as a sequence file Print a report Print a report containing selected information Produce a backup file Safeguard the data by saving it ina secondary location Start tools O pen another software application when the processing is complete Note All of these tasks with exception to the production of a backup file can also be accessed from ALFwin Sequence Analyser File Information Conditions Sample Information Post un Actions Notes m Common post run actions _ Application specific post tun actions i Browse IV Auto process data Sep Backup filename ackupt at IV Export Setup Setup Backup path C Pr
69. Click on the Manual window to make sure that it is activated 2 Select View Properties The Manual View Properties dialogue is shown Select the Window Contents tab bianual View Froportios E Window Contents Sr Balaci korr bo lech che ir aire Figure 4 34 Manual View Properties dialogue Window Contents tab for ALFexpress II Note that if you are using ALFexpress Transmittance is expressed as the Laser value 3 Select the items in the list to be displayed in the Manual window Click to activate or de activate each item 4 Click on OK The selected items are displayed in the Manual Window 4 8 15 Changing the font in the Manual window Changing the font in the Manual window adjusts the size of the fields in the window The field for each parameter changes size according to font size ALFwin Control 4 To change the font 1 2 Click on the Manual window to make sure that it is activated Select View Properties The Manual View Properties dialogue is shown Manual View Properties x Window Contents Style Font Select font for controls Figure 4 35 Manual View Properties dialogue Style tab Select the Style tab Click on the Font button and use the Font dialogue to select the Font Font style and Size Click on OK Click on Apply to view the results without closing the dialogue or click on OK to change the font and close the dialogue 4 8 16 Changing the font in the Logboo
70. Curves window for a clone you can easily view the data and directly edit base nucleotides on the screen BAD ACT ALFwin Sequence Analyser 5 When you work with data processing you will be working with five types of curves raw data before processing no bases called raw data after processing bases called shifted raw data processed data shifted processed data 5 7 1 Processing clones To process raw data 1 With a result file open select Analysis Process T he Process Status dialogue opens oO Drak MT T i 1M aasa ta rm jaf Clonee At omnei a ba Lt Goes MT ad Em Eo bt Core aaa bol nol F LP wae ore HI Useeocecred co ie LT LT Cira Hi Lapas wa ro Lt Lt Corel MT rid w iE LT MIJ Uaid m L Fr qo abo bres T Mera tiop tren Mea hasura Ceara et Hama Etap Tr r ee im o j Figure 5 18 Process Status dialogue Select the clone s to be processed This is not necessary if you are going to process all the clones at the same time To select more Click on the first clone hold down the than one clonein lt Shift gt key and click on the last clone in a sequence the column 5 31 5 ALFwin Sequence Analyser 5 32 To select a H old down lt Ctrl gt and click on the number of clones required separate clones Note It is useful to select all the clones of interest in the Result window Then these will be preselected in the Process window Select the type of process
71. DEN SCF format acombination of the above Y ou should open all of the result files containing sequence data for the clones and also import any other sequences before you create a sequence alignment item All sample sequences from open Result windows in ALFwin Sequence Analyser are then automatically added to a new alignment item Sequence Alignment 6 Note Raw data without sequences cannot be used in sequence alignment The raw data must first be processed in ALFwin Sequence Analyser 6 1 1 Creating anew sequence alignment item Select File New Alignment Item T he Alignment Item window is shown containing a list of all sequences from the open Result windows E hesAlgrmenit 24 4 6 Foe H Figure 6 2 Alignment Item window The sequences are displayed in a sequence alignment list Label Clone Name _ File Name Length ch Seq1 CloneO3 7 fabc alx 513 et Seq2 CloneO 7 7abc alx 419 5Seq3 Clonef8 7 7abc alx 466 Figure 6 3 Sequence alignment list as one part in the Alignment Item window containing sequences for fragment 3 of the p53 gene Various information is presented for each sequence which includes Icon The icon to the left of each sequence gives its status in Sequence Alignment In the following table examples are given for the type of effect seen on an icon dependent on its status An icon can have more than one of the effects described in the right column and the icon will change a
72. Export 2 Click on Setup The Export Settings dialogue is shown ALFwin Control 4 z e a al 4 a i Eat F am z0 7 ar aa 34 4 a i ao Figure 4 19 Export Settings dialogue O pen the Format menu and select the export format you require GCG STADEN SCF PC GENE ASCII EMBL DNAsis The seq format for GCG the sequence analysis program from Wisconsin University The scf format for the Staden sequence analysis package SCF format includes curve data TheN prefixed format for PC GEN E a sequence analysis program by IntelliG enetics from the Oxford M olecular group ASCII txt format that will load into many standard commercial applications The eml format for Bioresearch the sequence analysis program by Fujitsu Ltd The dna format of DN Asis a sequence analysis program from Hitachi Software 4 31 7 win Control 4 By default all clones are selected Click to remove the check mark from any clones in the Clone field that are not to be included in the export 5 Select the Range All bases Upright bases From base to 6 Select the File name s Clone name clone no Result name clone no New name clone no All bases in the selected clones will be exported Only upright bases will be exported Selects the part of the sequence to be exported for example from base pair number 12 to base pair number 123 The file will be named using t
73. LFwin Control from ALFwin Sequence Analyser see Chapter 5 3 4 2 Launching ALFwin Sequence Analysis 1 From the Available applications field of the ALFwin Administrator dialogue select Sequence Analysis 2 00 2 Click on Start to launch the application ALFwin Control 4 4 ALFwin Control ALFwin Control is used to control sequencing runs using oneor two ALF family instruments While a run is being performed the data is monitored in real time allowing you to confirm that the run is proceeding normally All important information needed to perform a run in ALFwin Control is specified within the casebook A casebook specifies the experimental run conditions that are used to control the instrument You can make changes to the run conditions before or during a run Any changes you make are automatically recorded in the logbook The casebook stores important information about your samples and allows you to define the storage folder for the generated result files At the end of a run defined procedures called post run actions are automatically executed A typical run consists of the following steps Start ALFwin Control You start ALFwin Control from ALFwin Adminstrator or Sequence Analyser Usean existing casebook file You name your result file specify the or create a new casebook run conditions add sample You can create your new information specify data storage casebook from an existing folders add prerun notes and specify
74. Shift when you want to see the shift from the processing in all the curve windows or when you want to apply manual shift see Section 5 7 5 5 To effect the changes without closing the dialogue click on Apply To close the dialogue and effect the changes click on OK The changes are applied to all open Curve windows 5 17 5 ALFwin Sequence Analyser Y axis scaling To change the scale for the currently selected clone Curve window Command View Scale Separately T View Scale Together Al View Scale Fixed Zl Effect This automatically scales each of the curves separately so that the displayed amplitude scale for each curveis adjusted to best fit the window displayed This may be useful in discerning the peaks in a curve that had a relatively low detection signal during sequencing N ote that selection of separate scales can be misleading when for example trying to establish if a peak is a true peak or background noise This automatically maximises the size of the largest peak visible in the current view of the Curves window The amplitude scale for the curve containing this maximised peak in the current view is used as the basis to scale the remaining curves Thus all peaks are cor rectly proportioned in relation to the largest visible peak This allows you to look at the relative signal detection sensitivity of the curves for the area of the result that you are viewing in the curve view This displa
75. alyser 5 48 2 Select the clones that you want to include in the result file s If this dialogue is called while the Curve or Gel View is active all the clones will be selected by default If the dialogue is called while the Result view is active only the clones selected in the Result view will be selected by default in the Split File Settings dialogue Click the tick boxes to deselect or re select clones O pen the Format menu and select the format required Normally the alx format is used alf is an older format used in the O S2 version of the program for ALF family instruments Click on Browse and use the Save As dialogue to select the destination folder for the file Select the way in which the clones are saved As a Single file Use this option to save the selected clones in onefile Enter the name for the file in the Single file name field As Multiple files Use this option to save each of the selected clones in a separate file Y ou must select the naming scheme to be used Clone Name clone no Use the name of the clone from the casebook the clone number Result Name clone no Use the name of the result file the clone number New Name clone no Use the name typed into the field the clone number The raw data for the selected clones is always saved Activate the Processed data button to include processed data Select the Include sequence button if you want to include the base sequence s in the sav
76. amic programming and involves setting several different parameters The parameters of the algorithm affect the final alignment and depending on the quality of the sequences to align different settings of the alignment algorithm parameters should be used ALFwin Sequence Analyser 2 00 is licensed under U S Patent N 0 4 558 302 and foreign counterparts License from Unisys to use TIFF LZW graphics E 3 E Reference list E 4 Index Index Symbols unit 4 46 A add column 4 24 Add counter no to filename 4 16 add file 5 62 add user 2 3 3 2 adding tools 5 59 Administrator introduction 3 1 launching Control and Analyser 3 7 ALF and ALX convert to TIFF 4 37 alf file decompressing 4 37 5 61 saving files as alf 5 60 ALFexpress automatic settings 4 5 ALFexpress and ALFexpress II differences in use 4 5 ALFwin Control interface 4 5 introduction 1 1 key features 1 1 linking to the instrument 2 3 on linehelp 1 3 Sequence Analyser interface 5 5 starting 3 1 suitable printers A 1 system requirement A 1 uninstalling i use on network B 1 ALFwin Control contents of workspace 4 5 introduction 4 1 screen contents 4 6 starting 4 3 ALFwin Sequence Analyser introduction 5 1 starting Sequence Analyser after a run 2 12 algorithm base calling overview 5 30 configuring alignment algorithm 6 36 configuring options 6 36 for alignment 6 36 Index align how the base calling algorithm works C 1 sensitivity to heterozy
77. an see the zoomed time interval in this display This provides the opportunity to measureintervals distances between peaks If you point at a base letter the information window displays the base number To switch this information window off or on again select View Show Info Window Using the vertical cursor line To display a vertical cursor line select View Show Line The line moves left and right with the mouse pointer The vertical cursor line allows more precise positioning of the cursor in relation to the curves and the time axis and can beused to position a base when editing the sequence 5 5 4 Orientation view The orientation view at the bottom of the window shows the whole result as curves sequence bars or sequence text 3 5 or 10 letter format For further information refer to Section 5 5 17 The rectangular cursor in the orientation view indicates the area of the result that is displayed in the curve view 5 13 5 ALFwin Sequence Analyser 5 14 To alter the size of the orientation view 1 Position the mouse pointer over its top border so that the pointer becomes a bi directional arrow up and down 2 Press down and hold the left mouse button and then drag the top border to increase or decrease the size of the orientation view 3 Release the mouse button 5 5 5 Navigating the Curve window Use the orientation view to select different areas of the result to be displayed in the curve view 1 Place the mouse
78. and temperature indicator lamps on the instrument are continuously illuminated On the ALFwin Control screen check the laser value and the temperature in the Manual window The laser Transmittance value should have a reading over 70 Click on the Start button in the toolbar The Start Run dialogue is shown Check that the filename and path are correct and press on OK A message is displayed on the screen The sampling will start after 16 seconds please wait A green lamp in the title bar and in the Windows 95 T askbar confirms the run has started The electrophoresis lamp on the instrument will be illuminated W ait until you can see curves in the Curve window to confirm the electrophoresis is running The electrophoresis lamp should be continuously illuminated Follow the run in real time in ALFwin Control The result file is automatically generated at the end of the run and can be opened in ALFwin Sequence Analyser 2 Making your first run 2 3 Analysing the results of your first run At theend of the sequencing run a result filewas automatically created with the filename M yresult alx This contains the raw data from the run in addition to a copy of the casebook which now includes the logbook and an Analysis log ALFwin Sequence Analyser gives you a wide variety of useful analysis and presentation tools for improving data processing or the presentation of your results Y ou will now perform an analysis of th
79. ar in ALFwin Control 4 7 in Sequence Analyser 5 6 M etafile exporting curves as M etafiles 5 49 min field 4 29 mismatches in alignment sequences 6 12 mouse activating items with the mouse B 4 dragging and dropping B 4 mouse pointer showing time and curve amplitude 5 13 Index right mouse button menus B 4 selecting using mouse B 3 using in Windows 95 B 3 moving bases 5 40 5 43 M SF export format 6 2 Web site 6 3 M SF sequence alignment export format 6 32 my computer in Windows 95 B 2 N Name column 2 8 network B 1 network neighborhood in Windows 95 B 2 new alignment item 6 5 new casebook 4 11 notes adding Analyser notes 5 45 Notes tab in casebook 4 9 4 37 sequence alignment notes 6 29 nucleotide sequence extraction by processing raw data 5 30 O one letter amino acid codes 6 27 on line help 1 3 open casebook file 4 9 Curve window 5 11 opening Curve windows 5 11 result filein Analyser 2 13 result file in Sequence Analyser 5 8 orientation view changing the mode 5 20 using and adjusting 5 13 view curves 5 21 view sequence bar 5 21 view sequence text 5 21 overview display of alignment overview 6 10 display of alignment table 6 12 P parameters estimate by base calling algorithm C 2 pausing arun 4 43 PC GENE export format 4 31 5 50 E 1 xvii Index xviii Amersham Biosciences Web address 1 2 phone number E mail 3 3 PHY LIP export format 6 2 6 32 Web site 6 3 post processing of raw d
80. ata 4 29 post run actions automating backup file 4 36 automating data export 4 30 automating post processing of raw data 4 29 automating report print 4 32 automating tools selection 4 36 overview 4 9 tab in casebook 4 9 tab in casebook detail 4 28 power electrophoresis 4 18 pre result 4 45 pre run notes 4 37 Pre run notes field 2 8 presentation options in Analyser overview 5 2 preset 4 38 4 39 temperature 4 5 presetting the instrument 2 9 print alignment report 6 30 automating report print 4 32 curve and gel views 5 57 headers 4 33 5 54 printers suitable for ALFwin A 1 printing a casebook 4 14 printing a report 2 18 printing all views 5 57 recalling a report layout 5 53 report 5 53 selecting items to be included in report 4 33 5 54 setup 4 14 supported printers A 1 Process All 2 14 processed data in result file 5 7 viewing 5 12 processing ambiguities expected 4 30 auto shift 5 32 auto start and auto stop time 5 32 clones 5 31 halting processing 5 33 improving result by using manual shift 5 37 Index improving result by using manual start stop times 5 38 introduction 5 30 mode 4 29 observing process status 5 33 parameters overview 5 30 post processing of raw data 4 29 process type 4 30 reviewing the results 5 33 run data 5 30 selecting the clones for processing 5 31 selecting the type of processing 5 32 when to adjust the time shift manually C 5 when to set the start or stop point manually C 4 pr
81. atest used and saved layout file in a casebook is automatically selected when you open the Report Settings dialogue 4 Select the Headers Casebook and Groups buttons in turn and select items to be included in the report from the lists shown The white button must be checked to print the items selected in each group Headers Thisallows you to select the name current user current filenameand current date in the present result file that are to be included in the header of the report Casebook This allows you to select items from the casebook and logbooks that are to be included in the report 4 33 7 win Control Groups This allows you to select the clones or groups to be included in the report Note Groups must be selected to affect the following 5 Select the Range Entire curve Print curve from the first to the last minute of the run All bases Print curves for the called bases Upright bases Print curves for the upright bases From base to Print curves for the range of bases you enter in the From to fields From time mins to Print curves for the range of time you enter in the From to fields 6 Select the Scaling to be applied to the print of the curves Together The amplitude scale for the curve containing the largest peak is used as the basis to scale the remaining curves Thus all peaks are correctly proportioned in relation to the largest visible peak Separately Each of the c
82. automatically e the temperature of the gel is preset to 40 C and the temperature begins to increase until this value is reached e the light on the panel is switched on when the lid is open M oreover depending on which of the two analysers you are performing a run the following also apply ALFexpress I ALFexpress the laser is switched on and the laser is switched on and aligned when you press the adjusted after you have closed Realign Laser button in the and opened the lid on the M anual window of ALFwin instrument Control see Section 4 5 1 therun conditions can be stepped the run conditions can be stepped or ramped to different values to different values during the during the course of the run See course of the run Section 4 4 2 4 2 Introducing the ALFwin Control interface TheALFwin Control interface contains the Curve View Manual and Logbook windows together with the Title Menu Tool and Status bars 4 5 4 a win Control There is also a run light indicator in the Windows 95 taskbar Title bar Be J Menu bar mia rati Ma Tool bar F Curve View window Manual window Logbook window e e Status bar ae Run light indicator a ae c Windows 95 taskbar U EE Figure 4 2 ALFwin Control screen controlling a run on an ALFexpress Il instrument Note that you can maximize the display of any window or change the size of the windows by dragging the window borders The fonts size of
83. cale method Ml Sa Ea Scale all lanes together Scale each lane separately Default Settings Cancel Apply Help Figure 5 17 Appearance tab of the Gel Properties dialogue 4 Select the Scale region to apply the contrast adjustment either to the Visible view on the screen or to the Whole run 5 Select the Scale method the mode of calculation for the contrast adjustment either Scale all lanes together This applies an adjustment in contrast to thelanes in the selected region see step 4 based on contrast data from all of the lanes Scale each lane separately This applies an adjustment in contrast to individual lanes based on their own contrast data 6 Adjust the contrast by dragging the slide bar up or down for increased or decreased contrast respectively 7 Click on Apply to effect the change R eadjust the contrast level if required or click on Default Setting to return all the settings to their default conditions 8 When you are satisfied with the adjusted contrast level click on OK 5 29 5 ALFwin Sequence Analyser Showing and hiding the Gel View axes and bases Y ou can adjust the Gel View to show or hidea vertical time axis and a horizontal clone number axis Y ou can also show or hide the base positions evaluated by data processing 1 Select View Gel Properties The Gel Properties dialogue is displayed 2 Click on the Appearance tab 3 Select or deselect Time and Clone numb
84. cation using the mouse 5 27 adjusting the contrast 5 28 copying images into documents 5 52 navigating the Gel View window 5 27 selecting raw or processed data 5 26 showing or hiding the axes and bases 5 30 using the Gel View window 5 26 viewing raw data or processed data 5 26 working in the Gel View window 5 26 group deleting 4 53 editing 4 52 naming 4 51 H help button 1 3 help option from Windows 95 start button B 3 help topics 1 3 heterozygote adjusting processing for expected heterozygous positions 5 32 low medium and high expectancy 5 32 modes in base calling C 5 hiding identical bases in sequence alignment 6 24 hiding or showing alignment table gridlines 6 14 home folder creating 3 2 I icon cloneicons 5 9 edited cloneicon 5 41 result file icons 2 13 xiii Index xiv imperfections colour coded cells 6 13 examining 6 12 import ASCII 6 38 ASCII sequence data 6 4 configuring alignment import format 6 38 STADEN SCF 6 38 include driver trace 3 6 indicator light in the taskbar 4 7 indicator electrophoresis lamp 2 11 inserting bases 5 40 5 41 instrument 2 15 changing the name of the instrument 3 6 confirming stable instrument operation 2 15 connecting a second instrument 3 6 connecting to ALFwin Control 3 5 connecting to the demonstration instrument 3 6 Control connecting an ALF family instrument 3 5 demonstration instrument 3 6 differences in use of ALFexpress and ALFexpress II 4 5 displaying instrume
85. ccording to changing status 6 5 6 Sequence Alignment 6 6 Default icon and status AT C i N on aligned sequence AT CG Source Result file for the sequence open in ALFwin Sequence Analyser AT CG Raw data for the sequence is available AT CG Sequence is imported into Sequence Alignment AT CG Source file for the sequence in its original folder Effect on icon and status ACGT AGT The sequence has been aligned Tes Top right edge folded over Source Result file for the sequence not open in ALFwin Sequence Analyser i e a saved alignment item has been opened but not the source Result file AT Lear Grey background to icon Raw data for the sequence is unavaila ble which usually indicates an imported sequence format This means that raw data curves can not be viewed for this sequence Red background to icon Sequence has been edited in ALFwin Sequence Analyser but not re imported into Sequence Alignment Greyed icon Source file for the sequence is not in its original folder This usually occurs when an alignment file is moved between computers Label Clone Name File Name Length Sequence Alignment 6 Each sequence is given a numbered sequence label e g Seq1 Seq2 etc The sequence numbers allow sequences with the same name to be included in the sequence alignment item This is useful when sequences with the same name are selected from different result files T
86. clear view of the curve for the selected base 4 Open any other curves you need by repeating the above procedure Y ou can select opened curves for viewing by opening the Window menu and selecting the curve name from thelist Alternatively use Window Tile All Curve Windows to compare the curves 5 Edit any bases in the Curve view as described in Section 5 8 6 22 Sequence Alignment 6 6 Click on thealignment item If you have edited any of the original data you will receive a message asking if you want to reimport the edited sequence Click on OK to re import the sequence A second message is then displayed asking if you want to align the re imported sequence If you click on OK the sequence is automatically aligned using the Align to Previous function see Section 6 2 2 6 7 4 Finding Ambiguities or Special Sequences The Edit Find command in Sequence Alignment allows you to search for specific objects in the alignment after an alignment has been performed 1 Click on the Alignment Item window to make it active 2 Select Edit Find or lt Ctrl F gt The Sequence Alignment Find dialogue is displayed ALFwin Sequence Alignment Find Ea A Sequence to Search For 7 Search Attributes Base Sequence X Search Consensus C Search Selected Sequences Find Prev _Find Next Close V Exclude Gaps In Search Figure 6 10 Sequence Alignment Find dialogue 3 Select the appropriate search object from
87. cording to the four bases Any excluded lanes are represented by grey boxes labelled X To excludea lane Select the lane and click on Exclude or double click on the lane N ote that lanes can only be excluded before the run is started To re install a lane Select the lane and click on Undo or double click on the lane ALFwin Control 4 Click on OK Excluded lanes are marked with an asterisk in the Lanes view and are grey in the Detector view during arun Clones are rearranged automatically in the Lanes view When lanes are excluded the remaining lanes are rearranged so that each clone is allocated the four lanes it requires Y ou can use the Lanes view to see which lanes are associated with each clone For example Lane 2 1 means that lane 2 is included in clone 1 Excluded lanes are marked with for example Lane 3 means that lane 3 is excluded and is not included in any clone T he excluded lanes settings will not be stored in the casebook file Casebook Seqdem1 cbk File Information Conditions Sample lr Lane 1 1 Lane 2 1 Clone 1 lanes 1 5 ane 3 excluding lane 3 ane 4 1 ane 5 1 ane 6 ane 7 2 ane 8 2 Clone 2 Lanes 6 11 excluding lanes ane 9 6 and ane 10 2 ane 11 2 ane 12 3 ane 13 3 ete ane 14 3 ane 15 3 1 ane 16 4 Lane 3 Lane 4 4 Lane 7 2 Lane 9 Lane 41 2 Lane 43 3 Lane 45 3 Lane 16 4 Clones anes Figure 4 16
88. ctual DN A sequences for each of the clones and these generated sequences can bealigned for comparisons Present your results on the screen in gel or curve view and print out a report of the analysis that you have performed Finally export the results for direct use or for further analysis in other software applications 1 1 Key features of ALFwin Sequence Analyser M odularised software The software is modularised for convenient control analysis and administration of sequencing runs and sequence data e Programmed casebooks Programmed casebooks contain all information for total control of each run Include the settings for experiment run conditions notes on samples and other experimental factors post run actions data file handling and full documentation logging Simultaneous control and analysis ALFwin Control allows simultaneous control of two ALF family instruments while also evaluating results from other runs e Real time monitoring Sequencing runs are monitored in real time on the screen e Intuitive interaction The graphical interface has been designed for intuitively interactive control and analysis Work with the data in several modes including the Curve view and the Gel fluorogram view Full data processing with accurate base calling Processing involves the automatic extraction of the sequence from the run data The base calling algorithms are extremely accurate providing user confidencein the results and advanc
89. d maximum amplitudes and the differences in amplitude for the part of the curves currently displayed This should allow you to gauge the minimum and difference in amplitude in one curve against the maximum peak amplitude in another curve 5 8 Editing sequence bases This section describes how you can edit the processed data to replace insert move or delete bases All the changes you make are logged in the Analysis Log in the casebook Y ou can edit bases using a combination of mouse and keyboard or you can use the mouse with the Edit Toolbar 5 39 5 ALFwin Sequence Analyser 5 40 A Warning Care is needed when selecting an edit mode An active modeis indicated in the Edit menu by a black dot marker Selecting a mode toggles it on or off If you select a mode that is already active you can inadvertently turn it off and return to the default Replace mode Check by looking in the status bar which shows the current edit mode The Edit Toolbar is displayed on screen when you select Edit Edit Sequence or press the lt Ctrl E gt keys Y ou can move the Edit Toolbar to another position on screen if any toolbar buttons are obscured Click and hold on a non button part of the toolbar and drag it to the position required This toolbar allows you to replace insert move and delete bases without calling these functions separately Clicking the appropriate button on the Edit Toolbar executes action on the selected base Use a button
90. d press Continue Exclusion of lanes have no effect Problem Excluded lanesin the Sample Information tab in the casebook do not seem to have any effect in the Curve window Explanation Y ou have closed the casebook and opened it again The excluded lanes information is not saved when the casebook is closed Troubleshooting or Y ou have probably ended the run opened the casebook again and restarted the run The excluded lanes information is not saved between runs Solution End the run open the casebook again re enter the excluded lanes information and restart the run Run Conditions set values are not reached Problem Run conditions such as Voltage Current and or Power in the Manual window do not reach the set values Explanation Due to the chemistry of the electrophoretic separation only oneof the parameters will set the limit of the system This onewill reach or almost reach the set value but the other two will not Solution Check only the parameter that limits the system ALFwin can not find the new run Problem After the completion of a run the ALFwin Sequence Analyser module can not find the file for that newly completed run Explanation Possibly due to too heavy loading on the computer during the end of the run Solution Start the sameALFwin Control module as the run was made in and log in as same user as before After this treatment the file will be possible to open from ALFwin Seq
91. d the taskbar turn red post run action and then grey not running Run data are saved in the result file You can read the casebook but not make any further changes To start the next run open or create a casebook file File Open Casebook or File New Casebook To use the same casebook file as the previous run you can select it from the list at the bottom of the File menu 4 8 Changing the screen layout This section describes how to configure the layout of your screen 4 8 1 Arranging open windows Y ou can decide how to arrange all the open windows using commands in the Window menu To arrange the windows on the screen select Window Cascade To position all open windows on top of each other Window Tile as Default To position the windows in the default tiled configuration Refer to Appendix B Short guide to Windows 95 for a description of how you move and resize the Curve Manual and Logbook windows 4 8 2 Saving window positions on exit To save the present positions of the windows on the screen and also the font settings in the Manual and Logbook windows for the next time you open ALFwin Control 7 win Control 4 48 1 Position the windows as you want them on the screen 2 Select Window Save Window Positions on Exit The windows will be in these positions the next time you open ALFwin Control O peration of this command does not affect the default tiled positions that can still be recalled using the Win
92. demonstration instrument 1 From the ALFwin Administrator dialogue select an instrument and click on System Setup to open the ALFwin Edit System Settings dialogue ALFwin Administrator 3 ALFwin Edit System Settings x m System Name instrument as demo Comment m Instrument M Demo instrument COM port AlFexpress ll Demo m Tracing P Include driver trace I Instrument trace window cont_ oee Figure 3 4 Edit System Settings dialogue 2 Check the Demo instrument option box 3 Select ALFexpress Demo or ALFexpress II Demo 4 Click on OK to return to the ALFwin Administrator dialogue 5 Click on OK to start ALFwin Control with the demo instrument connected 3 4 Launching the ALFwin applications ALFwin Control and Sequence Analyser are launched directly from ALFwin Administrator 3 4 1 Launching ALFwin Control 1 To open ALFwin Control select the instruments in the Available applications field and click on Start 2 Control islaunched and a splash screen is shown for a few seconds 3 7 SS er Administrator 3 8 3 Aninformation panel advises that initialisation is taking place If the instrument has been switched on since ALFwin Control was started the instrument program is loaded 4 An information panel advises that the system is searching for the connection to the instrument 5 ALFwin Control is launched N ote Y ou can also launch A
93. dow 2 Select View Properties and click on the Data Set tab in the Gel Properties dialogue 3 Select the dataset required Raw data or Processed data ALFwin Sequence Analyser 5 4 To effect the change without closing the dialogue click on Apply To close the dialogue and effect the change click on OK 5 6 2 Navigating the Gel View window Usethe scroll bars to display different portions of the gel The position of the mouse pointer is indicated in the status bar The vertical position is shown as time into the run The horizontal position is shown in the form Clone number base type eg Clone7 A 5 6 3 Using the Zoom function in Gel View Y ou can alter the magnification of the whole gel view or select particular portions of the gel using the mouse for increased magnification Altering the magnification of the whole gel To alter the magnification of the whole gel use the following commands Command Effect View Zoom Time In x2 Increases the magnification of the lt 7 gt key time scale by a factor of 2 View Zoom Channels In x2 Increases the magnification of the Shift F7 gt keys gel lanes by a factor of 2 View Zoom Time Out x2 Decreases the magnification of lt 8 gt key the time scale by a factor of 2 View Zoom Channels Out x2 Decreases the magnification of Shift F8 gt keys the gel lanes by a factor of 2 Increasing the magnification using the mouse To magnify a specific portion of the Gel View window using the mouse
94. dow Tile as Default option 4 8 3 Hiding and showing the toolbar and status bar Command buttons on the Toolbar help you speed up your work The status bar gives you information about what is currently happening Y ou can toggle these bars on and off by selecting View Toolbar or View Status Bar Toolbar buttons cannot be changed 4 8 4 Changing the look of the curves This section explains how you can change the way the curves are shown in the Curve window The run must be started before these commands can be used Show the Curve or Detector view N ormally the result from therun is shown asa series of curves that plot the signal level against time Curve view Y ou can also choose to view the momentary signal level for all lanes at the same time Detector view These views are automatically updated after each signal sample To change from Curve view to Detector view 1 Click on the Curve window to make sure that it is activated 2 Select View Detector View or click on the Detector View button The detector view is shown in the Curve window To return to curve view select View Curve View or click on the Curve View button Thesignal levels are normally very low compared to the dynamic range maximum detector values You can zoom in on the signals by entering a lower value for maximum signal level 1 Click on the Curve window 2 Select View Properties 3 Select the Settings tab ALFwin Control 4 4 Enter a val
95. e Figure 6 4 Alignment Item window after alignment of all sequences 6 2 2 Aligning additional sequences to the previous alignment After you have performed an alignment you may later wish to add more sequences to the sequence alignment item and add them to the previous alignment This is achieved using the Analysis Align to Previous function whereby the new sequence s are aligned with the consensus sequence that was generated in the previous alignment i e thereis no new alignment involving all of the existing sequences which contrasts with the Align All function The consensus sequence is automatically updated to reflect the alignment with respect to the new sequence s If asa result of the Align to Previous function the consensus sequence is changed in any way this will in turn be reflected in the other sample sequences used in the original alignment Y ou may also at any time perform a full alignment by selecting Analysis Align All whereby all of the sample sequences in the sequence alignment list are aligned and contribute to the generation of a new consensus sequence 6 Sequence Alignment Y ou can align additional sequences to the previous alignment 1 Open the result files or import other sequence files 2 Select each newly opened Result window in turn and select the sequences to be added Select Analysis Add To Alignment Sequences are added to the sequence alignment list in the Alignment Item window 3 Select the seq
96. e ALFwin Administrator dialogue select the user name you want to delete 2 Click on the Delete User button A dialogue asks you to confirm the deletion 3 Click on Yes to confirm the deletion Folders and files are not deleted by this command 3 4 ALFwin Administrator 3 3 3 Connecting an instrument to ALFwin Control To makea sequencing run it is necessary that the software is connected to at least oneALF family instrument After the connection is made an identification can be added for the serial COM port on the back of the computer to which an instrument is connected Two instruments can be connected and used simultaneously Tracefunctions can beinitiated by ALFwin Administrator which allow service technicians to examine the operation of the instrument control 3 3 1 Connecting an ALF family instrument To define the connection of an instrument 1 From the ALFwin Administrator dialogue highlight a User name and select an instrument in the Available applications field 2 Click on System Setup The ALFwin Edit System Settings dialogue opens ALFwin Edit System Settings x m System Name Instrument 1 Comment Po m Instrument l Demo instrument COM port ALFexpress Demo f Tracing T Include driver trace T Instrument trace window coc e Figure 3 3 Edit System Settings dialogue 3 5 SS er Administrator 3 6 The name of the instrument is shown in the
97. e clone name changes to the E processed data icon if the processing is successful The Status column shows Normal type of processing no heterozygocity expected e The Start Time and Stop Time columns show the times where the processing of data starts and stops 2 14 Making your first run 2 e The Shift A C G T column shows Auto indicating that automatic correction for smiling has been applied The Upright bases Ambiguities column contains two figures separated by an oblique stroke The first figure shows the reading length of the sequence for each clone The Called bases Ambiguities column contains two figures separated by an oblique stroke The first figure is the reading length and this number includes a further 300 bases beyond the point where the certainty of the result fell below 99 The second figure to the right of the oblique stroke is the number of ambiguities T hese are the positions where the base calling algorithm had two or more alternative interpretations 2 3 4 Analysing the processed data There are several analysis options that are open to you to confirm the quality of the results J ust some of these options are described here although as you become experienced with using ALFwin Sequence Analyser you will discover a wide range of analysis and presentation possibilities to get the most out of your data see Chapters 5 Examples of analysis options include e checking the upright bases fig
98. e field Y ou can use up to a maximum of 256 characters 3 Your default Home Folder is created automatically when you enter a user name It has the same name as the user and is located in the Program Files AP Biotech ALFwin H ome user name folder 4 Your Backup Folder is also created automatically when you enter a user name 10 ALFwin Administrator 3 Th ALPwin Add of Edit Waar E Pigmea folder F 4 ade Ekiris chiA Paini Hara Caa Sechup folder F VAP Boots chal Famin rma Liar Aria Dupatmgai i address a Paced mirabe g mail l Ce Carel Henle Figure 3 2 Add or Edit User dialogue If you want to change the location of your home folder click on the Browse button adjacent to the home folder field The Browse for folder dialogue opens Select the path to an existing folder or use the Create New Folder button to generate any new folders you require To open the new folder double click the folder name or select the name then click on Open Click on Save to create your home folder The Add or Edit User dialogue returns If you wish to change the name or location of your Backup Folder click on the Browse button adjacent to the backup folder field and use the Browse for folder dialogue to select a path and name for your backup folder Use the Create New Folder button to generate any new folders you require Choose if possible an alternative drive for this backup folder but be aware tha
99. e heat on after run The maximum electrophoresis voltage The maximum current in milliA mperes The maximum power in Watts The time of the electrophoresis run in minutes The thermostatically controlled water temperature in degrees centigrade The interval between data samples in seconds Select this option when you plan to start a new run soon after the current run Activating stepped or ramped pre set changes for ALFexpress Il If you are making a run using ALFexpress II you use the Automatic condition control to set a stepped change to the set conditions during the course of therun or to introduce a ramped changein conditions A ramped change differs from a stepped change in that the change is made gradually over a specified period of time Y ou can preset a change in the electrophoresis voltage current power temperature ALFwin Control 4 To set the condition control I 5 Click Edit to open the Edit Step Ramp Values dialogue CE Pe E ee E wa e respa Ti 2 Hep ising y i ran Daai in ran rhat Grae i D the arrari rachad of T Laie Hel Figure 4 10 Edit Step Ramp Values dialogue O pen the menu to view and select the parameter that you want to step Edit the target value in the Change to field If a step change is required select the Step change at button and enter the time at which the change is to occur If a ramp change is required select the Ramp starting a
100. e is shown Tools Setup x Tools Convert ALX ALF to TIFF Command C Program Files 4P BiotechsALFwinCommons lf2T iff exe Arguments Initial directory E Program Files AP Biotech 4LFwin Common Coreei __ He Figure 4 21 Tools Setup dialogue ALFwin Control 4 3 Open the Tools menu and select the tool required There are two available tools supplied with ALFwin Convert ALX ALF to TIFF and Decompress ALF file Please refer to Section 5 14 The path too the selected tool will be automatically given 4 Enter if required parameters to start the program in the Arguments field For example A filename 5 Click on OK to complete setting up the automation of tool Selection 4 4 5 Notes tab This tab allows you to make your prerun run and analysis post run notes The casebook manages note taking allowing access only to the appropriate type of note according to the stage reached in the experimental procedure The Notes tab of the Casebook dialogue provides fields where you can write experimental notes There are three fields in the Notes tab but only one is accessible at a time according to the stage in the procedure Pre run notes Run notes Analysis Notes Use this field to make notes before you start the run The casebook is still accessible at this stage and you have the option to save prerun notes in the casebook file When you start the run the Pre run notes field is closed against any f
101. e is to be saved and enter a name for the file 3 Click on Save Making your first run 2 Congratulations You have now completed your first run 2 Making your first run ALFwin Administrator 3 3 ALFwin Administrator ALFwin Administrator is used to create your user name which can then be used to open the ALFwin Control or Sequence Analysis modules When you create your user name additional information is given such as the storage and backup storage folders for result files ALFwin Administrator is used to connect one or two ALF family instruments or demonstration virtual instrument s to the computer for simultaneous control capability This chapter is divided into the following sections 3 1 Starting ALFwin Administrator 3 2 Creating a user name and folder 3 3 Connecting an instrument to ALFwin Control 3 4 Launching the ALFwin applications 3 1 Starting ALFwin Sequence Analyser To start ALFwin Administrator locate and select ALFwin software from the Windows 95 Start button menu options The ALFwin Administrator dialogue is shown ALFwin Administrator x m User information Add User Default Edit User Delete User m Available applications Sequence Analyser 2 00 Instrument 1 Instrument 2 System Setup Figure 3 1 ALFwin Administrator dialogue 3 1 e eer Administrator Note If you have also obtained the DNA fragment separation software from Amersha
102. e more than one base component was called by thealgorithm SeeAppendix C for information of the ambiguity codes The sequence also comprises different styles for representing the base nucleotides Bases are written in different styles depending on how the algorithm has evaluated the base position Please refer to Appendix C which describes the operation of the base calling algorithm ALFwin Sequence Analyser 5 CAPITAL letter The algorithm estimates that it is likely that the base has an uncertainty of less than one percent Italic letter The algorithm estimates that it is likely that the base has an uncertainty greater than one percent small lower case letter The algorithm is uncertain whether or not there is a base at that position 4 Usetherectangular cursor in the orientation view beneath the curves to select an area of the run and adjust its magnification Section 5 5 5 describes how the rectangular cursor is used to adjust the curve view 5 Usethe lt F7 gt and lt F8 gt keyboard keys to adjust the magnification of the view or use the mouse pointer to drag a box around the area of the curves to be magnified Finding special sequences or ambiguities To find the positions of special sequences or ambiguities 1 Click on the Curve window to make sure that it is activated 2 Select Edit Find or press the lt Ctri F gt keys The Find dialogue is shown Find Demo 001 alx Clone01 M13 x we F
103. e name typed into the field the clone number 5 51 5 ALFwin Sequence Analyser 5 52 8 Click on OK to complete the Export Settings dialogue The sequences are exported to files with the selected format one file for each sequence 5 11 3 Copying a clone a sequence or a Gel View window into another computer application Y ou can copy clones sequences and Gel View windowsso that they can be pasted directly into DTP or graphics applications on thePC If your computer has a reasonably high resolution screen a high quality graphic can be produced for a report or ohp transparency 1 Set theresolution of the computer screen to the maximum possible 2 Copy the information as follows To copy a clone click on the Curve View window and select Edit Copy Clone or press the lt Ctrl C gt keys To copy a gel click on the Gel View window and select Edit Copy Gel or press the lt Ctrl C gt keys To copy a sequence click in the sequence bar or sequence text and select Edit Copy Sequence or press the lt Ctri C gt keys 3 Open the target application and paste the copied image This is usually achieved by pressing the lt C trl V gt keys while the application is active ALFwin Sequence Analyser 5 Figure 5 27 Curve window from where clones or sequences can be copied 5 12 Printing a report A report is a print that can include items from the casebook curves sequences and headers Y ou select the items you wa
104. e one character to all text The cursor is posi the right tioned at the text end M ove to the cell above Validate the cell If it is valid move to the cell above M ove to the cell below Validate the cell If it is valid move to the cell below M ove to the top left cell Validate the cell If itis valid move to the top left cell M ove to the bottom right Validate the cell If it is cell valid move to the bot tom right cell M ove up one page Validate the cell If it is valid move up one page M ove down one page Validate the cell If itis valid move down one page Copy selected bases Activate Find Sequence dialogue Toggles Edit mode between Insert and Replace mode N ew alignment item O pen file Undo last edit operation Deletes a highlighted set of bases or deletes a sequence in the alignment item list Reference list E E Reference list E 1 References for export formats Format Description Further information GCG The seq format http www gcg com for GCG the sequence analysis program from Wisconsin Univer sity STADEN SCF The scf format Staden Rodger 1982 An interac for the Staden tive graphics program for com sequence analysis paring and aligning nucleic acid package SCF for and amino acid sequences N ucl matincludescurve Acids Res 10 9 2951 2961 data http w ww mrc Imb cam ac uk pubseq PC GENE TheN prefixed http w ww oxmol com f
105. e run results in the ALFwin Sequence Analyser module This involves a basic introduction to some of the major functions needed to analyse your first run to confirm that it was successful Full description of the features mentioned and many others besides are detailed in Chapters 5 and 6 of this User M anual 2 3 1 Starting ALFwin Sequence Analyser To start ALFwin Sequence Analyser 1 Restart ALFwin Administrator by locating and selecting ALFwin Software from the Windows 95 Start button menu options The ALFwin Administrator dialogue is displayed 2 In the ALFwin Administrator dialogue select your user name or the name Default and then double click on Sequence Analyser 2 00 under Available applications The Sequence Analyser module is started as indicated by the splash screen 3 The Tip of the Day dialogue is superimposed on the ALFwin Sequence A nalyser screen Tip of the Day provides tips about using the program Click on Close in this dialogue to proceed further Dij yau krie Th si iay lea ap rrn ieat g Curve r ie rash F eium To Figure 2 9 Tip of the Day dialogue a Making your first run 2 2 3 2 Opening the result file Open your result file 1 2 Select File Open or click on the Open button T he Open dialogueis shown In the Open dialogue locate the M yresult alx result file Select the file and click on Open Look in E David gt El c File name fr alx alf F
106. e the Realign Laser button and the Transmittance is expressed as the Laser value To see more of therun conditionsin the Manual window resize the window seeAppendix B Short guide to Windows 95 scroll the window contents using the scrollbars or change the font size using View Properties Style Font To align realign the laser using ALFexpress instruments close and open thelid of the instrument to start the laser If you are using an ALF or ALFred instrument you must click ON at Laser in the Manual window Check the laser Transmittance in the Manual window Transmittance is the value used in ALFexpress II to express the percentage of the laser light transmitted by the gel Adjust the laser if necessary If you are performing the run on an ALFexpress instrument Transmittance is instead expressed as the Laser value which is a measure of the accuracy of alignment of the laser beam 6 ALFwin Control 4 Fill the upper and lower buffer reservoirs with the appropriate running buffers W ait for the selected temperature to be reached in the Manual window Prepare the instrument remove the comb rinse the wells load the samples connect the electrodes Depending on which of the two ALFexpress instruments you are using the laser is realigned by ALFexpress I ALFexpress clicking on the Realign gently pulling the cassette Laser button in the Manual forward using the window thermostatic circulator connections and re
107. e window 5 5 21 Configuring the sequence display 5 5 22 Saving or opening a layout for the Curve window 5 6 Presenting the run in the Gel View window 5 6 1 Viewing raw data or processed data 5 6 2 Navigating the Gel View window 5 6 3 Using the Zoom function in Gel View 5 6 4 Adjusting the G el View window properties 5 7 Processing run data 5 7 1 Processing clones 5 7 2 Reviewing the data processing results 5 7 3 Viewing the data curve after processing and finding special sequences 5 7 4 Assigning upright status to bases 5 7 5 Shifting the curves manually 5 7 6 Using manual start or stop times 5 7 7 Displaying the signal levels of lane curves 5 8 Editing sequence bases 5 8 1 Replacing a base 5 8 2 Inserting a base 5 8 3 Moving a base 5 8 4 Deleting a base 5 8 5 Showing or hiding edited bases 5 8 6 Cutting clone curves 5 9 Viewing casebook information and adding evaluation notes 5 10 Saving the result file 5 10 1 Saving a complete result file 5 10 2 Saving part s of a result file 5 11 Exporting data 5 11 1 Exporting curves 5 11 2 Exporting sequences Contents 5 20 5 22 5 23 5 24 5 24 5 25 5 26 5 26 5 27 5 27 5 28 5 30 5 31 5 33 5 34 5 36 5 37 5 38 5 39 5 39 5 41 5 41 5 43 5 43 5 44 5 44 5 45 5 46 5 47 5 47 5 49 5 49 5 49 5 11 3 Copying a clone a sequence or a Gel View window into another computer application 5 12 Printing a report 5 52 5 5
108. ed 5 Check that Demo instrument is disabled unchecked 6 Click on OK 2 2 4 Starting ALFwin Control Start ALFwin Control 1 Select the instrument for your run in the Available applications field and click on START 2 ALFwin Control starts 3 A splash screen is shown for a few moments 4 Theinformation panel advises that initialization is taking place If the instrument has been switched on since Control was started the instrument program is loaded 2 4 Making your first run 2 Theinformation panel advises that the system is searching for the connection to the instrument Thegreen and red indicator lamps on theALFexpress instrument remain illuminated and the three yellow indicator lamps flash in series W hen the connection has been established only the green indicator lamp remains illuminated on the instrument The Tip of the Day dialogue is superimposed on the ALFwin Control screen This dialogue provides tips about using the program Click on Close in this dialogue to proceed 2 2 5 Mounting the gel cassette into the instrument Leave the computer for a few moments and mount the prepared gel cassette into the instrument 1 Lift the gel cassette and place its lower edge into the lower buffer reservoir Locatethe two holes on the cassette on to the appropriately placed fixed screws protruding from the front of the instrument The gel cassette should be pressed in against thetwo microswitches located
109. ed base calling functions can be programmed to accommodate different levels of heterozygocity Raw data obtained from the sequencing run can be processed either as post run actions directly after the run or later within ALFwin Sequence Analyser 1 1 1 Introduction Sequence alignment The sequences can be aligned with one another for comparison of different sequences to locate similarities and differences between the samples Compatible export formats DNA sequence export formats are ASCII DNASIS EM BL and GCG DNA sequences with data curves included can be exported as STADEN SCF Data curves can also be exported as pictures by saving as Windows M etaFiles and using the copy clone and copy to clipboard functions Aligned sequences can be exported in M SF and Phylip formats Additionally you can convert the data to TIFF files for direct use in other application software e Customised reports Customised reports can be created from the result file the aligned sequences and also from the casebook e Windows 95 The software has a modern user interface developed for Windows 95 Updates on the Web For the latest information user methods run conditions and more for ALFwin Sequence Analyser contact the Amersham Biosciences internet home pages www amershambiosciences com 1 2 Using ALFwin Sequence Analyser 1 2 1 Windows 95 operating system ALFwin Sequence Analyser has been designed to use the features and benefits of the
110. ed data If processed data is selected the sequences will always be saved 8 Click on OK to save the file s ALFwin Sequence Analyser 5 5 11 Exporting data 5 11 1 Exporting curves Y ou can export curve graphics in a standard M etafile format that can be imported by a number of applications on PC and M acintosh computers This allows you to include curve graphics in reports being prepared in for example M icrosoft Word on aPC or M acintosh The M eta File format can also be imported by many standard graphics applications that you can use to edit an image before it is drafted into a document Curves and sequence data can also be exported in SCF format Please refer to Section 5 11 2 To export a curve graphic as a wmf file 1 Click on the Curve window to make sure that it is activated 2 Using the zoom controls and the scroll bars adjust the curve window so that it displays the part of the curve you require 3 Select File Export to Meta File The Save As dialogue is shown 4 Usethe dialogue to select the destination folder for the file 5 Enter a name for the file 6 Click Save The picture is saved as a wmf file 5 11 2 Exporting sequences Y ou can export sequences to files so that they can be loaded into other applications that allow further evaluation of the data Files can be saved in a range of formats appropriate to various sequence analysis applications They can also be saved in ASCII format that is accepted b
111. ed on the top of the cascaded windows Window Tile All Windows This displays all open windows on the ALFwin Sequence Analyser workspace without overlapping Window Tile All Curve Windows This displays all open Curve windows on theALFwin Sequence Analyser workspace side by side This option is particularly useful for comparing Curve windows and can be used with the link function see Section 5 5 11 5 5 11 Linking Curve windows for comparison of clones To aid in the comparison of curves several Curve windows can be linked to compare regions of the result and simultaneously respond to a single command function 1 O pen the Curve windows that you want to compare and close all other Curve windows Select Window Tile All Curve Windows to display all of the Curve windows on the screen Adjust the position and magnification of each curve to display the regions you wish to compare Select Window Link Work with one Curve window by zooming or moving within the curve and all the linked Curve windows will respond in the same way ALFwin Sequence Analyser 5 5 5 12 Changing the axes X axis units 1 Select any open Curve window 2 Select View Properties and click on the Settings tab in the Curve Properties Layout dialogue Curve Properties Layout lt default gt Figure 5 10 Settings tab of the Curve Properties Layout dialogue 3 Select the relevant X axis Units either Bases or Time 4 Select
112. ed sequences one base position to the left select Edit Shift Left or click on the Shift Left button Conversely to shift the selected sequences one base position to the right select Edit Shift Right or click on the Shift Right button 6 21 6 Sequence Alignment If the alignment is affected by the shift the consensus sequence is automatically recalculated to reflect the new alignment Note Sequences cannot be shifted in any direction that would cause the extreme base positions to be lost from the alignment table 6 7 3 Viewing and editing bases from Sequence Alignment using the Curve window Provided that a selected sequence originates from an ALFwin ALX ALF data file you can move directly from a basein the alignment table and view its position in its Curve window 1 From the alignment table double click the base that you want to fas investigate or select the base and click on the View Curves button 2 Theraw data Curve window for the selected sequence opens with the selected base highlighted Double clicking the base in the Alignment Table HI rel iy f ii i aT i M ji in Ws I AU MAC N e of AN W opens the ates alle Te aaa aa appropriate r k Belge soy Curve window Te and highlights the base selected Figure 6 9 Showing how selecting a base in the alignment table opens the appropriate Curve window locates and highlights the base 3 Adjust the Curve window so that you can obtain a
113. ell where the cursor is visible A cell that has the black box around it but no selected text or visible cursor is not an activated cell The action of keyboard keys depends on the activated or deactivated status of the cell s Tab Return Home End Delete Escape Left arrow With deactivated cells M ove to the next cell Activate the cell select all textin thecell and position the cursor at the end of the selected text Activate the cell and posi tion the cursor at the beginning of the text Activate the cell and posi tion the cursor at the end of the text Remove all cell contents and activate the cell N othing Activate the cell and select all text The cursor is posi tioned at the beginning of the text With activated cells Confirm the cell con tents If valid move to the next cell Confirm the cell con tents If valid move to the next cell Position the cursor at the beginning of the text Position the cursor at the end of the text Remove all selected text in the cell If none is selected remove the character right of the cursor Cancel any cell edits and deactivate the cell M ove one character to the left D Keyboard shortcuts Right arrow Up arrow Down arrow Ctrl Home Ctrlnd Page up Page down D 6 Sequence Alignment D 8 Ctrl C Ctrl F Ctrl 1 Ctrl N Ctrl 0 Ctrl Z Delete Activate the cell and select M ov
114. ent shown A consensus sequence is also generated that represents the combined alignment between the sample sequences which allows base positions with imperfect alignment to be easily identified and if necessary edited The full graphical presentation provides you with a direct link to sequence curves and statistical data giving you a powerful qualitative and quantitative analysis tool for sequence alignment 6 1 6 Sequence Alignment K ey features include Alignment of areas of similarity between sequences Automated generation of a consensus sequence Location of mismatching sequence base positions Location of sequence gaps Generation of amino acid sequences Alignment export facilities Statistical summary Sample sequences are compared in the forward and complementary reverse directions and the best alignment is displayed This is useful for comparing similar sequence data from the same or different runs or locating overlapping regions between neighbouring sequencing fragments Thisis generated from the consensus of the aligned sample sequences which allows you to view the overall result Sequence base mismatch positions between aligned sample sequences are automatically located and colour coded This is useful for comparing similar sequence data from the same or different runs and editing the sequence bases Gapsin the aligned sequences are easily identified in the sample sequences and consensus sequence
115. ent statistics 6 18 6 7 Editing sequence bases from sequence alignment 6 20 6 7 1 Editing a base position 6 20 6 7 2 Shifting sequences 6 21 6 7 3 Viewing and editing bases from Sequence Alignment using the Curve window 6 22 6 7 4 Finding Ambiguities or Special Sequences 6 23 6 7 5 Hiding identical bases 6 24 6 7 6 Disabling the editing functions 6 25 6 7 7 Adjusting the number of the first alignment position 6 25 6 8 Amino acid sequences 6 26 6 8 1 Generating amino acid sequences 6 26 6 8 2 Recalculating amino acid sequences 6 27 6 8 3 Changing the translation table 6 28 6 9 The alignment log 6 28 6 10 The alignment notes 6 29 6 11 Saving the sequence alignment file 6 30 6 12 Printing an alignment report 6 30 6 13 Exporting sequence alignment data 6 32 6 14 Opening a previously saved sequence alignment item 6 34 6 15 Configuring the sequence alignment module 6 34 6 15 1 Configuring the consensus mode 6 34 6 15 2 Configuring the amino acid display 6 35 6 15 3 Configuring the operation of the alignment algorithm 6 36 6 15 4 Configuring data import 6 38 6 15 5 Changing the colour settings 6 40 6 15 6 Changing the font settings 6 41 7 Troubleshooting vii Contents A System requirements A 1 Computer A 1 A 2 Supported printers A 1 B Short guide to Windows 95 B 1 Starting Windows 95 and logging on B 1 B 2 What is on your screen B 1 B 3 Getting started in Windows 95 B 3 1 Start button B 3 2 Basic mouse functions in Windows 95
116. equence Alignment Advanced dialogue opens 6 37 6 Sequence Alignment 6 38 ALFwin Sequence Alignment Advanced EQ Match Score Settings Default settings No gaps expected except in the beginning Small gaps expected G Cancel Help Figure 6 20 Sequence Alignment Advanced dialogue Use this dialogue to select the algorithm settings used by the alignment algorithm Y ou can select the algorithm according to the number of gaps you expect in the sequences Default settings e No gaps expected except in the beginning Small gaps expected The method used to align any two sequences is suggested by Needleman and Wunsch 1970 M ol Biol 48 444 It is based on dynamic programming and involves setting quite a few different parameters The parameters of the algorithm affect the final alignment and depending on the quality of the sequences to align different settings of the alignment algorithm parameters should be used 6 Click on OK to close this dialogue 6 15 4 Configuring data import Data can be imported in the following formats Amersham Biosciences alf or alx result files e STADEN SCF files only the sequences are imported e ASCII files ALFwin data files include information about thestatistical significance of bases they contain and you can configure the import procedure to import all the bases or just the upright bases Sequence Alignment 6 SCF and ASCII files do not include information ab
117. equence Analyser 5 5 ALFwin Sequence Analyser After you have obtained the run data in ALFwin Control the results can be evaluated in ALFwin Sequence Analyser ALFwin Sequence Analyser should be used to achieve four main goals e To extract a nucleotide sequence from the raw data using the automated data processing algorithms To present the data on paper by printing either direct from screen or by use of the powerful Report function e To export the data in appropriate formats for direct use in other software applications e To check that the run was successful e To analyse the sequence data and make judged improvements to the quality of the data DRG n LDO a tassel aoe 2a a ed Sd a aE tai 3 M f j i i Ny il 4 ii i 5 K J MA tii UN AMAL HK a Figure 5 1 Typical screen display in ALFwin Sequence Analyser In order to achieve these goals ALFwin Sequence Analyser has been designed with a wide range of powerful tools including Data processing options ALFwin Sequence Analyser contains many processing options for example altering the stringency of the data processing based on the degree of heterozygocity in the 5 1 5 ALFwin Sequence Analyser 5 2 Editing facilities Sequence alignment Presentation tools samples automatically or manually defining the start times of the run edit shift in raw data curves to account for heterogeneous running speed in the gel Powerful editi
118. equences at a time which means that all the multiple alignment operations are performed in pairs The included sequences can thus be paired in different ways and depending on what sequences that are aligned during the calculation the final alignment may become different There are a few different theories about how to Sequence Alignment 6 combine sequences in the alignment procedure and they are designed to work with different amounts and qualities of the sequences Some of the methods suggested work quickly but produce reduced quality of the final alignment Other algorithms use iterative procedures that take more time but generate better final alignments For more details concerning multiple alignment algorithms consult the paper Comprehensive study of iterative algorithms of multiple sequence alignment by M akoto H irosawa CABIOS Vol 11 no 11995 where comparisons are made between the effect of different multiple alignment algorithms Multiple Sequence Alignment Algorithms x Simple Tree Based i good fin Align Align Align Align Align Figure 6 19 Multiple Sequence Alignment Algorithms dialogue In ALFwin 2 00 the Simple Tree Based algorithm is applied This algorithm works quickly and is designed to work best when the quality of the included sequences is about the same T he algorithm is straight forward and teams up the sequences in a binary tree fashion Click on the Advanced button The ALFwin S
119. er according to the axes you want to display on the borders of the Gel View 4 Select or deselect Show bases When this option is selected the base positions are superimposed in a different colour on the Gel lanes of processed data 5 Click on Apply to effect the change without closing the dialogue 6 When you are satisfied with the settings click on OK 5 7 Processing run data 5 30 The key purpose of the ALFwin software application is that a nucleotide sequence can be extracted from the raw sequencing data The algorithms used to calculate the nucleotide sequence see Appendix C are extremely accurate in calling the correct order of bases as they were detected on the gel but also sensitive in being able to sort a real peak out from base signal levels and to differentiate individual peaks from close running fragments in the gel ALFwin Sequence Analyser allows you to process the raw data with various parameters applied to improve the quality of the result Such parameters include being able to set the stringency level of the data processing based on the degree of heterozygocity expected in the samples or to manually set the start and stop time for the data included in the processing Following data processing various numeric values are given that give information and clues about the success of therun Viewing the data curve directly from the processing dialogues requires only a double click of the mouse button Working in the
120. er bases 5 35 cascade curve windows 5 16 casebook adding notes after a run 2 17 adding sample information after a run 2 17 analysis notes 4 37 cbk casebook files 4 8 conditions 4 8 conditions detail 4 17 conditions tab 2 8 creating a new casebook 4 11 discard 4 13 edit again 4 13 editing 4 13 editing during a run 4 9 entering information 2 6 file information 4 8 file information detail 4 15 in result file 5 7 introduction 4 1 4 8 modifying parameters 4 15 new 4 11 notes 4 9 notes tab 4 37 open casebook file 4 9 opening a casebook file 2 6 post run actions 4 9 post run actions detail 4 28 prerun notes 4 37 printing 4 14 run notes 4 37 4 46 sample information 4 9 sample information detail 4 20 sample information retention 4 25 sample information tab 2 8 Index vi saving 4 13 viewing casebook information 2 16 5 45 cbk casebook files 4 8 cells adjusting height 4 25 adjusting height of row in alignment table 6 14 adjusting the width of a column in alignment table 6 14 adjusting width 4 24 gridlines hiding or showing 6 14 moving and copying cell contents 4 23 selecting in the sample information tab 4 22 Change to 4 19 4 20 check both directions 6 36 check current direction 6 36 clones adding for alignment 6 7 adding sample information 4 21 altering the lane order 4 21 comparing by linking curve windows 5 16 copying images into documents 5 52 cutting clone curves 5 44 exporting 4 32 icons in the Result windo
121. es include manual adjustments you make and step adjustments that have been programmed into the casebook The Curve window is active throughout the course of the run so that you can observe the output of the light detectors and assess the quality of the raw curve data see Section 4 6 At the end of a run ALFwin Control automatically saves the result file and implements any post run procedures that you programmed in the casebook see Section 4 7 4 5 1 Preset and prepare the instrument To preset the analyser 1 M ount the gel cassette into the instrument and connect the thermostatic water circulator connectors 2 Select Run Preset or click on the Preset button T he run conditions are sent to the instrument The preset order is logged in the Logbook Y ou can also see the set conditions in the Manual window on your screen Black numbers are set values Green numbers are actual values Note You can set the temperature in the Manual window before you open or create the casebook The temperature increases while you work with the casebook and saves you time later T ype the selected temperature and click on Set The ALFexpress family instruments switch the heater on automatically but if you are using an ALF or ALFred instrument you must click ON at the Heater in the Manual window 4 39 7 win Control 4 40 Figure 4 23 Manual window in ALFexpress II Note that if you are using ALFexpress the Manual window does not hav
122. es included multiplied by the match score value Maximum total score Alignment Ratio lt _ eee il Minimum langth Maleh soore W here Total score is the resulting alignment score obtained by aligning two sequences according to the N eedleman Wunsch algorithm Maximum total score is the maximum score from forward and reverse complement alignment of two sequences Minimum length is the length of the shortest sequence in an alignment of two sequences 6 18 Sequence Alignment 6 M atch score is the score assigned to a single match of identical bases in two aligned sequences This is a crude measure but generally gives an overall quality measure of the alignment The optimal Mean of Pairwise Alignment Ratio is 1 which is obtained if both aligned sequences are identical Therefore the closer the mean value is to 1 the better the quality of thealignment If the Minimum and Maximum Pairwise Alignment Ratios are not close it indicates a lower degree of similarity between the sequences especially if the minimum ratio is very small In such a case it is advisable to remove the sequence s that appear not to align well with the others Conversely if the minimum and maximum ratios are close all of the included sequences show a high degree of similarity It is not possible to get the specific alignment ratios between any two sequences since most of the alignment operations are actually performed on virtual sequences whic
123. es tab 3 Type your notes into the Notes field 6 29 6 Sequence Alignment 4 Toprint thenotes copy them by pressing the lt Ctrl C gt keys then paste them into a text application Usethetext application to print the notes 5 Usethe close button to remove the dialogue or select another window 6 11 Saving the sequence alignment file A sequence alignment result can be saved on file The file contains all of the alignment data the alignment log and notes The file is recognised as an alignment file by the extension sqa name activate the Sequence Alignment window and select File Save or click on the Save button fell To save the alignment file in the same location using the existing file To save using a different name or location 1 Select File Save As The Save As dialogue opens 2 Usethe dialogue to select the folder where the file is to be saved 3 Enter a name for the file 4 Click on Save 6 12 Printing an alignment report An alignment report is a printout that includes items about selected sequences present in the current alignment item Y ou select the items you want to include by means of the Alignment Report Setup dialogue 6 30 Sequence Alignment 6 Sequences r Alignment item data a IM Alignment item table Mv Seq1 V Seq2 T Log M Seq3 Pi Seq 4 I Notes M Seq5 I Alignment settings Alignment results I Statistics M Overview T Result view i m Prin
124. etter you want at that position Replace mode is the default editing mode indicated by the check mark beside the Edit Replace option Replace mode toggles with the Insert mode aje To insert a base sample sequences only PeT To delete a base To undo the last edit action Sequence Alignment 6 To insert a base first activate the Insert mode by selecting Edit Insert or clicking on the Insert Mode button Select the base to the right of the insert position Type the letter you want to insert and the baseis inserted All bases to the right are shifted one position An active Insert mode is indicated by the check mark beside the Edit Insert option Insert mode toggles with the R eplace mode Click on the base that you want to delete Select Edit Delete or press the D e gt key Deletions are emphasised by a bold left border to thecell from which the base was deleted Select Edit Undo or press the lt Ctrl Z gt keys Only the last edit operation can be undone Note By default you will receive a warning when you edit a base in the consensus sequence If you do not want to see this warning deselect uncheck the View User Preferences Warn on Consensus Editing command 6 7 2 Shifting sequences It is possible to shift one or several sequences in the alignment table to the left or right of the current position 1 Select the sequences that you want to shift in the alignment table 2 To shift the select
125. ew Details are provided in Section 5 5 3 To configure these options on or off 1 Click anywhere in the appropriate Curve window 2 Select View Properties and click on the Style tab in the Curve Properties Layout dialogue 3 Select the items required from the Style options Deselect those items not required 4 To effect the changes without closing the dialogue click on Apply To close the dialogue and effect the changes click on OK 5 5 21 Configuring the sequence display Y ou can configure the way in which base sequences are shown and edited in the Curve window 1 Click anywhere in the appropriate Curve window 2 Select View Properties and click on the Sequence tab in the Curve Properties Layout dialogue ALFwin Sequence Analyser 5 3 Select deselect the options available Edit Sequence to allow disallow editing of sequence bases Show Edited Bases to show hide underlining of edited bases Show Italics to show italic bases or display them as upright bases Complement to show the complement normal sequence Reverse to show the reverse forward sequence 5 5 22 Saving or opening a layout for the Curve window Y ou can save a Curve window layout for later use All user definable properties of the curve window are saved To save a layout or open a saved layout 1 Select the relevant Curve window 2 Select View Properties and click on the File tab in the Curve Properties Layout dialogue 3 Use the followin
126. f each logged action the User name the location of the original file of each sequence included in the alignment the stages performed in producing the alignment the statistics of the alignment editing actions performed on the sequences and bases alignment algorithm data The log is saved in the sqa sequence alignment file on completion of the alignment procedures To view the log 1 From the Sequence Alignment dialogue select View Alignment Log Notes T he Log Notes dialogue opens Select the Log tab to view the logged information Use the close button to remove the dialogue or select another window Sequence Alignment 6 AL Fein Ai gee L cog hpi e EHEH ES spao CPvo gina F ke Bha Fan Hava E PHEA SE Hapa Cn Fih AL Fai Hl IHd S i Ca F y Bhi Al Faia Harra EHG jd E SG sip Prigen F iri Bahi Al Foe Hir mie S70 4 SE eguna Proce Fier jeh A Fen Horne BAA Oo gerd oe al cere imisi aff Figure 6 14 Alignment Log Notes dialogue Log tab 6 10 The alignment notes Y ou use the notes facility to record your own observations while using the sequence alignment module When you save the alignment file the notes you make are saved with the rest of the data To make notes 1 From the Sequence Alignment dialogue select View Alignment Log Notes T he Log Notes dialogue opens ALF win Alignment Log Notes x Log Notes Figure 6 15 Alignment Log Notes dialogue Notes tab 2 Select the Not
127. g functions from the File tab Save Layout As button Allows you to save the layout for later use as a lyt file The last used layout file will be applied by default the next time ALFwin Sequence Analyser is started by the same user Open Layout button Allows you to open a saved layout 4 Click on OK 5 25 5 ALFwin Sequence Analyser 5 6 Presenting the run in the Gel View window 5 26 ye To open the Gel View window select View Gel or click on the Show Gel button em sac D0 wets reel Wee Figure 5 15 Gel View window The Gel View window gives a pictorial representation of the bands in each lane in the sequencing gel These can be used to check the primer peaks background noise and signal levels Primer peaks are shown as black bands in the lower part of the gel whereas the sample bands are shown higher up in the gel in different grey scale densities dependent on the strength of the signals 5 6 1 Viewing raw data or processed data If the data have already been processed it is possible to toggle between viewing the processed data and the raw data To view the raw data select either View Raw Data press the lt F5 gt key or click on the Raw Data toolbar button Conversely to view the processed data select either View Processed Data press the 3 6 gt key or click on the Processed Data toolbar button By default the gel view first shows the raw data Y ou can change this default 1 Select the Gel View win
128. gn the sequences 1 Open result files containing sequences and or import sequences 2 Create a new analysis item 3 Align the sequences 4 If necessary edit the sequence alignment result 5 Generate the amino acid sequence in the desired reading frame Saving and printing Save File Save lt Ctrl S gt Save As File Save As Print File Print lt Ctrl P gt Print all File Print All lt Ctrl Shift P gt Report File Report Report alignment File Report Alignment Export to metafile File Export to Metafile Export File Export Export alignment File Export Alignment Split file File Split File Copy Edit Copy Clone Sequence Gel lt Ctrl C gt Amersham e Biosciences 18 1125 90 Edition AA Printed in Sweden by Tryck Jouren 9803
129. gous positions 5 32 settings 6 38 align all 6 8 align sequences 6 8 align to previous 6 10 alignment adding clones 6 7 adding more sequences 6 9 algorithm configuring 6 36 alignment log 6 28 alignment notes 6 29 areas of similarity between sequences 6 2 automated generation of a consensus sequence 6 2 blue line 6 10 configuring data import 6 38 disabling the editing functions 6 25 displaying the sequence alignment results 6 10 export facilities 6 2 exporting sequences 6 32 generation of amino acid sequences 6 2 imperfections 6 11 imperfections colour code 6 13 interactive editing features 6 3 investigating imperfections 6 12 location of mismatching sequence base positions 6 2 location of sequence gaps 6 2 overview display 6 10 printing report 6 30 red line 6 10 saving the sequence alignment file 6 30 selecting sequences for alignment 6 4 selecting the sequence orientation 6 36 sequence directions 6 10 showing ambiguities 6 12 showing gaps 6 12 showing mismatches 6 12 statistical summary 6 2 using clones from Evaluation 6 4 viewing amino acid sequences 6 26 viewing base position in curve window 6 22 alignment table adjusting 6 14 adjusting the height of a row of cells 6 14 adjusting the number of the first base 6 25 adjusting the width of a column of cells 6 14 colour settings configuring 6 40 configuring font 6 41 Index display 6 12 display of data in table 6 12 font 6 41 gridlines hidi
130. h are sequences that make up a consensus between two or more other sequences T hese intermediate consensus sequences are not registered anywhere Only the final alignment and the final consensus are listed This quality measure should not be subject to any deeper study of the alignment since it is quite crude It gives some clues as to whether the sequences are similar or not and it can also be useful when trying out different algorithm settings The setting that gives the highest mean alignment ratio is in most cases the setting that gives the best final alignment Sequence specific statistics The statistics give the following information about alignment imperfections Number of mismatches This is the number of resolved base component mismatches in the sample sequence s Number of Ambiguities This is the number of unresolved base component mismatches ambiguities in the sample sequence s Number of gaps This is the number of gaps in the sequence s Number of edit operations This is the number of base positions that have been edited in the sequence s of the alignment 6 19 6 Sequence Alignment 6 7 Editing sequence bases from sequence alignment 6 20 Itis possible to edit individual bases in all of the sample sequences and also in the consensus sequence to improve the overall quality of the data This is achieved by editing the bases directly in the alignment table Provided that a selected sequence originates from
131. he character that you want to use to replace the perfectly matched bases Sequence Alignment 6 Alternatively type another character that you want here 4 Select OK to close the dialogue and make the changes in the alignment table To return the normal display of bases repeat the above procedure but select Default from the options available in the Replace Identical Character drop down list When you close ALFwin the setting is retained and becomes the default when the application is run again 6 7 6 Disabling the editing functions The sequence alignment module is supplied with the editing functions enabled To disable or re enable the base editing functions select View User Preferences and deselect or select Allow Sequence Editing When you close ALFwin the setting is retained and becomes the default when the application is run again 6 7 7 Adjusting the number of the first alignment position By default the first position in the alignment table is labelled as number 1 Y ou can adjust this start label to allow for bases earlier in the sequences which are not part of the alignment To change the starting base number 1 Select View Properties to open the ALFwin Alignment Properties dialogue 2 Select the Alignment Item Properties tab 2 Typein the Start Label For Bases field the number of the first base 3 Select OK to close the dialogue and make the changes in the alignment table 6 25 6 Sequence
132. he alx files for different runs Help Starts Windows 95 Help Y ou can then use the H elp Contents Index or other tabs to find out how to performatask in Windows 95 Run Starts a program or opens a folder when you type an M S DOS command Shut Down Shuts down or restarts your computer or logs you off If you have installed and later closed other applications it can be useful to shut down and restart the computer to clear memory B 3 2 Basic mouse functions in Windows 95 Use the mouse to select items activate items drag and drop items or view right mouse button command options Selecting items Place the mouse pointer over the item of interest and click once with the left mouse button to select it note the grey shading of the selected item To select several items on the desktop either hold down the lt Ctrl gt key on the keyboard when you select items as described above Alternatively place the mouse pointer anywhere outside of the icons of interest and click and hold the left mouse button M ove the mouse pointer to drag a B 3 B Short guide to Windows 95 B 4 Activating items Dragging and dropping items Right mouse button menus box around the icons of interest and release the mouse button All of the items are selected To view the contents of a folder on the desktop double click on it with the left mouse button Similarly to launch a software application double click on its icon Y ou can
133. he Start button in the lower left corner on the Windows 95 taskbar to launch a software application open a document change the system settings get help find items on your computer and more see Section B 3 1 The Start button is located on the taskbar for launching programs The taskbar also shows an icon for each of the applications that you open To switch between applications click on the icon for the application you want W hen you close a window its icon disappears from the taskbar Start p Taskbar button rogram dgona control s Recycle Bin The Recycle Bin is a temporary storage oy Recycle Bin B 3 Getting started in Windows 95 place for deleted files You can use it to retrieve files deleted by mistake This section gives you a quick overview of getting started in Windows 95 B 3 1 Start button The Start button is in the lower left corner of your screen located on the Windows 95 taskbar When you click on the Start button a menu cascade is displayed containing everything you need to begin using Windows 95 B 2 Short guide Windows 95 B Programs Displays a list of programs you can start Documents Displays a list of documents that you have opened previously Settings Displays a list of icons that can be opened to allow you to change the settings for your computer Find Enables you to find a file folder file shared computer or mail message H ere you can search for example t
134. he active view By this means you can obtain a print of a Curve or Gel View or a sequence 1 2 Click on the view required to ensure that it is activated Adjust the window size and contents to display the region that you want to print If you want to check that the print is appropriate to your requirements select File Print Preview A preview of the printed document is shown on screen Use the buttons to adjust the view Next page and Previous page to navigate a multipage table not applicable in this instance One Page Two Page to adjust the number of table pages displayed not applicable in this instance Zoom in and Zoom out to adjust the magnification of the print image To print from the print preview click on the Print button Otherwise click on the Close button to close the preview To print from a selected window select File Print The Print dialogue opens Use the dialogue according to standard Windows 95 conventions 5 13 2 Printing all views The Print All command allows you to print all the open Curve windows 1 O pen the Curve window s and make adjustments so that they display the region you want to print 5 57 5 ALFwin Sequence Analyser 5 14 Using tools 5 58 2 If you want to check that the print is appropriate to your requirements select File Print All Preview A preview of the printed document is shown on screen Use the buttons to adjust the view Next page and Prev
135. he alignment algorithm or that not enough memory exists for the alignment to be performed Solution Try to set another algorithm setting or to release memory by closing other applications or closing files in Sequence Analyser 7 5 Troubleshooting 7 6 System requirements A A System requirements A 1 Computer Computer RAM memory Recommended free hard disk space Operating system Graphics Serial ports A 2 Supported printers Compaq PC Pentium 233 M H z minimum Pentium 75 M H z for one instrument 64 M B minimum 32 M B 300 M B if running two ALFexpress systems simultaneously Windows 95 international edition including Service release 2 4 00 950b 800 x 600 pixels with 64k colours minimum 800 x 600 pixels SuperV GA graphics adapter 256 colours 2 serial ports are recommended allows two instruments to be run Thefollowing printers have been tested for compatibility with ALFwin Sequence Analyser 2 00 HP LaserJet 4M and 5M P HP DeskJet 870Cxi and 890CXi A 1 A System requirements A 2 Short guide Windows 95 B B Short guide to Windows 95 This appendix gives a brief introduction to Windows 95 for beginners For further information you are recommended to read the M icrosoft publications for using Windows 95 and other associated literature The following topics are covered e Starting Windows 95 and logging on e What ison your screen e Getting started in Windows 95 e If
136. he program 1 6 Place the mouse pointer anywhere on the Windows 95 desktop but not on an icon and click on the right mouse button A pop up menu is displayed Select New and then Shortcut The Create Shortcut dialogue is displayed Click on the Browse button to display the Browse dialogue Locate and select the program filefor which you want to create a shortcut Click on Open to return to the Create Shortcut dialogue Click on the Next button to display the Select a Title for the Program dialogue Enter a name for the program if different to the one displayed Click on Finish and the dialogue closes A shortcut icon to the program will appear on the desktop To start the program using the shortcut double click on the icon B 4 If you have used an earlier version of Windows B 6 For those of you who have used an earlier version of Windows typically Windows version 3 1 a few of the common functions have been changed Windows 3 x x function Windows 95 equivalent Program M anager Start button on the Windows 95 taskbar File M anager Windows Explorer located in the Start button menu under Programs MS DOS Prompt Control Panel Print M anager Run Command Short guide Windows 95 B MS DOS Prompt located in the Start button menu under Programs Control Panel located in the Start button menu under Settings Printers located in the Start button menu under Settings Run located in the Start butto
137. heclone name the clone number in the Sample Information tab Thefilewill be named using the result file name the clone number Type a name Files are saved using this name the clone number 7 Usethe Path field to specify thelocation for the exported file T ype the path or click on Browse to select the required folder 8 Click on OK to complete setting up the automation of export sequence Automating the printing of a report The printed report consists of result curves and information from the casebook Y ou select the type of information that you require in the report 1 From the Post run Actions tab in the Casebook dialogue select Report 2 Click on Setup The Report Settings dialogue is shown Hepsi atispa a ee ee apari ai Deepa maar D Hesden F cae Eo ine 5 Taba D Seeded curves Le Curve radh i Sh F meri iF Do FP ihre sidham Figure 4 20 Report Settings dialogue ALFwin Control 4 Farge C Dima Alb ipj beet 7 Fem bee E A eime fF i io hata Eih Turas m inaha Fa Fe r Pome Ee FF F ei ka jopa E Foi C Corpin C Ma O Cope La 3 You can use a report setting that was created and saved in a previous analysis see step 10 below To recall this setting click on Open Layout and use the Open dialogue to select the path and the rpt filename Click on OK to load the settings Y ou can usethe following procedures to edit the settings Note The l
138. hese Labels can be edited by clicking on thenameto highlight it and typing in the new name Thisis the clone name and number for the sequence as displayed in the source Result window N ote that sequences imported from other formats are placed into a Result window and are given a clone name and number This displays the source file name for the sequence This shows the length of the sequence 6 1 2 Adding further sequences before alignment If you want to add further sequences to the sequence alignment list before performing an alignment 1 Open the result files or import the files containing additional sequences 2 Select each newly opened Result window in turn and select the sequences to be added Select Analysis Add To Alignment Sequences are added to the sequence alignment list in the Alignment Item window 6 1 3 Changing the order of sequences To change the order of the sequences in the sequence alignment list click a sequence name in the sequence alignment list and drag it to the required position 6 1 4 Removing sequences To remove sequences from the sequence alignment list 1 Select the sequence s from the list that you want to remove Press the lt Ctrl gt key to select several sequences 2 Select Edit Remove sequences The sequences are removed from the list 6 7 6 Sequence Alignment 6 2 Aligning the sequences 6 8 This section describes how to perform an alignment 6 2 1 Aligning all sa
139. hether it is raw data or if the data have been processed Raw data the run data received from the I F instrument mE z Processed data these data have been processed i e the bases in the sequence have been called by the base calling algorithm ae Edited processed data these data have been processed and then manually edited These icons are displayed in other windows with respect to the clone data being presented 5 9 5 ALFwin Sequence Analyser 5 4 2 Viewing the lane information The sequencing lanes for a clone can be viewed by clicking on the symbol beside the clone or conversely hidden from view by clicking on the corresponding symbol Information about the base component for each lane and the lane number is shown EB SeqDemo alx Result of x nan SeqD emo alx Ses Clone Smt DNA A Lanell C Lanef2 G LaneO3 T Lanel4 AT Clonel2 Smt DNA AT Clone03 Smt DNA RT CloneO4 Smt DNA AT Clone05 Smt DNA RT CloneQ6 Smt DNA RT CloneO Smt DNA Open Open all Help Figure 5 7 Result window showing the sequencing lanes for the first clone H H A 5 5 Presenting curves in the Curve window 5 10 The ability to view and manipulate the raw data curves and processed data curves in the Curve window is essential to the successful evaluation and editing of data using the various features of ALFwin Sequence Analyser The Curve window gives a graphical represe
140. ich describes this procedure 5 3 1 Contents of a result file A result file contains the following Raw data Raw data curves for each of the clones included in the run Casebook This contains all documentation that you included in the casebook in the Control modulein addition to the logbook and evaluation log information This information cannot be changed although new notes regarding the evaluation process and sample information can beadded in addition to the automatic generation of an evaluation log Processed data The processed data curves for each of the clones included in the run sequence are included if they were generated by post run action or added here after processing has been performed and the result saved Sequence The bases called by the base calling algorithm 5 7 5 ALFwin Sequence Analyser 5 8 5 3 2 To open a result file To open a result file Ej t Select File Open or click on the Open button The Open dialogueis shown Alternatively you can directly open one of the eight most recently saved files from the list in the File menu In the Open dialogue select the drive and folder containing the appropriate alx or alf result file Select the file and click on Open Y ou can open several files by pressing the lt Ctrl gt key while you select the files In this case all the files selected will be opened when you select Open Open 21x Look in E David z al cl i ja DEMOrund 002 alx File
141. icon only or M aximise Return to previous size Drag the border of the window by pointing at the window border pressing and holding the left mouse button and dragging the window border to appropriate size B 3 5 Closing a window To close a window or exit a program click the close button button with a cross in the upper right corner of the window next to the Minimise and M aximise buttons B 3 6 Moving windows Y ou can move windows on the screen by pointing at the window title bar pressing and holding the left mouse button and dragging the window to its new position B 3 7 Using toolbars Toolbars bars with buttons appear in many application software and provide quick ways to perform tasks M ost toolbar buttons correspond to available menu commands B 3 8 Scrolling windows If a window is not large enough to display all the information a scroll bar appears at theside and or bottom of the window Y ou can drag the scroll box or click on the scroll arrows B 5 B Short guide to Windows 95 B 3 9 On line Help Use the on line help function if you need help Y ou can find help for Windows 95 under the Start button M ost software applications also have extensive help facilities accessed either from the Help menu in the program or by clicking on a Help button in a dialogue B 3 10 Creating a shortcut Y ou can create a shortcut to a program thus enabling you to click on an icon on the Windows 95 desktop to start t
142. ignment calculation determines the best direction of the sequences for alignment purposes Y ou can manually change the direction of all sequences by selecting checking Edit Rev Comp Sequences This reverses the direction of all displayed sequences This is especially useful in determining the correct sequencing direction with respect to the amino acid sequence see Section 6 8 Original alignment display Complementary and Reverse alignment display Blue line for sequences in the forward direction becomes red line in complementary reverse direction Red line for sequences in complementary reverse direction becomes blue line for sequences in the forward direction 6 3 2 Consensus line A black consensus sequence line is displayed above the sample sequence lines The consensus sequence is automatically generated during the alignment process and is a representative sequence of all aligned sample sequences Further details are given in Section 6 5 6 3 3 Rectangular cursor A rectangular cursor spans several or all of the sequence lines corresponding to the visible number of sequences in the alignment table above The position and width of the rectangular cursor relates to the area of the spanned sequences for which specific alignment details are shown in the alignment table Use the mouse pointer to drag the cursor rectangle to any desired position to display a new view in the alignment table or double click on the desired porti
143. igure 5 20 Find dialogue 3 Select Ambiguity or Sequence If Sequence is selected type in the Sequence field the sequence required For both forward and reverse curves start with the base 5 35 5 ALFwin Sequence Analyser 5 36 5 6 with the lowest number in the sequence Click Find Previous to search to the left in the curves or click Find Next to search to the right The first found ambiguity sequence is highlighted in the Curve window Click Find Previous or Find Next again to continue the search Click Close to end the search 5 7 4 Assigning upright status to bases If you want to assign upright status to bases that the processing algorithm has calculated with less certainty namely italic bases or small bases 1 Display the raw or processed data curves and sequences for the appropriate clone either from the Process Status dialogue or the Result window Select Edit Mark Upright Bases The Mark Upright Bases dialogue is shown Click in the Start field and enter the number of the first base to be marked by typing or by clicking on the start point in the Curve window Click in the End field and enter the number of the last base to be marked or click on the end point in the Curve window Click OK to mark all the selected bases as upright bases Note Asan alternative to steps 3 4 in the above procedure bases can be marked directly in the Curve window Hiding the italic status of bases Y ou can ch
144. ile saved at the end of arun This includes the record of all events which is stored in the run and evaluation logs In the casebook copy you can check which Post run Actions were selected read the Run conditions read add or change Sample Information read the result File Information read the Pre run and Run notes write Analysis notes read the Logbook and Analysis Log 5 45 5 ALFwin Sequence Analyser To view or edit the copy of the Casebook in the result file 1 Select Edit Casebook or click the Edit Casebook button EE The Casebook dialogue is shown File information Conditions Sample Information Post Run Actions Notes Logbook Evaluation Log gt Name Casebook name C Date C Name ey Iv Add cournter no tahleneme File path Home m Information Operator David Run date f36 08 1 5 Last modified by Modification date For system _ ALFexpress Casebook Demo cbk Cancel Figure 5 24 Casebook dialogue Help 2 Select the Sample Information or Notes tabs to change or add information in the casebook 3 Use the remaining tabs to access read only information in the Casebook 4 Click on OK and the changes and additions will besaved when you save the result file 5 10 Saving the result file ALFwin Sequence Analyser allows you to save the result file as one complete file or in two or more parts N ormally you save as one file but you may want to
145. iles of type ALF files alf alx 7 Cancel Figure 2 10 Open dialogue The Result window is displayed in the Sequence Analyser workspace The Result window contains a tree structure of all the clones contained in the opened result file All clones in this result file have the raw data icon E Myresult 001 alx Result OF x det TOT M13 d Clone02 M13 d Clone03 M13 d Clone04 M13 d Clone05 M13 d CloneO6 M13 dar CloneO M13 d Clone08 M13 d Clone09 M13 d Clonel10 M13 Open Openal Help Figure 2 11 Result window H 2 Making your first run 3 To view the lane information for a clone click on the symbol beside the clone Conversely to hide lane information from view click on the corresponding symbol Myresult 001 alx 4 Clone01 M13 A Lanell C Lane02 G LaneO3 T Lane04 4e Clone02 M13 4 Clone03 M13 d Clone04 M13 za F Figure 2 12 Result window lane information 2 3 3 Processing the data 1 Select Analyse Process The Process Status dialogue opens fe H paH as Peon Shade vopna Lja HEBER E E mmi me Ue ppd ah een te m aair i taguad pertal 7 Ube haa pt Taga Figure 2 13 Process Status dialogue 2 Click on Process All 3 When processing is complete observe the following T e Theicon to the left of th
146. in Control 4 3 Release the mouse button Resetting the magnification To reset the magnification factor to the default level select View Reset Zoom or press the lt 9 gt key 4 8 7 Selecting the group to be shown To choose the group of lanes to be shown in the Curve window open the list of Groups in the toolbar and select the group required Alternatively double click in the Curve window to show the next By default Group 1 displays the curves for all of the lanes and Group 2 displays the lanes containing external standards The remaining default groups Group 1 to Group 5 show groups of eight curves together The next section describes how you can create your own lane groups 4 8 8 Creating your own lane grouping To create your own lane group 1 Click on the Curve window to make sure that it is activated 2 Select View Properties The Curve Properties dialogue is shown Carve Pompesties Laem cepa Ven seange sim Fae Figure 4 29 Curve Properties dialogue View tab for ALFexpress II If you are using ALFexpress Transmittance is instead expressed as Laser value 7 win Control The Group name menu lists the names of existing groups The Items in group field lists the lanes included in the group currently selected in the Group name list The contents of the present group are highlighted Any instrument data included with the curves is highlighted in the Instrument data field 3 Click on Add Gro
147. in Sequence Analyser 2 00 is supplied with a default casebook Sequencelst cbk located in the Default folder Run conditions voltage current power sample interval temperature and time for different experimental applications can be obtained from the Recommended O perating Procedures located on the internet page www amershambiosciences com Casebook contents The contents of the casebook file are separated into several tabs File Information After a run the data logbook and a copy of the casebook are saved as a result file This tab allows you to specify the name and location of the result file so that saving can proceed automatically Conditions These are the run settings for the ALF family instrument controlled by ALFwin Control These settings specify the run time gel temperature the voltage current and power limits and thesampling interval These settings can be automatically stepped or ramped to different values during the course of arun ALFwin Control 4 Sample Information This tab allows you to store information about the clones and the lanes in the gel Post run Actions This tab allows you to specify activities that will proceed automatically when the run is completed T hese include data processing export printing a report launching tools and file backup Notes Y ou use the casebook to make your pre run run and analysis post run notes The casebook manages note taking allowing access only to the ap
148. ing to be applied using the options available in the Type of processing field O nly one type of processing is allowed at a time Select Normal when no heterozygous positions are expected O therwise select one of the heterozygote modes depending on how sensitive the algorithm should be to ambiguity codes Low Heterozygote There is a low expectancy of finding ambiguity codes Medium Heterozygote Thereisa medium expectancy of finding ambiguity codes High Heterozygote There is a high expectancy of finding ambiguity codes Select Auto Start Time so that processing starts after the highest found primer peak or after a stop or where the raw data curves have a normal form Select Auto Stop Time so that processing stops after the last found peak If the program is unable to make a normal evaluation using the auto settings you can try using manual start time Y ou can also use manual stop time The procedure is described in Section 5 7 6 Select Auto Shift so that the software calculates the shifts to be used If the shift of lanes in the gel is too large to be corrected automatically by the program it will be necessary to shift the curves manually This procedure is described in Section 5 7 5 Click on Process to execute base calling on the selected clones or click on Process All to process all the clones To halt processing at any point press the lt Esc gt key The progress of the raw data processing can be observed in the Statu
149. ious page to navigate a multipage document One Page Two Page Zoom in and Zoom out to adjust the number of pages displayed to adjust the image magnification 6 To print from the print preview click on the Print button Otherwise click on the Close button to close the preview 7 To print from a selected window select File Print All The Print dialogue opens Use the dialogue according to standard Windows 95 conventions Tools are shortcuts to other programs Add shortcuts to programs that you often use in connection with ALFwin Sequence Analyser for example Microsoft Excel or Word HLA Sequityper M utation Analyser etc Use tools to open these programs directly from ALFwin Sequence Analyser The tools supplied with ALFwin Sequence Analyser ALFwin Sequence Analyser is supplied with two tools Convert ALX ALF to TIFF Tagged Image File Format is a Decompress ALF File commonly used bitmap format that allows graphical images to be imported into other applications such as Image M aster ALFwin Sequence Analyser accepts alx and alf file formats The alf format preceeded the present alx format If the alf files have been saved in compressed format they must be decompressed before they are opened by ALFwin Sequence Analyser ALFwin Sequence Analyser 5 Viewing settings for the supplied tools The Tools dialogue allows you to define up to six tools that can be launched directly from the Tools menu in ALFwi
150. itle bar and in the Windows 95 taskbar confirms the run has started Wait until you can see curves in the Curve window to confirm that the electrophoresis is running 4 6 Activities that can be performed during a run 4 42 There are various activities that you can perform during a run These include Pausing the run see Section 4 6 1 M anually ending the run see Section 4 6 2 Reloading the gel see Section 4 6 3 Pause Continue End ALFwin Control 4 e Changing run conditions see Section 4 6 4 e Analysing running samples see Section 4 6 5 e Viewing curve amplitude information see Section 4 6 6 Writing run notes in the casebook see Section 4 6 7 e Changing the screen layout see Section 4 8 Starting a run on another instrument see Section 4 6 8 e Analysing other result files see Chapter 5 4 6 1 Pausing the run Y ou may need to pause the run to check or adjust the instrument Do not pausefor morethan a couple of minutes otherwise the samples will diffuse O pening the lid on the instrument pauses the run until the lid is closed again To pause the run Select Run Pause or click on the Pause button Therun stops and the green lights turn red N ote that the laser and the heater are not affected by Pause To continue the run select Run Continue or click on the Continue button A message counting down the time before the run resumes is displayed the time delay allows the instrument to check
151. k window Y ou can select the font to be used in the Logbook window The font settings are saved for the current user and are used when ALFwin Control is next started To change font 1 Click on the Logbook window to make sure that it is activated 2 Select View Properties The Logbook View Properties dialogue is shown 4 59 7 win Control 7 Logbook View Properties x Style Font Select font for logbook window Figure 4 36 Logbook View Properties dialogue Style tab Click on Set Font The Font dialogue is shown Select the Font Font Style and Size Click on OK Click on Apply if you want to view the results without closing the dialogue Click on OK The font is changed 4 9 Exiting ALFwin Control To exit ALFwin Control when you are not performing a run i e when the indicator light in the Windows 95 taskbar is grey I Check that no samples are running The lights should be grey and the last line in the Logbook window should begin Run ready at followed by three more lines confirming that the default values have been sent to the instrument The instrument is ready for a new run If you try to exit ALFwin Control while a run is in progress you are warned that you cannot quit during a run Press OK and therun continues normally Select File Exit If Control was entered from Sequence Analyser you are returned to Sequence Analyser Otherwise ALFwin Control is closed ALFwin S
152. l sample sequences extending to that region have the same base component Y ellow cell There is a gap at this position Blue cell Thereis an ambiguity at this position Red cell Thereis a single base component mismatch at this position compared to the consensus 6 13 6 Sequence Alignment 6 4 5 Adjusting the alignment table W hen all the required data is displayed you may wish to adjust the rows and columns in the table To adjust the height M ove the mouse pointer into the grey of a row of cells sequence column and position it on the horizontal border you want to move The mouse pointer becomes an up down arrow Click and drag the border to the new position To adjust the width M ovethe mouse pointer into the grey header of a column of cells column and position it on the vertical border you want to move The mouse pointer becomes an left right arrow Click and drag the border to the new position To display or hidethe Use View User Preferences to select or gridlines deselect Display Gridlines 6 5 The consensus sequence 6 14 The consensus sequence is a product of the alignment process which gives you an overall result representing the contribution from all the sequences This allows you to easily assess the quality of alignment The consensus sequence is automatically generated after all of the sample sequences have been aligned The algorithm achieves this by comparing the base components between the aligned seque
153. lable applications 3 8 Available applications field 4 3 available applications field 2 3 3 4 axes changing the X and Y axes 5 17 B backup file automating 4 36 purpose of backup file 3 2 base calling algorithm assigning bases C 2 background subtraction C 2 different types of processing C 5 estimate parameters C 2 how it works C 1 how to use C 4 manually adjusting time shift C 5 sharpen peaks C 2 start and or stop point set manually C 4 start point C 2 stop point C 3 update parameters C 3 bases adjusting the number of the first base in the alignment table 6 25 ambiguity in alignment 6 13 assigning upright status 5 36 deleting 5 40 5 43 deleting bases in the alignment table 6 21 editing in sequences 5 39 editing options in Analyser overview 5 2 editing sequence bases in sequence alignment 6 20 exporting 4 32 hiding identical bases in sequence alignment 6 24 in Curve view 5 12 in the alignment table 6 13 inserting 5 40 5 41 inserting bases in the alignment table 6 21 italic 5 34 meaning of capital italic and small base letters 5 34 mismatch in alignment 6 13 moving 5 40 5 43 Index replacing 5 40 5 41 replacing bases in the alignment table 6 20 selecting for export 4 32 showing or hiding edited bases 5 44 showing hiding edited bases 5 24 upright 5 34 blue cell in the alignment table 6 13 buffer filling the reservoirs 2 6 C called base ambiguities 5 33 Called bases A mbiguities column 2 15 capital lett
154. lack and white printouts which are useful if you wish to photocopy or fax the print Different types of lines are used to differentiate between curves If you want to save these settings for later use click on Save As in the Report Settings dialogue and use the Save Layout dialogue to define the path and file name Click on Save 4 35 7 win Control 11 Click on OK to complete setting up the automation of print report Automating the production of a backup file This function allows you to automate the creation of a backup of the result file that is generated at the end of a run The backup copy should be saved in a drive different to that used for your result files for example a server drive N ote that a diskette may not have sufficient capacity for the files created by a run 1 From the Post run Actions tab in the Casebook dialogue select Backup By default your backup folder will beselected if there was no other path stored in the casebook file from an earlier run 2 Click on Browse and use the Save As dialogue to select the location and file name for the backup file 4 Click on OK to complete setting up the automation of backup Automating the selection of tools A tool isa shortcut to another program The program you specify will be opened automatically when the run and post run actions are completed 1 From the Post run Actions tab in the Casebook dialogue select Tools 2 Click on Setup The Tools Setup dialogu
155. ly to All Store table IV information in casebook file Lane 16 4 Figure 4 14 Casebook dialogue Sample Information Lanes tab 2 The default settings provide three columns Name Column and Legend Text and you can add up to four more columns Click on the Table Settings button to add further columns as required and name their headers Use the same procedure as you used for clones 4 25 7 win Control Excl Lanes Note You can rename all of the headers including the first three columns but you cannot move or remove the first three columns 3 Enter lane information into the cells of the table 4 Select Store table information in casebook file if it would be useful to retain the sample information as part of the casebook If this option is not selected the table contents are only saved in the result file for this run and you will have to rewrite the table the next time you use this casebook file If you have added or edited the sample information but not selected Store table information in casebook file a warning is shown when you close the Casebook dialogue Excluding lanes Lanes can be excluded from the data collection process 1 To exclude lanes either in the Clones or Lanes mode click on the Excl Lanes button to show the Edit Lane Configuration dialogue Figure 4 15 Edit Lane Configuration dialogue The four lanes for each clone are default colour coded ac
156. m Biosciences you will also see an option called Fragment Analysis 1 00 in the Available applications field For further information on DNA fragment separation please refer to the ALFwin Fragment Analyser 1 00 User M anual or contact your local Amersham Biosciences representative 3 2 Adding a user 3 2 To make it easier to administrate documents and files you should create your own user name user folder and backup folder The folders are used to store the casebook and run files including layout files report files and alignment item files that are created during use of the ALFwin Software When you install the software into your computer a Default user and Default folder are created The Default folder contains some useful standard routines which you can copy into your own folder using Windows Explorer If you use the normal installation routine the Default folder can be found in c Program Files AP Biotech ALFwin H ome Normally you use this same location for your own user files The facility for creating a backup file allows you to save a copy of the result file that is generated at the end of arun in alternative drive location usually on a network server 3 2 1 Adding a new user Adding a new user allows you to specify your user name and thus a location for your user and backup files 1 From the ALFwin Administrator dialogue click on Add User The Add or Edit User dialogue is shown 2 Type your name in the User nam
157. m thelist provided Please refer to Section 3 2 if you need to add or edit a user Select an instrument in the Available applications field The colour of the icon in front of the instrument name indicates the current status Grey Unused system instrument Y edlow Instrument connected to Control but not running Green Instrument connected to Control and running Sauna irahan 7 iN lainat 7 lariumar 4 Figure 4 1 ALFwin Administrator dialogue 4 3 7 win Control 4 4 If you need to set up the link between the computer and the instrument please refer to Section 3 3 4 Click on Start The ALFwin Control splash screen is shown for a few moments Y ou can close this splash screen more quickly by clicking on it or by pressing any Key 5 If you selected the demonstration instrument from ALFwin Administrator a warning is shown advising how you must proceed to reconnect a real instrument To continue with the demonstration instrument click on OK 6 If areal instrument is connected and switched on the following messages are displayed Initializing program looks for the COM port Looking for instrument searches for the instrument Loading instrument program loads theinstrument program to the instrument Connecting to the instrument connects to the instrument 7 Themain ALFwin Control screen opens with a superimposed Tip of the Day dialogue that provides tips about using the program To
158. ment and Lane Order Select the header to be moved and click on Move Up or Move Down Click on OK to view the changes to the columns in the table of sample information Add information to the new columns as appropriate Adjust the width and height of the cells to accommodate the sample information To adjust the width of cells M ove the mouse pointer into the top margin of the table and on to the cell boundary The mouse pointer becomes a horizontal double arrow Click and drag the cell margin ALFwin Control 4 To adjust the height of M ove the mouse pointer into the cells left hand margin of thetable and on to the cell boundary The mouse pointer becomes a vertical double arrow Click and drag the cell margin 7 Select Store table information in casebook file if it is useful to retain the sample information as part of the casebook file If this option is not selected the table contents are only saved in the result file for this run and you will haveto rewrite the table the next time you use this casebook file Adding sample information for the lanes The procedure for adding information on thelanesis similar to that for clones and the same cell editing procedures are used 1 From Sample Information select the Lanes tab Casebook Seqdem1 cbk x File Information Conditions Sample Information Post un Actions Notes Comment Legend Text Table Settings Excl Lanes App
159. move icons around by placing the mouse pointer on an icon pressing down and holding the left mouse button moving the mouse button together with theicon to a new place and releasing the mouse button to drop theicon Thisis useful for moving files between folders and to or from the Recycle Bin Investigate various command options for an icon and an active window by clicking on it with the right mouse button M ake the appropriate selection from the menu that is displayed These command options are the same as those offered in the menu View Properties options in ALFwin B 3 3 Using a dialogue box Windows 95 displays a dialogue box when you must supply information or specify options or properties T here are different types of options Radio buttons Circular buttons allowing you to select one option only at a time Check option boxes Square boxes that you can click in to place a check mark for theonemore options you want Short guide Windows 95 B Text fields Empty boxes allowing you to insert either free text or parameters within a defined range Option buttons Buttons allowing you to execute or retract the implementation of the selected options within a dialogue B 3 4 Sizing windows Y ou can adjust the size of a window to accommodate viewing several windows at the same time There are two methods you can use to size windows e Use the buttons in the upper right corner of the window to M inimise view the taskbar
160. mple sequences The usual way to perform an alignment is to create a new alignment item containing all of the sequences that you want to align and then use the Analysis Align All function This function aligns all sequences with respect to one another testing the alignment in the forward direction and also the reverse complementary direction Asa result the best alignment of the sample sequences is displayed A consensus sequence is also produced see Section 6 5 which isa representative sequence for all aligned sample sequences The consensus sequence and aligned sample sequences are linked If you edit a base position in a sample sequence the corresponding position in the consensus sequence will change in accordance with the edit Conversely if you edit a base in the consensus sequence the corresponding base position in all aligned sample sequences will change to be the same as in the consensus sequence To initiate the alignment process 1 Click on the Alignment Item window to ensure that it is active 2 Select Analysis Align All or click on the Align All button The results are shown as an alignment overview see Section 6 3 and as an alignment table see Section 6 4 Sequences included Sequence Alignment 6 EE emdim iae Cis hi Fi Chep 7 Ter te Cima Tes aio Ches Tii Alignment table Conon Seq r z Alignment Beg Ea x ESE overview Rectangular cursor showing the region included in the alignment tabl
161. n adding for the clones 4 21 adjusting the height of the cells 4 25 adjusting the width of the cells 4 24 on clones 4 21 on lanes 4 25 sample information tab in casebook 4 9 sample information tab in casebook detail 4 20 sample int 4 18 saving casebook 4 12 4 13 clones in a single split file 5 48 clones in multiple split files 5 48 curve layout file 4 57 layout for report 4 35 5 56 xxi Index xxii naming split clone files 5 48 part s of a result file 5 47 result file 2 18 5 46 saving window positions on exit 4 47 sequence alignment file 6 30 scaling Use current scale area 4 55 Y axis 5 18 scaling Y axis 4 53 screen configuring screen layout 4 47 second instrument connecting 3 6 Select System 4 4 selected sequences exporting 6 33 selecting cells in the sample information tab 4 22 seq icon 6 5 seq number 6 7 sequence in result file 5 7 Sequence Alignment as an analysis tool 2 16 configuring the Sequence Alignment module 6 34 introduction 6 1 overview of Sequence Alignment facilities 5 2 statistics 6 17 typical session 6 3 Sequence A nalyser launching from Administrator 3 4 3 8 sequence orientation selection 6 36 sequence text view options 5 21 sequences adding more to an alignment 6 9 aligning 6 8 direction 6 13 editing bases 5 39 exporting 5 49 6 32 finding position of special sequences 5 35 finding special sequences 5 34 selecting for alignment 6 4 set manual start time 5 38 set manual stop time 5
162. n Appendix A and that you are using M icrosoft Windows 95 operating system To install the software Check that all analysis instruments are switched off Turn on the computer Windows 95 should start automatically Insert Disc 1 into drive A Click on the Start button in the desktop taskbar and select Run Enter a setup at the command prompt Click on OK Follow the instructions on the screen Remove the final diskette from drive A before performing the computer restart To uninstall the software 4 Locate and select Settings Control Panel from the Start button menu Select Add Remove Programs Select ALFwin Sequence Analyser 2 00 in the list and click on the Add Remove button Follow the instructions on the screen Note When you uninstall the program only the program is removed All the files and folders created by users will NOT be removed If there are any shared files you will be asked to retain or discard them If you are unsure retain the files and these will not interfere with the normal running of your computer Installing the software Introduction 1 1 Introduction To obtain the best sequencing results using the ALF family instruments Amersham Biosciences has developed a control and analysis sequencing software called ALFwin Sequence Analyser 2 00 ALFwin Sequence Analyser gives you total control over the sequencing run and collection of the run data Data can then be processed to generate the a
163. n Curve view 5 15 in Gel View 5 28 result file contents 4 9 4 15 5 7 counter number 4 16 formats 5 7 icons 2 13 name in casebook 4 8 naming the result file 4 16 4 42 open 5 8 opening 2 13 opening in Sequence Analyser 5 7 opening several result files 5 9 saving 2 18 5 46 saving part s of a result file 5 47 Result window clone icons 5 9 contents 2 13 working in the Result window 5 9 results evaluating 2 12 getting the most out of your data 2 15 quality assessment 2 15 reverse sequence 5 24 reverse sequence direction 6 10 reverse sequencing direction symbol 6 13 reviewing the results of data processing 5 33 run activities during a run 4 42 before starting a run 2 1 changing run conditions during run 4 44 Index ending 4 39 ending a run manually 4 43 evaluating during a run 4 45 logbook records 4 39 making your first run 2 1 manual adjustments during a run 4 38 manual intervention 4 38 monitoring during run 4 38 notes 4 37 4 46 observing run conditions 4 40 overview of run activities 4 2 pausing a run 4 43 performing a run 4 38 preparing the instrument 4 39 preset 4 38 recording changes during a run 4 39 starting 2 11 4 38 starting a run 4 41 starting another instrument 4 47 starting another run 4 47 typical stages 4 1 using the manual window 4 40 run time 4 18 S safety consultation 2 1 hazardous chemicals 2 1 sample denaturing 2 10 loading 2 10 storing samples 2 10 sample informatio
164. n Sequence Analyser To open the Tools dialogue select Tools Edit Tools If you want to view the default settings open the Items list and select the appropriate tool Information is presented in the Tools dialogue for Command This is the command line to launch the tool Argument This shows any extra arguments relating to the command line Initial Directory This is the folder that contains other important components needed by the tool Editing the settings for the supplied tools 1 Open the Items list and select the appropriate tool Click on the Edit Tool button and the Edit Tool dialogue is displayed 2 Edit the appropriate information Command This is the command line to launch the tool Argument This shows any extra arguments relating to the command line Initial Directory This is the folder that contains other important components needed by the tool 3 Click on Save Tool Adding new tools Y ou can define new tools programs which can be launched from within ALFwin Sequence Analyser Note You can only define a maximum of six tools including the two tools supplied with ALFwin Sequence Analyser 5 59 5 ALFwin Sequence Analyser 5 60 1 Click on the Add Tool button and the Define Tool dialogue is displayed 2 Enter the appropriate information Name Enter a name for the tool Command Enter thecommand lineto launch the tool Use the Browse facility to help you Argument Enter any extra argumen
165. n menu B 7 B Short guide to Windows 95 B 8 The base calling algorithm C C The base calling algorithm This appendix describes the base calling algorithm used to process the raw data from a sequencing run C 1 How the base calling algorithm works The algorithm is described in the following flow chart C 1 C The base calling algorithm C 2 Each position in the flow chart represents the following Search for start point Background subtraction Estimate parameters Sharpen peaks Assign bases The algorithm searches automatically for a suitable start point A start and a stop point can also be defined manually The raw data signal contains a slowly varying offset value often referred to as the background At this stage the background component is calculated and subtracted from each lane to simplify further processing The algorithm makes an initial estimation of the parameters e g time shift peak shape and peak distance This is done by analysing the region after the start point The signal is filtered through a digital filter that enhances the resolution of the signal This separates individual peaks into peak clusters groups of peaks The bases within a limited region are assumed to be fairly equally spaced The algorithm is ableto estimate wherethe next base will appear The symbol of the base in each position is chosen according to the following rules If there is a peak in a certain
166. n the toolbar 4 gt alv fa cla t Figure 5 21 Shift toolbar Y ou can move the Shift Toolbar to another position on screen if any toolbar buttons are obscured Click and hold on a non button part of the toolbar and drag it to the position required Select the appropriate lane A C G or T button M ove the selected lane curve by clicking the left or right arrow button adjusting until the distances between adjacent peaks are even From the Process Status dialogue select Use applied shifts 5 37 5 ALFwin Sequence Analyser 5 38 7 Click on Process to process the selected clone Recalling previously applied shifts M anual shifts that have been applied to a curve are recorded in the Analysis Logbook Y ou can recall these records using Edit Casebook and selecting the Analysis Log tab To reproduce the same shifts 1 From the Process Status window select Use these shifts 2 Enter the values for the shifts in the A C G and T fields 3 Click on Process to process the selected clone Please refer to Appendix C for further information about shift 5 7 6 Using manual start or stop times If a normal sequence evaluation can not be obtained using auto selection of start and stop times it may help if you manually set these times before processing the raw data You may use a combination of auto and manually set start and stop times 1 Select the clone or clones and select Manual start time 2 Click on the Curve wi
167. nces at each base position This produces a consensus base for each position that is used to build up the consensus sequence It is possible to select different modes for calculating the consensus and these will affect the content of the consensus sequence 6 5 1 The consensus modes The consensus sequence can be configured in either of two modes Least Common Denominator This is the default mode and uses the simplest IUPAC base code which incorporates all the contributions from the sequences A small letter is used if any of the contributions are blanks or if any of the sequences have no contribution Majority Sequence Alignment 6 An ambiguity in any of the sequence bases is conferred on the consensus Least common denominator mode is illustrated by the following example Consensus Seq 1 Seq 2 Seq 3 B CTG K GT R AG V ACG Y CT Consensus uses the base code letter which appears most frequently in the sequences If thereisno letter in majority the appropriate ambiguity code is used Gaps are ignored unless they occur ina majority in which case the remaining base letters are represented by a lowercase letter in the consensus M ajority mode is illustrated by the following example Consensus Seq 1 Seq 2 Seq 3 Seq 4 Seq 5 R AG K GT_ Y CT Changing the consensus mode The following procedure is used to configure the consensus mode These settings
168. nctions such as comparing sequences from different files e checking for base specific differences between clones e calculating the consensus sequence e displaying amino acids This powerful feature is not described here but instead in Chapter 6 2 3 5 Other options Y ou can e edit the sequence data e check the gel view e search for specific sequences e display complementary and reverse variants of your sequence e print all of your data export the sequence for further analysis in other application software 2 3 6 Viewing casebook information and adding analysis notes The casebook information is included in the result file saved at the end of arun In the casebook you can E3 Making your first run 2 read the result File Information read the Run conditions read add or change Sample Information read the Post Run Action settings e read the Pre run and Run notes e write Analysis notes read the Logbook and Analysis Log To view or edit the Casebook 1 Select Edit Casebook or click the Edit Casebook button The Casebook dialogue is shown File Information Conditions Sample Information Post un Actions Notes Logbook Evaluation Log J name Comment Lane Order Table Setings Evel Lenes Apply to All Store table IV information in casebook file Clones Figure 2 14 Casebook dialogue Sample Information tab 2 Selec
169. nd 4 2 3 The light indicator The light indicator provides a quick visual check of the operation of the instrument and is visible in the Windows 95 taskbar and in the ALFwin Control title bar Grey light The instrument is not running but is connected Green light The instrument is running Red light The run is paused or performing post run actions Grey light with red cross ALFwin Control is not in contact with the instrument 4 7 7 win Control 4 3 Working with casebooks 4 8 4 3 1 Introducing casebooks A casebook is needed to perform arun A casebook is a file that contains the parameters for the experimental conditions used by ALFwin Control to control the run The casebook also defines the storage locations of results and allows various data to be stored such as sample information and experimental notes M oreover various post run actions can be selected which are procedures that are automatically executed at the end of the run Casebook files cbk A new casebook file can be created within ALFwin Control for each run that you want to perform H owever you may find it more useful to open an existing casebook file and use it directly or make some appropriate modifications according to the current experiment Casebook files must be saved before starting a run By saving modified casebooks under new file names you are able to build up your own library of casebooks tailored to your own application requirements ALFw
170. ndow and select View Show Line to switch on the vertical line that allows more precise positioning of the cursor in relation to the curves and the time axis 3 Click the Set Manual Start Time button and click in the Curve window at the position where processing is to start The time reading is transferred to the Start Time column for the selected clones To change the selected time click again in the Curve window Y ou can usethe time display that accompanies the cursor to help position it correctly 4 Repeat the above procedure to set the Manual stop time 5 Click on Process to process the selected clones or click on Process All to process all the clones ALFwin Sequence Analyser 5 5 7 7 Displaying the signal levels of lane curves Signal levels are measured as a percentage of the maximum possible amplitude To display the amplitudes of the lane curves 1 Click on the Curve window to make sure that it is activated 2 Adjust the Curve window to display the part of the curve required 3 To display the Signal Level table for only the currently selected Curve window select View Signal Levels or press the lt C trl Shift S gt keys To display the Signal Level table for all Curve windows select View Properties to open the Curve Properties Layout dialogue Select the Settings tab and check the Show signal levels option Signal L Demo 001 alx Clone01 M13 EG Figure 5 22 Signal Level table The table shows the minimum an
171. ndow borders 5 2 2 Bars The following bars are displayed on the screen Title bar This shows information about the name of the open result file and the name of the selected user Status bar This gives you detailed information about for example the position of the mouse pointer and current mode selected on the screen Y ou can hide the status bar by deselecting unchecking the active View Status Bar command M enu bar Click on any menu option to view and select the specific commands 5 6 ALFwin Sequence Analyser 5 Toolbar The toolbar buttons allow you to quickly implement the most commonly used commands The range of active buttons is dependent upon the currently active window in the workspace Unavailable buttons are greyed out or not shown Use the mouse pointer to get a description of each button Y ou can hide the toolbar by deselecting unchecking the active View Toolbar command e eel iss fl Fal SS fm wl B S 2 Sjal jii 5 3 Opening a result file This section describes how to open one or several result files after a sequencing run and select to view the data in different presentation modes ALFwin Sequence Analyser uses the current alx result file format but can also read data in the earlier alf format If the alf data have been saved in compressed format it is necessary to decompress the files before they are used by ALFwin Sequence Analyser Please refer to Section 5 14 2 wh
172. ndscape or Portrait The default is portrait 6 If you want to close the Alignment Report Setup dialogue without printing a report click on Cancel 7 To view theappearance of thereport before printing click on Print Preview 8 Click on Print to send the report to the printer The next time the same user opens the Alignment Report Setup dialogue the settings will be the same as in the last used setup 6 13 Exporting sequence alignment data 6 32 Y ou can export alignment sequences to files so that they can be loaded into other applications that allow further evaluation of the data The formats available depend on the type of data being exported Consensus and amino acid sequences can be exported in ASCII format which is accepted by many standard spreadsheet and word processing applications In M SF or PH Y LIP format see page 6 3 you can export all or only the selected base sequences Sequence Alignment 6 To export alignment sequence s 1 In the Sequence alignment window select the base or amino acid sequences to be exported If you want to export all the sequences leave the sequences unselected 2 Select File Export Alignment The ALFwin Sequence Alignment Export dialogue is shown ALFwin Sequence Alignment Export Ea Exp C Selected sequences C Consensus Amino acid sequences for selected sequences Reading frame 1 Y Format MSF File 7 Cancel Help
173. ng facilities are provided that enable you to directly replace insert or delete bases in the sequence Sequence alignment is a separate module within ALFwin Sequence Analyser that allows you to align sequences within the same or different result files This enables you to view base specific differences between aligned sequences and directly edit the sequence data accordingly Sequence Alignment is presented in Chapter 6 In order to properly analyse the data ALFwin Sequence Analyser has very flexible data presentation options both in curve view and also in gel fluorogram view Y ou are able to view one or several sets of clone data simultaneously to zoom in on selected areas of arun and to move between views for raw data and processed data All data is colour coded and presentation preferences easily changed for maximum comfort M uch of the editing work is performed in the curves view All of the actions that you perform in ALFwin Sequence Analyser are logged so that you can always look back on what you have done M oreover you can print out a wide selection of the results of your evaluation either direct from screen or in a report ALFwin Sequence Analyser 5 This chapter is divided into the following sections 5 1 5 2 5 3 5 4 5 5 5 6 5 7 5 8 5 9 5 10 5 11 5 12 5 13 5 14 5 15 Starting ALFwin Sequence Analyser Introducing the ALFwin Sequence Analyser interface O pening a result file Presenting results in
174. ng or showing 6 14 navigating using the rectangular cursor 6 12 alx and alf file formats 5 7 5 60 alx alf to TIFF 5 58 5 61 ambiguities display in alignment sequences 6 12 finding position of ambiguities 5 35 Amersham Biosciences W eb address 4 8 4 9 amino acid configuring the display 6 35 configuring the reading frame 6 35 amino acid sequences configuring the display 6 26 enabling disabling automatic recalculation 6 27 export 6 33 manually initiated recalculation 6 27 recalculating 6 27 selecting the reading frame 6 27 viewing in the alignment table 6 26 amplitude information 4 46 analyse align all 6 8 align to previous 6 10 process 2 14 5 31 Analyser adding Analyser notes 5 45 adding analysis notes 4 37 base editing options overview 5 2 choosing the user 5 3 data processing options overview 5 1 data processing options overview 5 1 editing facilities overview 5 2 introduction 5 1 logging actions 5 2 presentation options overview 5 2 presentation tools overview 5 2 sequence alignment overview 5 2 starting 5 3 ASCII adding for alignment 6 4 configuring for import 6 38 export format 4 31 sequence alignment export format 6 32 sequence export format 4 31 5 50 E 1 auto display of statistics popup table 6 18 Index auto process data 4 29 auto shift 5 32 auto start and auto stop time 5 32 automatic condition control 4 18 4 19 automatic recalculation of consensus 6 16 automatic settings for ALFexpress 4 5 avai
175. nt data 2 15 filling buffer reservoirs 2 6 fitting the electrodes 2 11 indicator lights 2 5 instrument data shown with curves 4 51 presetting 2 9 problem identification 2 15 running two instruments 4 4 saving the instrument ID 3 6 selecting real or demonstration 4 4 status indicators 4 3 temperature indicator light 2 9 trace function 3 5 trace window 3 6 water level 2 5 instrument data 2 15 including instrument data in curves view 5 22 reading values in the Curve window 5 23 selecting the curve to view 2 16 internet Amersham Biosciences address 4 8 introduction to ALFwin 1 1 italic bases assigning upright status 5 36 at the end of a run 5 34 definition 5 35 hiding the italic status 5 36 showing hiding italic bases 5 24 Index items in group 4 51 IUPAC ambiguity codes C 6 K keyboard shortcuts in ALFwin Control D 1 in ALFwin Control Curve window D 1 in ALFwin Control M anual window D 2 in ALFwin Evaluation D 2 in ALFwin Evaluation Clone view D 3 in ALFwin Evaluation Curve window D 4 in ALFwin Evaluation Curve window in edit mode D 6 in ALFwin Evaluation Gel View D 3 in ALFwin Evaluation Process Status window D 6 in ALFwin Sequence Alignment D 8 L lanes adding sample information 4 25 altering lane order 4 21 creating lane groups 4 5 1 excluding 4 26 matching lanes to clones 4 27 reinstalling 4 26 reinstalling excluded lanes 4 26 selecting the group to be shown 4 51 viewing lane information 5 10 laser
176. nt replace by selecting Edit Undo Replace Mode or by pressing the lt Ctrl Z gt keys or by clicking the Undo button on the Edit Toolbar To exit edit mode select Edit Edit Sequence to deactivate it or click on the Close button in the Edit Toolbar 5 8 2 Inserting a base To insert a base in the sequence 1 Click on the Curve window to make sure that it is activated 5 41 5 ALFwin Sequence Analyser 5 42 2 Select Edit Edit Sequence or press the lt Ctrl E gt keysto activate the edit mode The Edit Toolbar is shown 3 Select the base to the right of the position where you want to insert a base 4 Click the Insert New Base button on the Edit Toolbar and use the ACGT buttons to set the new base type If you prefer to use the keyboard select Edit Insert Base Mode if it is not already activated then type the base letter 5 You can undo the most recent base component insertion by clicking the Undo button on the Edit Toolbar or by selecting Edit Undo Insert or press the lt Ctrl Z gt keys 6 To exit edit mode select Edit Edit Sequence to deactivate it or click on the Close button in the Edit toolbar To insert a base using the mouse 1 Select Edit Insert Base Mode 2 Select Edit Show Line if the vertical cursor line is not already activated This will help you locate the base correctly 3 Select the base required on the Edit Toolbar then double click on the position on the curve where you wish to make the base
177. nt to include by means of the Report Settings dialogue The settings can be saved as a report layout file that allows you to recall a report configuration The report layout file does not include information about selected clones Before printing the clones must be selected as described below 1 Select File Report The Report Settings dialogue is shown 2 If you want to use a report setting that was created and saved in a previous evaluation click on Open Layout and use the Open dialogue to select the path and the filename of the layout rpt you wish to use To use the default report layout open the Default folder and select Report rpt 5 53 5 ALFwin Sequence Analyser 5 54 Repo Sehir CUP tage PAP Mirisch Al Fees C Peguiher T irham F Winisapag 0 arma ninckove Tars th 1 S20 F peal E Cia F Shee pin brm a ha Uae ipi Arga T Enit nre T aema E iph bom F Feonbm E tT T fonai e Da F pa Carer e inch Fa F T Prad Fe FT F ohited Figure 5 28 Report Settings dialogue Select the Headers Casebook and Groups buttons in turn and select items to be included in the report from the lists shown Headers Casebook Allows you to select the items that are to be included in the header of the report Y ou may include any of Current user Current date and File name Allows you to select items from the casebook to be included in the report Allows you to select the clones to be included in
178. ntation of the curves for a selected clone Curve windows can be opened for more than one clone at a time from within the same result file and from other opened result files The Curve window contains an orientation view that shows the whole length of the run and allows you to quickly select any part of the run for closer viewing of the curves If you have processed the data by default the generated nucleotide sequence is displayed in this orientation view beneath the peaks of the curves ALFwin Sequence Analyser 5 5 5 1 Opening Curve windows To open Curve windows 1 3 Select the Result window and double click on a clone or select a clone and click on Open To select a number of clones hold down the lt Ctrl gt key while clicking on the clones required then click on Open Alternatively you can simultaneously open Curve windows for all of the clones by clicking on Open All The Curve window s for the clone s are displayed one upon the other If you want to view all the opened curves select Window Tile All Curve Windows If you have more than one result file open in the ALFwin Sequence Analyser workspace you can open any number of Curve windows from the Result windows Select the appropriate Curve window from the Window menu or arrange the display of all the curves see Section 5 5 10 Figure 5 8 The Curve windows after selecting Till All Curve Windows Use in conjunction with window linking to compare corresponding part
179. o which starts the processing after the highest peak corresponding to the primer To start the data processing at a fixed point along the time axis select Manual and enter the time in the min field 4 Select the End mode N ormally data processing is terminated when the peak quality is low the signal level decreases or when the 4 29 7 win Control resolution of the peaks is less see Appendix C This is the default Auto option To stop data processing at a fixed point select Manual and enter the stop time in the min field 5 Select Process type Normal Select this option when no heterozygote position is expected Heterozygote Low Select this option when there is a low expectancy of finding ambiguity codes Heterozygote Medium Select this option when thereisa medium expectancy of finding ambiguity codes Heterozygote High Select this option when there is a high expectancy of finding ambiguity codes 6 Click on OK to complete setting up the automation of post run data processing Automating the export sequence Y ou can export a sequence result in a format that is compliant with other applications The options available are described below Note Itisnot possibleto obtain export files from raw data that has not been processed By default the export function creates a file for each sequence clone but you can deselect any that are not required 1 From the Post run Actions tab in the Casebook dialogue select
180. of the run M ove to the end of the run When curves are reversed move to the beginning of the run M ove to base one step to the left or right D 5 D Keyboard shortcuts D 6 Text mode Home M oveto thefirst basein the marked part of the sequence Always counts from the left End M ove to the last base in the marked part of the sequence Always counts from the left Arrow keys M ove to base one step to the left or right or up or down Ctrl C Copy the sequence to clipboard D 5 7 In the Edit mode after Ctrl E in the Curve widow Insert Switch between Replace and Insert mode Delete Delete marked base Ctrl Heft or right arrow keys M ovethe position of selected base to the left or right N ote that the curve view and not theorientation view has to be activated Ctrl Z Undo last entry or move D 5 8 Inthe Process Status window Ctrl nter O pen the selected clone s D 5 9 Inthe Casebook Shortcuts within the casebook Tab M ove forward between fields in the current page Shift T ab M ove backwards between fields in the current page Ctrl Tab or Shift Ctrl Tab M ove between pages in the casebook Shortcuts within the casebook Sample Information page ALT4C Switch to the clone tab ALT Switch to the lane tab CTRL C CTRL X CTRL V Keyboard shortcuts D Copy information from selected cells Cut information from selected cells Paste information into cells An activated cell is a c
181. ogram Files 4P Biotech ALFwin Home D avi IV Report M Tools Setup Figure 4 17 Casebook dialogue Post run Actions tab ALFwin Control 4 Each task can be customised to select the items to be included For example within Print a Report you can specify the parts of the casebook that areprinted T asks themselves can be enabled or disabled so that you can quickly alter the post run actions required for a particular run Y ou can save the post run action settings in the current casebook file before starting the run Automating the post processing of raw data It is possible to automatically process the raw data at the end of therun to generate the specific DN A sequence for each of the clones Processed data is stored in the result file This post run action uses the default automatic settings for raw data processing from ALFwin Sequence Analyser R aw data that has been processed using post run actions can be viewed and analysed in the SequenceA nalyser module and you can process the data again with using a wider range of processing options see Section 5 7 1 From the Post run Actions tab of the Casebook dialogue select Auto process data 2 Click on Setup The Process Setup dialogue is shown Start Auto C Manual min End Auto C Manual min Process type Normal x Cancel Help Figure 4 18 Process Setup dialogue 3 Select the Start mode The default is Aut
182. ograms option from Windows 95 start button B 3 R Ramp starting at button 4 19 range in export file 4 32 5 51 ratio of upright bases to the total of called bases 5 33 raw data viewing 5 12 reading frame selection for amino acid sequences 6 27 Realign Laser 4 5 Realign Laser button 2 11 4 5 recalculation of amino acid sequences 6 27 of consensus 6 16 rectangular cursor in orientation view 5 14 in sequence alignment 6 11 recycle bin B 2 red cell in the alignment table 6 13 reference list E 1 reloading the gel 4 43 replace identical character 6 24 replacing bases 5 40 5 41 report automating report print 4 32 bases to include 4 34 5 55 bases window 4 35 5 56 casebook items 4 33 5 54 colour or monochrome 4 35 5 56 complement sequence 4 34 5 55 curve width 4 35 5 56 curvepart to include 4 34 5 55 data to include 4 34 5 55 forward or reversed sequence 4 34 5 55 include headers 4 33 5 54 xix Index XX layout file 4 33 print with shift applied 4 34 5 55 printing 2 18 5 53 printout options 4 35 5 56 raw and processed data 4 34 5 55 saving the layout 4 35 5 56 scaling the curves 4 34 5 56 select curves 4 34 5 55 selecting items to be included in print 4 33 5 54 selecting the clones to include 5 54 selecting the clones groups to include 4 34 settings 2 18 show edited bases 5 56 time range to include 4 34 5 55 using a saved layout 5 53 windows page 4 35 5 56 reset zoom 4 51 resetting the magnification i
183. on The Open Casebook dialogue is shown Open Casebook Look in E Defaut l c E Sequencelst cbk Files of type Casebook files fbk cbk 7 Cancel Figure 4 3 Open Casebook dialogue 2 Usethis dialogue to navigate the filing system and select a casebook file Casebooks files for sequencing are always saved with the file extension cbk Note If you also have ALFwin Fragment Analyser 1 00 installed on your computer you will see that it is possible to select casebook files for the control of sequencing runs as denoted by the fbk file extension do not select these casebook files for sequencing runs 3 Double click the selected cbk file or select it and click on Open The Casebook dialogue is shown with the name of the chosen casebook at the top ALFwin Control 4 File hiaan Coar Sarake atoae Posi Arrions Pase 2 Eik rel F fas O Hm Add courier rm bo ia raa File pear CP Pay EP Bapkegh il PeoarH ome lured Erre Hire irda ti ir Dannes I Flas dade Lael ames by Hatin tne Fea geen Cansbork Ferk obk ume e E Ls Figure 4 4 Casebook dialogue File Information tab Note You can open a casebook that you have used recently by selecting it from the list at the bottom of the File menu The four latest saved files are shown 4 Ifyou want to usethe casebook without modification click on OK This will close the Casebook dialogue and load it into ALFwin Control
184. on of the alignment overview 6 3 4 Alignment imperfections Imperfections in the matching of specific base positions between aligned sequences i e detected single base components are indicated by short vertical tick marks on the sample sequence lines The tick marks are colour coded to indicate the type of imperfection 6 11 6 Sequence Alignment Note Thecolour codes can be redefined in the Alignment Properties dialogue see Section 6 15 5 No tick mark All the sample sequences that extend to that region have the same single base component Y ellow tick mark There is a gap at this position Blue tick mark There is an ambiguity at this position Red tick mark Thereis a single base component mismatch at this position Tick marks displayed on the consensus sequence are also dependent on the consensus mode being used see Section 6 5 1 Use the mouse to drag the rectangular cursor to a position that encompasses the tick marks you want to evaluate The alignment table changes to show the region of the base sequences covered by the cursor 6 4 The alignment table 6 12 The alignment table displays the actual sequence data for each of the aligned sample sequences and the consensus sequence The area of the aligned sequences shown corresponds to the position of the rectangular cursor in the alignment overview see Section 6 3 3 Y ou can view different areas of the alignment by moving the rectangular cursor or by using
185. on the front panel of the instrument Check that the cassette is operating both of these switches This is most easily seen by checking the indicator lamps on the front of the instrument since the yellow temperature lamp starts blinking when the microswitches are pressed in Check the water level on the right side of the instrument The water level should be between the max and min marks of the water tube If this is not the case attach a small funnel to the water tube and add sufficient M illiQ water to bring the level up to the max mark Connect the two push fit thermocirculator connectors of the instrument to the connectors on the gel cassette The yellow temperature indicator lamp on the instrument flashes while pre heat raises the temperature to the default 40 C When the default temperature is reached the yellow lamp illuminates continuously 2 5 2 Making your first run 5 To switch on the laser and initiate the automatic laser alignment routine close the lid of the instrument and open it again While alignment proceeds the yellow laser indicator lamp flashes W hen alignment has been completed successfully the laser indicator lamp illuminates continuously Check the laser beam for dust particles in the gel or on the glass in front of the detectors If dust particles are in evidence simultaneously adjust the vertical position of the gel cassette using the two adjustment wheels on the front panel of the instrument Keep
186. oose to view italic bases as upright bases by hiding the italic status 1 2 Click on the Curve window to make sure that it is activated Deactivate italic status by selecting View Show Italics This removes the check mark from this menu option meaning that it is not active ALFwin Sequence Analyser 5 All bases are shown as upright bases R epeat the above procedure to activate the menu command to show italic bases Note Theitalicstatusis only hidden from view despite all bases being displayed as upright bases For processing purposes italic bases are always so unless they are assigned upright status 5 7 5 Shifting the curves manually When there are few upright bases in a processed clone the cause may be excessive shift of one or more lanes in the gel For example the effect known as smiling is produced when samples in the first and last lanes move more slowly than those in the middle lanes This may be evident when viewing the primer peaks for the four curves of a clone Y ou can apply manual shift correction to make it easier for the program to analyse the run To shift a curve by applying manual shifts 1 2 A 3 4 6 Click on the Curve window to make sure that it is activated Zoom in to the curve so that adjacent peaks can be seen clearly Select either Edit Apply Shift or lt Ctri Y gt keys The Shift Toolbar with four arrow buttons and four lane buttons is added to the other buttons i
187. ooting 7 2 ALFwin Instrument Control Connection problem Problem Looking for instrument please wait does not disappear after a few minutes of starting ALFwin Control Explanation There is probably a problem linking to an instrument on the designated COM port Solution Click on Cancel in the message window A dialogue asks if you want to stop waiting Click on No Check the connections between the instrument and the computer If the connection is fine check the instrument setup in the ALFwin Administrator and changeCOM port setting Start ALFwin Control again If you click on Yes you can only edit a casebook but not start a run Comment If you answer No to the question Stop waiting the dialogue Looking for instrument is displayed again To be able to quit you must answer Yes Set temperature in casebook is not reached Problem Instrument temperature is 40 C but the set temperature in the casebook is different Explanation The experimental conditions in the casebook are not activated Solution Click on Preset and the temperature setting is sent to the instrument If not sufficient see instrument manual Flashing light on the Control icon Problem T hecoloured dot on the minimized Control icon is flashing Explanation Run is paused dueto some problem indicated by an error message Solution M aximize the window and view the displayed error message Take care of the problem an
188. operties 5 29 Casebook File Information tab 2 7 Casebook Sample Information tab 2 17 Conditions tab in the casebook 4 17 Curve Properties 4 51 Cut Clone 5 44 Data Set tab of Gel Properties 5 28 Edit Columns 4 23 Index Edit Lane Configuration 4 26 Edit Step Values 4 19 4 20 Edit System Settings 2 4 Export Settings 4 30 File Information tab in casebook 4 11 4 16 Find 5 35 Font 5 23 Gel Properties 5 30 Group 4 52 N otes tab in the casebook 4 38 open 2 13 Open Casebook 2 7 4 10 Open in Sequence Analyser 5 8 Post run Actions tab in casebook 4 28 Print Casebook 4 14 Process Setup 4 29 Process Status 2 14 5 31 Report Settings 2 18 4 32 5 53 Sample Information Clones tab in casebook 4 21 Sample Information Lanes tab in casebook 4 25 4 27 Save Casebook 4 12 Select Application 4 12 Sequence Alignment Export 6 33 Sequence Alignment Statistics 6 18 Settings tab in Curve Properties 4 49 4 55 Settings tab of the Curve Properties Layout 5 17 Signal levels 4 46 Split File Settings 5 47 Start Run 4 42 Style tab in Curve Properties 4 56 Style tab in Logbook View Properties 4 60 Style tab in M anual View Properties 4 59 Tip of the Day 5 5 Tools Setup 4 36 View tab in Curve Properties 4 5 1 Window Contents tab in M anual View Properties 4 58 disabling automatic recalculation of amino acid sequences 6 27 automatic recalculation of consensus 6 16 discard 4 13 DNAsis export format 4 31 5 51 E 2
189. ormat for PC GENE asequence analysis program by IntelliG enetics from the O xford M olecular group ASCII ASCII txt format that will load into many standard commercial appli cations E 1 E Reference list E 2 EMBL The eml format Information on the EM BL format for Bioresearch can be found at several web sites the sequence anal One exampleis ysis program by Fujitsu Ltd http w ww psc edu general software packages seq intro emblfile html DNAsis The dna format http www hitachi soft com gs of DNAsis a sequence analysis program from Hitachi Software Genetics Systems Group MSF M SF isthe multi http www embl heidelberg de ple sequence align predictprotein new D exa ment format of optin_msfDes html the GCG sequence analysis package PHYLIP PHY LIP the http evolution genetics PH Y Logeny Infer ence Package is a package of pro grams inferring phylogenesis evo lutionary trees washington edu phylip html Reference list E E 2 Other references 1 2 For more details concerning multiple alignment algorithms please refer to Comprehensive study of iterative algorithms of multiple sequence alignment by M akoto H irosawa CABIOS Vol 11 no 1 1995 where comparisons are made betw een the effect of different multiple alignment algorithms The method used to align any two sequences is suggested by N eedleman and Wunsch 1970 M ol Biol 48 444 It is based on dyn
190. out the quality of bases When you import these files all bases are always imported and the import configuration has no effect The statistical significance of the bases included in these files can thus not be guaranteed To configure the import scheme for ALFwin data files 1 Select View Properties to open the ALFwin Alignment Properties dialogue 2 Select the Alignment Item Properties tab ALF win Alignment Properties x Alignment item properties Display properties Amino Acid Translation m Algorithm Options Interpretation table Check both directions C Check current direction Start label for bases Advanced fi Algorithm r Consensus Mode Least common denominator Majority rule m Import Mode Import upright only ALX ALF Import all bases Cancel Help Figure 6 21 Alignment Item Properties tab of the Alignment Properties dialogue 3 To import only upright bases from alf and alx files select Import Upright Only O therwise select Import All Bases 6 39 6 Sequence Alignment 6 40 6 15 5 Changing the colour settings Ambiguities mismatches and gaps in the alignment table and the alignment overview are highlighted and identified by the use of colour To alter the colours 1 Select View Properties to open the ALFwin Alignment Properties dialogue 2 Select the Display Properties tab 4LPean Agea Praja iat Alignmenti
191. propriate type of note according to the stage reached in the experimental procedure Casebook files during a run and result files after a run The casebook can be fully modified before the run is started and the modifications are saved in the casebook file You can continue to edit some of theinformation in the casebook after the run has been started although these are not saved in the casebook file Changes made after the run has been started are saved in the result file alx that is generated at the end of the run A result file contains acopy of the casebook information including any information that you edited during the run e logbook information from the Logbook window e theraw data obtained from the run an analysis log empty until an analysis is performed This information can be accessed in ALFwin Sequence Analyser 4 3 2 Opening an existing casebook ALFwin is supplied with one standard casebook file appropriate for making your first sequencing run see Chapter 2 You may run this directly or else modify it to suit your own run requirements Remember to contact your Amersham Biosciences representative or visit the internet page www amershambiosciences com to obtain run conditions for different experimental applications 4 9 4 au win Control To open an existing casebook file Fj 1 From the ALFwin Control window select File Open Casebook or S press the lt Ctrl 0 gt keys or click on the Open Casebook butt
192. rees Information can be found at http evolution genetics washington edu phylip html A typical session with sequence alignment involves opening the appropriate result file s processing the sequences and or importing sequences in other formats creating a new sequence alignment item e aligning the sequences e viewing and editing the sequences generating the amino acid sequence in the desired reading frame e saving and or exporting the results e printing a report 6 3 6 Sequence Alignment This chapter is divided into the following sections 6 1 6 2 6 3 6 4 6 5 6 6 6 7 6 8 6 9 6 10 6 11 6 12 6 13 6 14 6 15 Creating a sequence alignment item Aligning the sequences The alignment overview The alignment table The consensus sequence Sequence Alignment statistics Editing sequence bases from the sequence alignment Amino acid sequences The alignment log The alignment notes Saving the sequence alignment Printing an alignment report Exporting sequence alignment data O pening a previously saved sequence alignment item Configuring the sequence alignment module 6 1 Creating a sequence alignment item 6 4 Sequence data from several sources can be used for sequence alignment Y ou can use e sequences from ALFwin data files obtained from processing raw data e sequences imported into ALFwin Sequence Analyser in ASCII format sequences imported into ALFwin Sequence Analyser in STA
193. rlier processing with a certain time shift C 2 3 The different types of processing In addition to the normal processing mode there are three heterozygote modes for processing Low M edium and High Using one of the heterozygote modes increases the tendency to call ambiguity symbols T his tendency is strongest in the High mode W e recommend you use one of the heterozygote modes when it is important that all ambiguous positions in the raw data are found such as when looking for heterozygocity in genes in DNA samples from diploid organisms The possible heterozygote positions are easily traversed by using the Find N ext Ambiguity search function N otes about the positions of interest can be written in Evaluation Notes in the casebook C 5 C The base calling algorithm C 3 Base ambiguity codes C 6 ThelUPAC ambiguity codes for unresolved base positions are listed below Code Unresolved bases M AC R AG W AT S CG Y CT K GT V ACG H ACT D AGT B CGT N ACGT Keyboard shortcuts D D Keyboard shortcuts D 4 ALFwin Control D 4 1 General Fl Ctri N Ctrl O Ctrl P Alt F4 Ctrl T ab or Ctrl Shift T ab Help Create new Casebook O pen an existing casebook The Open dialogue box is shown Print casebook The Print dialogue is shown Exit ALFwin Control Switch between Curve Manual and Logbook windows D 4 2 In the Curve window Ctrl 1 9 or 0 Ctrl Shift 1 9 or 0 Ctri Alt 1 9 or 0 Ctri
194. rvoir must be located into the front of the instrument 2 1 2 Making your first run a pair of electrodes must be available e the communication cable from the instrument must be connected to an available COM serial port on the computer 2 2 Making a run This procedure refers to the ALFexpress II instrument 2 2 1 Turning on the instrument and computer 1 Turn on the computer Windows 95 should start automatically 2 TurnontheALFexpress instrument The green and red indicator lamps on the bottom left side at the front of the instrument should illuminate steadily 3 Launch ALFwin Administrator by selecting ALFwin Software from the Windows 95 Start button menu options The ALFwin Administrator dialogue is shown fi ALFwin Administrator x m User information Add User Default Edit User Delete User Available applications Sequence Analyser 2 00 Instrument 1 Instrument 2 System Setup Figure 2 1 ALFwin Administrator dialogue 1 If you are unfamiliar with Windows 95 read Appendix B A short guide to Windows 95 or consult the appropriate user manual for the Windows 95 operating environment 2 2 Making your first run 2 2 2 2 Adding a user Y ou can enter details for each new user of ALFwin Sequence Analyser To add a new user 1 2 3 From the ALFwin Administrator dialogue click on Add User The Add or Edit User dialogue is shown
195. s of the curves A Curve window contains two main views the curve view see Section 5 5 3 and the orientation view see Section 5 5 4 Select to view either the raw data curves or processed data curves 5 11 5 ALFwin Sequence Analyser 5 12 Le 7 5 5 2 Viewing raw or processed data curves Curves can be viewed for clones that contain only raw data and also for clones that have been processed Y ou can tell whether a clone contains only raw data or has been processed by looking at the symbol in the top left corner of the Curve window see Section 5 3 1 If the clone contains only raw data some of the tools associated with the Curve window will not be available If the data have already been processed it is possible to toggle between viewing the curves for the raw data and the processed data To view the raw data curve select either View Raw Data press the lt 5 gt key or click on the Raw Data toolbar button Conversely to view the processed data curve select either View Processed Data press the lt F6 gt key or click on the Processed Data toolbar button See Section 5 7 for more information about processed data tei de Cie ee a Aer Sos se FSP oe eS A SE Ele Te ES AN d r aE celeste Coles oe Te Te RTT Figure 5 9 The two alternative views of the data curves raw and processed To obtain these two simultaneous views for a clone open the clone twice and then Tile All Curve Windows 5 5 3 Curve view
196. s column of the Process Status dialogue When the status of ALFwin Sequence Analyser 5 all the selected clones has changed raw data processing is complete 5 7 2 Reviewing the data processing results Y ou can view the results of the data processing and obtain confirmation that the run proceeded successfully T his review provides confirmation about the whole experiment including e thesample sample preparation e therun e data processing to produce the nucleotide sequence A particularly useful indicator is the ratio of upright bases to the total of called bases and also the total number of called bases between adjacent clones of the same sequenced region 1 In the Process Status dialogue look at thecolumn of data labelled Called base Ambiguities and the column labelled Upright bases Ambiguities Comparethe first values in each of the columns for a clone and compare with the values in an adjacent clone Compare how many of the total number of called bases are upright bases between the two clones Called Bases Ambiquities Upright Bases Ambiquities 1157 58 856 1 1037 56 737 10 1100 71 798 10 1096 60 794 2 1066 51 764 3 911 81 610 6 972 86 672 7 884 85 584 8 573 64 273 6 1021 89 720 10 Figure 5 19 Comparing called and upright bases in adjacent clones with different samples Called bases are the bases identified in the raw data by the base calling algorithm during processing Particularly at the ver
197. s in the alignment table of the alignment item 3 By default the amino acid sequences are displayed using the three letter codes If you wish to change the code select View Amino Acid Codes and select the Three Letter Code or the One Letter Code option as appropriate 6 8 2 Recalculating amino acid sequences When you make changes to the base sequences amino acid sequences which you have included in your display may need to be adjusted By default the necessary recalculations are performed automatically To disable the automatic Select View User Preferences and click recalculation of amino acid on Automatic Recalculation of Amino sequences Acids to deselect it To initiate a recalculation W hen automatic recalculation of amino manually acid sequences is disabled recalculate by selecting View Amino Acid Sequences Recalculate Amino Seq To restore automatic Select View User Preferences and click recalculation on Automatic Recalculation of Amino Acids to select it 6 27 6 Sequence Alignment 6 8 3 Changing the translation table Four different translation tables are provided and can beselected in the Alignment Item Properties tab of the Alignment Properties dialogue Standard Human M itochondria N eurospora M itochondria Yeast M itochondria 6 9 The alignment log 6 28 The alignment log is initiated when you operate the New Alignment Item command from ALFwin Sequence Analyser The log records the date and time o
198. split the file so that only the part s of interest are saved File splitting is also useful when you want to accommodate a large result file on media of limited capacity such as diskettes 5 46 ALFwin Sequence Analyser 5 5 10 1 Saving a complete result file To save the result file in the same location and using the existing file name select File Save To save using a different name or location 1 Select File Save As The Save As dialogue opens 2 Use the dialogue to select the folder where the file is to be saved 3 Enter a name for the file 4 Click on Save 5 10 2 Saving part s of a result file A result file can be split into its clone data components Y ou select which clone s are saved and whether they are saved in one file or ina separate file for each clone Files are normally saved in alx format but you can choose to save in alf format if required To save part s of a result file 1 Select File Split File The Split File Settings dialogue opens peme nn Fis cating Cate format F AL FE Procarsed F Gora 1 Ge DHG ford DHARAN 2 lw DHe mri HAHI RETRE THARHE a Sr Sard DOMAINE mi Set DHA Got Dian J 6 Ge DHS Feed DHAHI at Ha caren J St HS Mri DHANG FE Cias rais claret r ol Set ON Sard DART re Se DAs Sou MARE O erk rege dra m 79 Sve DHS Tani OMA P10 SADHA Sd DUNO Peet aot iene ea 4 es ie Figure 5 25 Split File Settings dialogue 5 47 5 ALFwin Sequence An
199. ssion in written form from the company Preface Preface This User M anual describes the use and operation of ALFwin Sequence Analyser 2 00 for the control of sequencing runs on the ALF family instruments and analysis of the sequencing results Specific information contained in the various chapters of the User M anual can be accessed using the Table of Contents or Index The contents of each chapter can be summarised as follows Contents Installing the software This instructs you how to install and uninstall ALFwin Sequence Analyser Chapter 1 This gives an introduction to the key features of ALFwin Sequence Analyser some general tips in using the software and an overview of the conventions used in the User M anual Chapter 2 This presents a step by step guide on how to make a first run Chapter 3 This introduces the ALFwin Administrator module and tells you how to create and edit user profiles Chapter 4 This introduces the ALFwin Control module and tells you how to perform a sequencing run including working with casebooks Chapter 5 This introduces ALFwin Sequence Analyser module and tells you how to process and present your data and prepare the data for export to other applications Chapter 6 This introduces the Sequence Alignment application that resides in the ALFwin Sequence Analyser module Chapter 7 This presents possible solutions to some of the problems that you may encounter using ALFwin Sequence Analyser
200. st the cassette back within one second If the laser lamp goes out restart the laser by closing the lid 10 Close the lid of the instrument The instrument is now ready 4 5 2 Starting the run To start the run L Start 3 Check that the laser and temperature lamps on the analyser are continuously illuminated Check temperature and the Transmittance value or Laser value for ALFexpress II or ALFexpress respectively in the Manual window Select Run Start or click on the Start button The Start Run dialogue is shown 7 win Control oO 1 ALFwin Control Instru m Casebook file File name Seqdem1 cbk In C4 WALFwin Home Default m Result file File name Seqdem1 017 alx In C N WALFwin HomeSD avid i Select Name Cancel Help Figure 4 24 Start Run dialogue Check that the correct casebook is used and that the result file will be stored in the correct folder under the correct name If you need to make changes click on Select Name and type a new filenamein the Enter result file name dialogue or select a new folder Click on Save and then OK Alternatively you can click on Cancel and edit the casebook to change the file name and folder Click on OK A message appears containing a time delay while the instrument checks that no residual daylight signal is saved from the detectors The sampling will start after x seconds please wait A green light in the t
201. t a diskette may not have sufficient capacity for the files created by a run If you require enter information in the Department Address and the Phone Number E mail fields Click on OK to save the information and to return to the ALFwin Administrator dialogue 3 3 SS er Administrator If you have completed the use of the ALFwin Administrator dialogue to add or edit user information you can proceed directly into ALFwin Sequence Analysis by selecting it in the Available Applications field To move into ALFwin Control you must first specify the connected instruments as described in Section 3 3 3 2 2 Editing a user Y ou can edit thelocation of the home and backup folders information in the Department Address and Phone Number E mail fields To edit a user 1 From the ALFwin Administrator dialogue select the user name and click on the Edit User button The Add or Edit User dialogueis shown 2 Edit the Department address and Phone number e mail fields 3 The Home folder and Backup folder name fields have a dark background to indicate that they can not be edited by typing Instead click on the adjacent Browse button to open the Browse for folder dialogue 4 Usethe dialogue to select the path for your folder Use the Create New Folder button to generate any new folders you require 5 Click on OK to complete the edit and to return to the ALFwin Administrator dialogue 3 2 3 Deleting a user 1 From th
202. t button enter the time at which the change is to occur and the duration of time over which the change occurs Click on OK to complete the Edit Step Ramp Values dialogue The change s you have entered are listed in the Commands field If you want to remove any of the condition changes select it in the Commands field and click on Remove Activating stepped pre set changes for ALFexpress Y ou use the Automatic condition control to set a stepped change to the set conditions during the course of the run This control allows you to preset a change in the e electrophoresis voltage current power temperature The change can be executed as a step in value to the new value over a period of time Each of the settings can have one step applied during the course of a run 4 19 7 win Control To set the condition control 1 Click Edit to open the Edit Step Values dialogue Eda Stew Y abre x Et Changs ic far a Fy C Gece i i F rimta n ha rari e mitad off E Carel Help Figure 4 11 Edit Step Values dialogue 2 Open the menu to view and select the parameter that you want to step 3 Edit the target value in the Change to field 4 Ifa step change is required select the Step change at button and enter the time at which the change is to occur 5 Click on OK to complete the Edit Step Values dialogue The change s you have entered are listed in the Commands field If you want to remove any of the
203. t layout that depends on height and width Solution Copy or export with maximized window Corruption of data during transfer from one drive to a network drive Problem After editing the result file it was saved to a network drive N ext time the file was opened it was found to be corrupt i e not possible to open Explanation This problem can occur if the disk space is limited on the drive where the file was attempted to be stored It is not possible to secure disk space on the network drive due to the fact that a lot of different network users may use up space at the same time Solution It is not possible to restore a file that has been destroyed in this way To avoid the problem always save the file to a disk with sufficient space Copy the file to the network drive Check that the copied file was correctly saved then delete the file on the computer hard disk Error message Too many samples in lane Explanation When trying to create an ALF or SCF file with too much data from the source file Solution Use the alx format for larger data quantities Sequence Alignment Error message Command failed Explanation No clear explanation is available Solution Restart ALFwin Sequence Analyser before applying the command again Error message An error occurred in the pair wise alignment algorithm check that the algorithm settings are OK Explanation An erratic algorithm setting may have been supplied to t
204. t the Sample Information or Notes tabs to change or add information in the Casebook 3 Usethe remaining tabs to access read only information in the Casebook 4 Click on OK and the changes and additions will be saved when you save the result file 2 17 2 Making your first run 2 3 7 Printing a report A report is a print out which can include items from the casebook curves sequences and headers Y ou select theitems you want to include by means of the Report Settings dialogue The settings can be saved as a layout file which allows you to recall a report configuration Specific report options and layouts are described in Chapter 5 1 Select File Report The Report Settings dialogue is shown TE CWP gia Flee ELE 1 brakajn kiii Farge Hia E Cire cores E Cuak Fiu ihj beer Fi over Flom base oor Choimnn oa i Duta pert Stipe curves Man Falhin it inet Poomed Fc Rr E O Wree O bemin FP bephate jopa Curve nec fl 50 F me E ii E Shore giint bamm Aill n C Mma O Comper L Maa Figure 2 15 Report Settings dialogue 2 Click on Print to send the report with default settings to the printer 2 3 8 Saving the whole result file To save the result file in the same location and using the existing file name select File Save To save using a different name or location 1 Select File Save As The Save As dialogue opens 2 Usethe dialogue to select the folder where the fil
205. tem properies Displey properties Amino Ard Sequencers C Feadag Fama 7 F Fosdiag Framo 2 M Feadag Frome J C ne Lester Gores i Thre Later Coden Figure 6 22 Alignment Properties dialogue Display Properties tab and open Colour dialogue 2 Select the appropriate item under the Colours list and click on the Set Colour button The Colour dialogue is shown 3 Select a colour Sequence Alignment 6 Note You should select different colours so that it is easy to distinguish different discrepancies Also select colours that allow the inverted colours used for the text to be clearly displayed 4 Click on OK to close the Colour dialogue 5 Click on OK to change the colour 6 15 6 Changing the font settings To configure the font used in the alignment table 1 Select View Properties to open the ALFwin Alignment Properties dialogue and select the Display Properties tab Click on the Set Font button The Font dialogue is shown 2 Select a Font Font Style and Size 3 Click on OK to close the Font dialogue 4 Click on OK to change the font If you want to restore the default colour and font settings click on the Default button 6 41 6 Sequence Alignment 6 42 Troubleshooting 7 Troubleshooting This chapter helps you identify and correct operational problems with ALFwin In all cases a reason for the encountered problem is suggested and if appropriate a course of action is proposed General hints using
206. ter the upright end point has been reached another 300 bases are read to a point called the stop point The bases that are read after the upright end point might have an error frequency higher than 1 and are therefore written in italics C 3 C The base calling algorithm C 2 How to use the base calling algorithm C 4 The base calling function is constructed to work with minimum interaction you can normally generate high quality data by using the standard settings In some cases however the quality of the data can be improved by overriding the default settings for the base calling process This is done by providing the base calling program with additional information The most important examples of user interactions are a setting the start and or the stop point for processing b manually adjusting thetime shifts and c choosing one of the heterozygote modes instead of the normal mode for processing These three user interactions are described in the following sections C 2 1 When to set the start and or stop point manually Processing can be set manually either by using the post run action function in ALFwin Control or the process function in ALFwin Evaluation Reasons for setting the start point manually include the data of interest is proceeded by another sequence e g by a vector sequence It may be easier to find the data of interest if the processing starts just before it e to make sure that the manually adjusted
207. th Resolution Compression Invert option box Image width height This allows you to set the sampling occasion of data points for generation of the TIFF file A larger value uses fewer data points i e data points are taken at every x interval Use of few data points reduces the size of the generated file although also reduces the resolution of the image In some cases you can generatea TIFF file based on the raw data or processed data This is the number of pixels separating the lanes in the image This is the width in pixels of each lane in the image In some cases you can select an alternative resolution for the TIFF file In some cases you can select a compression tool for the generated TIFF file This option produces the TIFF file as a negative image These closed fields show the width and height in pixels of the image to be generated based on the selections you make in other fields 5 To execute the conversion click on the Convert button The generated TIFF file is stored in the folder defined in the Initial Directory field of the Tools dialogue 5 14 4 Add file The Tools Add file command when active checked can for some programs automatically display the pathway and file name of the target file based on the path and name of the source file Only the file extension is changed 5 62 ALFwin Sequence Analyser 5 5 15 Exiting ALFwin Sequence Analyser When you have finished working wi
208. th the result file you can close ALFwin Sequence Analyser Select File Exit or press the lt Alt F4 gt keys or click the Close button in the upper right corner of the window Alternatively if you are going to use ALFwin Sequence Analyser again soon you can temporarily hide the ALFwin Sequence Analyser window by using the minimize button in the upper right corner of the window 5 63 5 ALFwin Sequence Analyser 5 64 Sequence Alignment 6 6 Sequence Alignment ALFwin Sequence Analyser contains an integral module for alignment of sample sequences Sample sequences for alignment can be obtained from a combination of sources by opening a result filein ALFwin Sequence Analyser and processing the raw data to produce a sequence for each clone by importing sequence data in other formats Ee traiks ses rae hiai Fie Pl are TS Crop Patera ade Cel Taber ae Crai Taber le Paii Ha Wit Hib H ie a i a a a a Sd ee T fa Consensus reading 3rd frames Figure 6 1 A typical alignment item including the sequences and the alignment table showing the aligned bases with the amino acid reading frames added The alignment overview indicates the area displayed in the alignment table Sequence alignment compares sample sequences from within the same result file or from different runs When the sequence alignment is calculated all sample sequences are aligned with each other and the best alignm
209. the Oxford M olecular group ASCII ASCII txt format that will load into many standard commercial applications ALFwin Sequence Analyser 5 EMBL The eml format for Bioresearch the sequence analysis program by Fujitsu Ltd DNAsis The dna format of DN Asis a sequence analysis program from Hitachi Software If the Export Settings dialogueis called while the Curve or Gel View is active all the clones will be selected by default If the dialogueis called while the Result view is active only the clones selected in the Result view will be selected by default Click on the tick boxes to select or de select clones Enter in the Path field the location for the exported file You can type the path or use Browse to open the Browse dialogue to select the folder Select the Range All bases All bases in the clones will be exported Upright bases Only upright bases in the clones will be exported From base to Specifies the part of the available clones range to be exported For example from base pair number 14 to base pair number 123 Select the Sequence mode this option is not available for Staden SCF format Forward or Reverse Export the forward or reverse sequences Complement Export the complement sequence Select File names Clone name clone no Use the name of the clone from the casebook the clone number Result name clone no Use the name of the result file the clone number New name clone no Use th
210. the lt F1 gt key on the keyboard Y ou can also access the help topics by selecting the Help Topics command in the Help menu This allows you to locate help topics based on the contents index or keyword search 1 3 Using this manual This User M anual provides you with step by step instructions on how to specify the experimental conditions of a run control a run process raw data align sequences analyse the results and prepare the data for export into other applications 1 3 1 Typographical conventions M enu commands the names of dialogue boxes and windows the contents of dialogue boxes windows and option buttons are written with a bold helvetica typeface M enu commands are written in the order of the menu name and then the command separated by a colon 1 3 1 Introduction For example Select File Save As to display the Save As dialogue Locate the destination drive and directory and enter a file name Click on Save This directs you to click on the File menu and select the command Save As A dialogue called Save As is displayed in which you must locate the destination directory for the saved file and give the file a name Y ou then click on the button called Save in the dialogue to execute the save command Some menu commands also have shortcut keys on thekeyboard which are written within lt gt marks Making your first run 2 2 Making your first run The aim of this chapter is to take you through your
211. the R esult window Presenting curves in the Curve window Presenting the run in the Gel View window Processing run data Editing sequence bases Viewing casebook information and adding evaluation notes Saving the result file Exporting data Printing a report Printing Curve and Gel Views Using tools Exiting ALFwin Sequence Analyser 5 1 Starting ALFwin Sequence Analyser 5 1 1 Starting ALFwin Sequence Analyser ALFwin Sequence Analyser is started from the ALFwin Administrator dialogue 1 Locate and select ALFwin from the Windows 95 Start button menu options The ALFwin Administrator dialogue is shown 5 3 5 ALFwin Sequence Analyser ALFwin Administrator x m User information Add User Default _EdtUser User _Delete User User m Available applications Sequence Analyser 2 00 Instrument 1 Instrument 2 System Setup Figure 5 2 ALFwin Administrator dialogue 2 Inthe ALFwin Administrator dialogue select your user name or the name Default and then double click on ALFwin Sequence Analyser 2 00 under Available applications The ALFwin Sequence Analyser module is started For more information on the Administrator module see Chapter 2 3 When ALFwin Sequence Analyser starts the splash screen is displayed for a few moments and is then replaced by the ALFwin Sequence Analyser screen The Tip of the Day dialogue is superimposed on the screen 5 4 ALFwin
212. the Search Attributes list Base Sequence This allows you to search for a specific nucleotide sequence The sequence should be entered into the Sequence to Search For field Gaps in the sequence can be included or ignored in the search Ambiguity This allows you to locate the next previous ambiguity Mismatches This allows you to locate the next previous mismatch 6 23 6 Sequence Alignment 6 24 4 Select to conduct the search in either the consensus sequence or the sequences that you have selected in the Alignment Item window 5 Locate the objects using the Next and Previous buttons as required 6 7 5 Hiding identical bases Sometimes it is useful to hide from view those bases which display perfect alignment ALFwin allows you to replace these positions with spaces or another character so that attention can be focused on the deviations To replace identical bases with a non specific character 1 Select View Properties to open the ALFwin Alignment Properties dialogue Apaadi p INE Dipi prarasti Sedan Colum Fret E mia Sas ia Foe ete were MMseelll Bisse Sarpe Aceria Mase Fir Ammi od Seequenoiri Papia kiiza Chere F Fosdieg Firan Reading Fone F Pendim Fera J Tine Loses Cadea VP Theme Later Corea Figure 6 11 Alignment Properties dialogue Display Properties tab 2 Select the Display Properties tab 3 Click on the Replace Identical Character list and select t
213. the gel level Fill the upper and lower buffer reservoirs with running buffer to the appropriate maximum level marks inside the reservoirs EEEa E D pa y e e pe F ETE eee a a nar if Eje op ae p e Coes toe Lerh iver jia ia Lya ems ia amama fi y D cm Figure 2 4 ALFwin control screen with Casebook dialogue open 2 2 6 Entering information into a casebook Casebooks are central to the operation of ALFwin Control as they contain the conditions for your run and also store details about the run and set up procedures for automatic post run actions ALFwin is supplied with one casebook file called Sequencelst cbk which contains defined experimental conditions used making this first sequencing run Casebooks are discussed in Chapter 4 1 2 6 In ALFwin Control select the command option File Open Casebook T he Open Casebook dialogue is shown Making your first run 2 Look in ja Default El cl sci El E Sequencelst cbk Files of type Casebook files fbk cbk 7 Cancel Figure 2 5 Open Casebook dialogue Browse to the Default folder and open it if the contents are not already displayed Select the file called Sequencelst cbk which contains the experimental conditions for this run Click on Open The File Information tab of the Casebook dialogue is displayed Fig inianmatin Cordem Sampla inforaston Perian Actors Hote Mara
214. the horizontal scroll bar beneath the alignment table fAignment Position 1 2 3 Figure 6 6 Alignment table in the Alignment Item window Sequence Alignment 6 6 4 1 Sample sequences The sample sequences are presented in tabular form Each sample sequence forms a row in thetable and the individual bases comprising the sequence are displayed in individual cells within the sequence row The sequence rows are positioned relative to one another based on the calculated alignment so that corresponding base positions are displayed in the same column 6 4 2 Consensus sequence The consensus sequence is displayed in a row above the sample sequence rows T he consensus sequence is automatically generated during the alignment process and is a representative sequence of all sequence data Further details are described in Section 6 5 6 4 3 Alignment direction The alignment direction of individual sequences is denoted by the symbol gt gt for forward and lt lt for complemetary reversed 6 4 4 Alignment imperfections Imperfections in the matching of specific base positions between aligned sequences with respect to the consensus sequence i e detected single base components are indicated by colour coded cells These correspond to the colour coded ticks on the sequence lines in the alignment overview The colour codes can be re defined in the Alignment Properties dialogue see Section 6 15 5 W hite cell Al
215. the number of windows to be printed on each page Enter the number of bases to be included in each window Enter the thickness for the curve lines Range is 1 50 pixels The default is colour Monochrome printouts are useful if you areplanning to fax or photocopy the print M onochrome prints use different line types to differentiate between curves Select this option to underline the edited bases 7 Select the Scaling that is applied to the curves Together Separately Stacked curves Apply the same scaling to all the curves so that only the highest peak uses the whole range of the y axis Scale each curve to use the whole range of the y axis Leave this option unchecked to print the curves on one axis Select the option to print the curves at four different levels in the curve window 8 If you want to save these settings for later use click on Save Layout and use the Save As dialogue to define the path and file name Click on Save 9 If you want to close the Report Settings dialogue without printing a report click on Cancel 10 Click on Print to send the report to the printer The next time the same user opens the Report Settings dialogue the settings will be the same as in the last saved and used setup ALFwin Sequence Analyser 5 5 13 Printing Curve and Gel Views and sequences Z 5 13 1 Printing the contents of a single view The Print command allows you to print the contents of t
216. the report By default the clones checked ticked in the result window arealso checked here N ote that the clone selection is not part of the report layout ALFwin Sequence Analyser 5 4 Select the Range of the curve to be printed Entire curve All bases Upright bases From base to From time mins to 5 Select the type of Data Raw or Processed Curves to include Shifted Include sequence Forward Reverse Complement Complement amp Reverse Curves are printed from the first to the last minute of the run Curves are printed for the called bases Curves are printed for the upright bases Print curves for the bases over the range you specify in the From to fields Print curves for the time period you specify in the From to fields Print curves of raw or processed data Select the base curves to includein the print Activate this option to print the curves with the shift applied during processing so that thepeaks arespaced at regular intervals Activate this option to print the base letters with the curves Print the sequences curves in forward order Print the sequences curves in reversed order Print the complementary sequences curves Print the complement of the reverse sequences curves 5 55 5 ALFwin Sequence Analyser 5 56 6 Select the Printout options Windows page Bases window Curve width Colour or Monochrome Show edited bases Enter
217. ting the instrument Before loading the samples and starting a run the instrument temperature must be preset to the value defined in the casebook 1 From the ALFwin Control toolbar on the screen click on the Preset button The temperature will now increase in the instrument to the value defined in the casebook 2 Monitor the increase in Temperature in the M anual window of ALFwin control Thegreen value is the current temperature within the instrument The black value is the one defined in the casebook 3 Check that the yellow temperature indicator lamp on the instrument flashes while the temperature increases to the set value W hen the temperature is reached the yellow indicator lamp stays lit 2 9 2 Making your first run 2 2 8 Denaturing the samples W e recommend that you use the Ready T o Go ALFexpress M 13 Ladder order no 27 4560 80 which you can obtain from your local Amersham Biosciences representative There are four colour coded and labelled tubes one for each of the four bases A C G T The samples should be stored at 20 C until use 1 Denaturethe DNA samples at 95 C for 2 3 minutes a thermocycler is recommended for this purpose 2 Snap cool the samples on ice for 2 minutes 3 Pulse spin the samples in a microcentrifuge at 13000 rpm 4 Return the samples to ice 2 2 9 Loading the samples Y ou will now load the samples and make the final preparations of the instrument before starting the run
218. top field of this dialogue Y ou can name your instrument here the name is shown in the Title bar of ALFwin Control during a run If you wish to change the name select it and typein the name you require If you includea nameor ID for the connected instrument it will help you to remember which instrument is connected to each COM port Type any comments in the Comment field Type the number of the COM port to which your instrument is connected The default at installation isCOM port 1 to instrument land COM port 2 to instrument 2 Click on OK An instrument is connected to the software Select the second instrument if required and proceed as above to connect it to the second port on the computer Note If required a service engineer can select the Instrument Trace Window option to view information about communication between theinstrument and the program Thetrace window resides on the Windows 95 taskbar If the instrument trace information is to include information from the instrument driver the Include Driver Trace option should be selected 3 3 2 Connecting to the demonstration instrument A demonstration run can be performed by connecting a demonstration instrument which operates asa virtual instrument T he benefit of being able to connect a demonstration instrument is the ability to run the software for learning or demonstration purposes without the need for a real instrument to be connected to the computer To install the
219. tout options Color i Print Preview C Monochrone Portrait c ancel Figure 6 16 Alignment Report Setup dialogue To configure and print an alignment report Z 1 Select the sequence alignment window to make it active and then select File Alignment Report or click on the Report button The Alignment Report Setup dialogue is displayed 2 Select the Sequences to include in the report Deselect uncheck unwanted sequences 3 Select check the Alignment item data to be included Alignment item table This includes the list of processed clones Log Notes Alignment settings present in the alignment list This includes information about the alignment automatically generated sequence alignment log This includes information about the alignment notes you have made This includes information about the alignment settings you selected 6 31 6 Sequence Alignment 4 Select check the Alignment results to be included Statistics This includes the alignment statistics Overview This includes the graphical information in the alignment overview Result view This includes the alignment table data as displayed on the screen for the whole sequence 5 Select the Printout options Colour or Monochrome Thedefault is colour See Section 6 15 5 M onochrome printouts are useful if you are planning to fax or photocopy the print Monochrome prints use different shades of grey for the alignment imperfections La
220. transmittance 2 11 4 40 value 4 40 value in M anual window 2 11 layout layout file for curve window 4 57 layout file for report print 2 18 opening Curve window layout 5 25 saving Curve window layout 5 25 least common denominator 6 14 leave heat on after run 4 18 letter codes for amino acid sequences 6 27 light indicator in the taskbar 4 7 linking ALFwin to the instrument 2 3 linking Curve windows 5 16 logbook altering the font 4 59 contents of logbook window 4 6 sequence alignment log 6 28 XV Index xvi M M 13 standard sample 2 1 2 10 magnification adjusting in curve view 4 50 5 14 adjusting in Gel View window 5 27 adjusting in Gel View with mouse 5 27 adjusting using the mouse 4 50 adjusting using the rectangular cursor 5 14 magnifying the signal level 4 49 resetting 4 51 resetting in Curve view 5 15 resetting in Gel View 5 28 using the zoom commands 4 50 majority consensus mode 6 15 making your first run before starting 2 1 casebook information 2 6 loading the samples 2 10 mounting the gel cassette 2 5 pre run activities 2 1 starting Control 2 4 starting Sequence Analyser 2 12 starting the run 2 11 turning on the instrument and computer 2 2 manual bold typeface entries 1 3 typographical conventions 1 3 using 1 3 manual shift 5 37 M anual window 4 40 4 45 adjusting contents 4 58 altering the font 4 58 contents of M anual window 4 6 mark upright bases 5 36 max signal level adjusting 4 49 Menu 4 7 menu b
221. trl N N ew alignment file Ctrl R Start ALFwin Control so that a new run can be made Tab M ove forward between fields in the active dialogue box Shift T ab M ove backwards between fields in the active dialogue box Ctrl T ab M ove between open windows Home or End Scroll moveto the beginning or end of view or list Page Up or Down Scroll move to next page up or down in view or list Arrow keys Scroll move one step in view or list D 5 2 In the Clone view in the Result window Up or down arrow key Moveup or down in the clone list Right or left arrow key Show or hide lanes in the selected clone Ctrl Enter O pen the selected clone D 5 3 In the Gel view in the Result window F5 Show raw data F6 Show processed data F7 Zoom time in F8 Zoom time out Shift F 7 Zoom channels in Shift F 8 Zoom channels out F9 Reset zoom D 3 D Keyboard shortcuts D 4 F10 Ctrl C Activate Zoom with M ouse mode Press again to deactivate this mode Copy Gel view to clipboard D 5 4 In the Curve window Ctrl T ab Ctrl 1 9 or 0 Ctrl Shift 1 9 or 0 Ctrl Alt 1 9 or 0 Ctrl Alt Shift 1 9 or 0 F5 F6 F7 F8 F9 Alt lt Ctrl Shift A C G or T Ctrl Shift S Ctrl Ctrl Y Ctrl Switch between open windows Clone 1 10 is displayed Clone 11 20 is displayed if it exists Clone 21 30 is displayed if it exists Clone 31 40 is displayed if it exists Show raw data Show processed data
222. ts relating to the command line Initial Directory Enter the folder that contains other important components needed by the tool Use the Browse facility to help you Deleting tools To delete a tool 1 Open the Items list and select the appropriate tool 2 Click on the Delete Tool button 5 14 1 The alx and alf formats ALFwin Sequence Analyser v 2 00 operates in the main with the alx file format ALFwin Sequence Analyser can also use files of the earlier alf type There are two reasons why you may wish to use the alf format in your work e You have data which were collected and saved before alx filetype became standard e You have computer applications which are designed to operate on files in alf format In the latter case you may wish to save data from ALFwin Sequence Analyser in alf format If you use the normal File Save option to save your result file ALFwin Sequence Analyser will save it in alx format To save as an alf file you must use the Split File Settings dialogue Please refer to Section 5 10 2 ALFwin Sequence Analyser 5 5 14 2 Decompressing alf files ALF files generated in earlier versions of ALF family instrument control software e g ALF M anager and AM could save ALF files in a compressed format To decompress an ALF file 1 4 Select File Open and proceed using the normal file opening procedure Please refer to Section 5 3 2 To open a decompressed ALF file click on Open
223. ue in the Max Signal Level field 5 Click on Apply to effect the change 6 Click on OK when the scale is satisfactory Curve Properties Layout lt default gt Figure 4 27 Curve Properties dialogue Settings tab 4 8 5 Showing stacked or overlapping curves By default the curves are displayed stacked above each other in the Curve window To view overlapping curves 1 Click on the Curve window to make sure that it is activated 2 Select the active checked View Stack Curves to deactivate the function or click on the active Stacked Curves button Overlapping curves are shown 4 49 7 win Control E Cae Vere Cleve 0 Figure 4 28 Curve view displaying stacked curves 4 8 6 Using the Zoom function with curves Y ou can alter the magnification of the curves view in the Curves window Changing magnification with the zoom commands Command Effect View Zoom In Successively increases the lt F7 gt key magnification lt Shift F7 gt Increases the magnification in small steps View Zoom Out Successively decreases the lt F8 gt key magnification Shift F8 gt Decreases the magnification in small steps Increasing the magnification using the mouse To magnify a specific portion of the window using the mouse 1 Position the mouse pointer on the left side of the area you want to magnify 2 Press and hold the left mouse button and drag the box to encompass the time interval to be magnified ALFw
224. uence Analyser ALFwin stops collecting data during a run Problem ALFwin stops collecting data during run Explanation A local language version of Windows 95 is used or resource conflict in the computer Solution We recommend using the International UK version of Windows 95 together with ALFwin since the UK version is the only one that ALFwin has been developed and tested against See also recommended system requirements section A 1 Error message occurs during an ALFwin run Problem During run a Control error message occurs such as This program has performed an illegal operation and will shut down If the problem persists contact the program vendor CONTROL caused an invalid page fault in module M FC42 DLL Explanation The operation system may sometimes interfere with ALFwin Control or vice versa 7 3 Troubleshooting 7 4 Solution During run start up the Control module again and log on as the same user If the instrument s green LED has not started to flash the data collection will continue Please note that on the screen it will look asif all data prior to the failureis deleted H owever all data prior to the error message will be added to the data collected after the failure when the run is ended That means that a complete file will be possible to open in ALFwin Analyser after the run ALFwin Sequence Analyser N o processing result Problem Peaks are seen but the algorithm failed to start
225. uences that are to be aligned to the previous alignment Press the lt Ctrl gt key to select several sequences 4 Select Analysis Align to Previous or click on the Align Sequence button The results are shown in the alignment overview see Section 6 3 and alignment table see Section 6 4 6 3 The alignment overview 6 10 Results of an alignment are displayed in the alignment overview and alignment table see Section 6 4 Thealignment overview shows a graphical representation of the whole result Each sequence is displayed as a line and the length corresponds to the number of bases in the sequence All lines are positioned relative to one another based on the results of the alignment The alignment overview includes a rectangular cursor that indicates the part of the result currently displayed in the alignment table Y ou use this cursor to navigate around the sequences Consensus Seq 1 Seq 2 I Seq 3 se a SS Figure 6 5 Alignment overview in the Alignment Item window 6 3 1 Sample sequence lines and alignment direction A blue sample sequence line going from left to right as denoted by the direction of the arrow head indicates a sequence that is aligned in the forward direction A red sample sequence line going from right to left as denoted by thedirection of the arrow head indicates a sequence that Sequence Alignment 6 is aligned in its complementary reverse direction The default settings for the al
226. up The Group dialogue opens 4 Inthe Group name field enter a name for the new group 5 Select the lanes to be included in the group Use lt Ctrl click gt to add items spread out in the lists Use lt Shift click gt to add items that are next to each other 6 Click on OK The new group is added to the list of Group names The name you entered is always added after Group Group name Lanes in group Aj Unselect All C G T C G Lane 8 T C G T C i Figure 4 30 Group dialogue 7 If required select the Instrument data oneor more to be displayed in all the curve windows To deselect all data lt Ctrl click gt on the selected data 8 Click on OK to exit the Curve Properties dialogue 4 8 9 Editing a group Y ou can edit groups that you have added T hese groups are marked with a before the name ALFwin Control 4 To edit a group 1 2 6 Click on the Curve window to make sure that it is activated Select View Properties T he Curve Properties dialogue is shown Select the Group to be edited from list of Group names Click on Edit group A dialogue is shown Change the name of the Group or lanes to be included in the group The lanes already included in the group are highlighted Click on OK The changes are made to the selected group 4 8 10 Deleting a group Y ou can delete groups that you have added T hese groups are marked with a
227. ure The same M 13 sample was loaded across the gel and there should be no great disparity between adjacent clones e checking for excessive shifts e checking the signal levels of the raw data Y ou can also confirm the stability of the instrument readings see below e check the sequence alignment see below Confirming stable instrument readings The instrumental parameters are logged during a run and can be displayed as curves 1 Click on the Curve window to make sure it is activated or open the curves for the appropriate clone 1 Smiling is the phenomenon produced when the samples in the first and last lanes move more slowly than those towards the centre of the gel 2 Making your first run 2 Select View Properties and click on the View tab in the Curve Properties Layout dialogue 3 Select the required Instrument data Y ou can select an individual item specific items if you hold down the lt Ctrl gt key while you select them or a range of items if you press the lt Shift gt key while selecting the last item 4 To effect the changes without closing the dialogue click on Apply To close the dialogue and effect the changes click on OK 5 Place the mouse pointer on the Instrument data curve of interest The value at the pointer is shown in the Status Bar Checking the sequence alignment It is possible to align the sequences within open runs This presents several useful analysis possibilities and fu
228. urther entry Use this field to make notes during the course of the run When the run finishes the Run notes field is closed against any further entry Use this field to make notes when using ALFwin Sequence Analyser 7 win Control Fis ivicerarien Cowie Saage mharan Paria Senge Neer Poeun ron F Steee pee ken cbet He Figure 4 22 Casebook dialogue Notes tab 4 5 Performing a run Y ou can start a run once you have selected a casebook made any modifications and saved the casebook file A run can be divided into four main stages Preset Preset is used before the start of a run to allow the gel and the buffer to achieve the correct temperature see Section 4 5 1 Starting the run Starting the run allows ALFwin Control to automatically control and monitor a sequencing run using the connected instrument When the run is started any modifications made to the casebook during the run are saved in the result file only see Section 4 5 2 During the run The instrument is controlled by the computer according to parameters contained in the casebook Y ou can use the Manual window to observe the target parameters and compare them with their measured values Adjustments can be made Preset Ending the run ALFwin Control 4 manually as the run proceeds or you can pause the run to make adjustments The Logbook is used to observe the progress of the run and automatically records any changes that are made Chang
229. urves is scaled separately so that the displayed amplitude scale for each curve is adjusted to best fit the print space available Stacked curves Print the curves at four different levels in the curve window 7 Select the type of Data to be included in the report Raw or Processed Print curves of raw or processed data Note In order to print processed data curves the raw data must first be processed as a post run action Curve A C G and T Include the selected base curve s 10 ALFwin Control 4 Shifted Print the curves with the shift applied during processing to space the peaks at regular intervals This only applies to processed data produced as a post run action Include Sequence Include the base sequence in the curve print This only applies to processed data produced as a post run action Forward or Reverse Complement Complement amp Reverse Select the Printout options Windows page Bases window Curve width Colour or Monochrome Click on OK Print the forward or reverse sequences curves Print the complementary of the forward sequences curves Print the reverse of the complementary sequences curves Enter the number of windows to be printed on each page Enter the number of bases to be printed in each window Enter the width required for the printed curve line Use the colour option when a colour printer is available for report printing Use the monochrome option to produce b
230. ve Casebook Seqdem1 x Select an action for the edited casebook Save ds ieee Edit Again Help Figure 4 6 Save Casebook dialogue 2 Select from the options available Save To save the casebook under the existing name and folder ALFwin Control 4 Save As To savethecasebook with anew name and or folder Selecting this option opens the Save Casebook As dialogue Use this dialogue to specify a name and location for the casebook or to rename an existing casebook that has been edited This is recommended when modifications have been made to an opened casebook Discard Available only after creating a new casebook This option removes the new casebook without saving or loading it Edit Again To leave the save procedure and return to the Casebook dialogue 4 3 5 Editing a casebook before a run As long as you have not started the run you can make changes to the casebook Changes made before the run is started are saved in the casebook file cbk file After the run has been started you can still change some items such as e post run action settings e contents of the sample information table run notes Any changes that are made during or after the run are saved in the result file alx file To edit the casebook 1 Select Edit Casebook or click on the Edit Casebook button The Casebook dialogue is shown 2 Make the changes see Section 4 4 3 Click on OK The Save Casebook dialogue is
231. w 5 9 naming the split files 5 48 saving in a single file 5 48 saving in multiple files 5 48 selecting for alignment 6 4 selecting for export 4 32 selecting the clones for a split file 5 48 Clones tab 2 8 colour changing colour in Curve window 4 55 5 23 column add 4 24 changing the order of columns 4 24 header deleting 4 24 header editing 4 24 COM port selecting 4 4 comb removing 2 10 Comment column 2 8 Comment field 2 4 complementart sequence 5 24 compressed files 5 7 computer specifications A 1 Conditions tab in casebook detail 4 17 overview 4 8 configuring algorithm options 6 36 alignment algorithm 6 36 Index alignment data import 6 38 alignment table colour settings 6 40 alignment table font 6 41 amino acid display 6 35 amino acid reading frame 6 35 amino acid sequences display 6 26 base curves in Curve window 5 20 colour in Curvewindow 5 23 consensus mode 6 34 dataset in Curve window 5 20 font in Curve window 5 23 instrument data curves in Curve window 5 22 presentation of curves in Curve window 5 19 scale in Curve window 5 18 sequence alignment module 6 34 sequence in Curve window 5 24 signal level table in Curve window 5 19 X and Y axes in Curve window 5 17 connecting ALF family instrument to ALFwin Control 3 5 instrument to ALFwin Control 3 5 to the demonstration instrument 3 6 consensus changing the consensus mode 6 15 configuring consensus mode 6 34 disabling automatic recalculation
232. will hold between sessions for each user If you want to alter the default startup configuration refer to Section 6 15 1 6 15 6 Sequence Alignment 6 16 1 From the ALFwin Sequence Alignment dialogue select View Consensus 2 Select Least Common Denominator or Majority according to your requirements 6 5 2 Recalculating the consensus sequence By default the consensus is automatically recalculated when you edit bases in the sample sequences within the alignment table or change the consensus mode To disable automatic Select View User Preferences and click recalculation of consensus Automatic Recalculation of Consensus to deselect it To initiate a recalculation W hen automatic recalculation of manually consensus is disabled recalculate by selecting View Consensus Recalculate Consensus To restore automatic Select View User Preferences and click recalculation on Automatic Recalculation of Consensus to select it 6 5 3 Viewing the IUPAC codes If you locate an ambiguity code in the consensus sequence you can display the IUPAC codes ALFwin IUPAC x a IUPAC Codes B gt CGT D AGT H gt ACT K gt G T M gt AC N gt A C G T R AG S CG V gt ACG W gt AT Y CT Figure 6 7 IUPAC dialogue Sequence Alignment 6 e ThelUPAC dialogue is automatically displayed when you click on an ambiguity code in the alignment table To keep the dialogue active on the desktop after
233. wing 1 2 Select any open Curve window Select View Properties and click on the View tab in the Curve Properties Layout dialogue Select the appropriate orientation view To effect the changes without closing the dialogue click on Apply To close the dialogue and effect the changes click on OK 5 5 18 Including instrument data as curves Figure 5 14 Instrument data curves shown below stacked data curves To include instrument data as curves I 2 Click anywhere in the appropriate Curve window Select View Properties and click on the View tab in the Curve Properties Layout dialogue Select the required Instrument data Y ou can select an individual item specific items if you hold down the lt Ctrl gt key while you select them or a range of items if you press the lt Shift gt key while selecting the last item ALFwin Sequence Analyser 5 4 To effect the changes without closing the dialogue click on Apply To close the dialogue and effect the changes click on OK To check values of the instrument data 1 Select the relevant Curve window 2 Place the mouse pointer on the Instrument data curve of interest The value at the pointer is shown in the Status Bar N ote that the scale of the instrument data is fixed 5 5 19 Changing the font and colours in the Curve window Y ou can change the colour of the curves and the background Y ou can also change the font and the curve width To change colours
234. y end of a sequencing run when the electrophoretic separation performs less well the bases are more difficult to detect by the base calling algorithm so are assigned as italic bases 5 33 5 ALFwin Sequence Analyser 5 34 2 Compare the total number of called bases between adjacent clones from the same sample If there is an obvious disparity between the values then you should check the data curves and the nucleotide sequence 5 7 3 Viewing the data curve after processing and finding special sequences Y ou can view the data curve for a processed clone directly from the Process Status dialogue It is important that you know how to work in the Curves window to make the best of your viewing and evaluation see Section 5 5 You can use the Curves window to find special sequences or ambiguities Viewing the data curves after processing base calling 1 Doubleclick on the clone icon in the Process Status dialogue The Curves window containing the data curves for the selected clone is displayed beneath the dialogue Use View Raw Data or click on the raw data button The base nucleotide sequence is displayed underneath Use View Processed Data or click on the Processed Data button to display curves for the processed data T he base nucleotide sequence is displayed underneath The sequence comprises the colour coded base nucleotide letters A C G and T in addition to any detected base ambiguity codes i e a base position wher
235. y many standard spreadsheet and word processing applications Note You can select the files for export in two ways They can be selected using the Export Settings dialogue as described below Alternatively you can select the files in the Result window and your selection is transferred into the Export Settings dialogue 5 49 5 ALFwin Sequence Analyser 5 50 To export sequences in a result file 1 Select File Export The Export Settings dialogue is shown m Export file settings Format m File names eros cinero Path bs Result name clone no C Program Files Biotech ALFwin Home Da PLIES Le r Range m Clones All bases Clone Name File Name E v4 M13 M13 01 prigm bases M 2 M13 M13 02 c to M3 M13 M13 03 From base fi fi 44 M13 M13804 V5 M13 M13 05 m Sequence mode Ve M13 M13 06 M7 M13 M13 07 Forward T Complement MS M13 M13 08 DE M 9 M13 M13 09 v10 M13 M13 10 Figure 5 26 Export Settings dialogue O pen the Format menu and select the format for the export file GCG The seq format for GCG the sequence analysis program from Wisconsin University STADEN SCF The scf format for the Staden sequence analysis package an interactive graphics program for comparing and aligning nucleic acid and amino acid sequences SCF format includes data curves PC GENE TheN prefixed format for PC GENE a sequence analysis program by IntelliGenetics from
236. ys the portion of the curve that corresponds to minimum and maximum amplitude limits that you have defined in the Properties dialogue see below To change the scale for the open Curve windows 1 Select any open Curve window 2 Select View Properties and click on the Settings tab in the Curve Properties Layout dialogue 5 18 ALFwin Sequence Analyser 5 3 Select the relevant Y axis Scale either Separately or Together see above or Fixed If you select Fixed you must enter a minimum and maximum amplitude value e g an entered range of 0 to 50 means that the portion of the curves with amplitude between 0 and 50 of the possible range is shown 4 Ifyou want the scale to be fixed according to the default scale area of the automatically calculated scales i e as calculated for Separately or Together check the Use current scale area box Whenever you select Fixed you can scroll along the result and view the peaks at their real height relative to the maximum amplitude for the whole run 5 To effect the changes without closing the dialogue click on Apply To close the dialogue and effect the changes click on OK 5 5 13 Changing the presentation of the curves within the curve windows 1 Select any open Curve window 2 Select View Properties and click on the Settings tab in the Curve Properties Layout dialogue 3 Select check Stack to stack the curves Leave this option unchecked to display overlapping curves

5ALFwin - GE Healthcare Life Sciences

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