Automatic Trace Calibration (ATC)
Contents
1. Dye2 Signal normalisation Dyes Estra data and headers E S Baselines peaks Fs Dyed Maxed out signal correction One trace name can be derived from the file name Bev _ Next gt Choose which trace and the method used pe Trace index not used v Method Star of file name v Finish All Cancel All Tabbed Config trace names Trace Parameters Load Save Some trace files contain the names of the traces used but the example does not instead just specifying Dye1 Dye4 These should be changed to recognized dye names as shown The Config trace names button lets you specify new trace names and lets you specify standard colours for existing and newly defined trace names 5 A trace name can also be derived from data file names which is beyond the scope of this document Trace selection Choose the traces that you want to load from the file Trace name in file 7 9961 data FAM Dyel 9961 data HEX Dye2 9961 data TAMRA Dye3 MV 9961 data ROX Dyed Maxed out signal correction lt Prey Finish Cancel Finish All Cancel Al Tabbed Trace Parameters Load Save Choose which traces to load In this example we are only interested in the size calibration ladder which is measured in the ROX trace so only ROX is checked Trace drawing colours Trace analysis parameters for D w_example6 Ladder_E10 fsz Le oe _ Parameters Choose the colours that you want to use to dr2. Sizes and calibration trace selection can be copied from the following predefined standards GeneScan 500 ROX no 250 340bp GeneScan 500 LIZ GeneScan 500 LIZ no 250 340bp GeneScan 600 LIZ Megabace ET ROX 550 ET400 F MapMarker 1000 MapMarker 400 Sizing standards can be read from a Genographer configuration file Pick configuration file Pick reer Cor In the list of predefined standards select the MegaBACE ET ROX 550 then click on the Copy button Click on the Trace type amp fragment sizes tab and confirm that the correct settings are now in place In order to set up some restrictions click on the Restrictions tab These restrictions while ideally not required can significantly speed up the ATC process Automatic Trace Calibration Trace type amp fragment sizes Import sizes Restrictions Calibration Advanced I Skip initial 0 00 A ts V Require minimum peak area 0 20 A C Absolute Relative Use largest peaks 50 Here Require minimum number of calibration points has been made active and the total number of sizes in the ET550 R standard 22 has been entered Note that if some measurements have been run short they may not contain all 22 standard sizes In this case the minimum number of sizes will have to be lowered Require minimum peak area has also been made active with a minimum peak area of 0 2 This filters out any really small peaks from consideration a
3. A g Sample_C04 FAM 4 r 4 ma 10000 Sample_C04 ROX 4 Sample_B04 FAM 4 s a 0 Sample_B04 ROX 4 rary A ar an Vs Pree aa E va l d m sa T T 0 200 400 DNA BP As always you can zoom in on the colour view by clicking and dragging the mouse The colour view window s View menu has a number of options that govern how things are displayed In the example above there is extra space between lanes each trace is displayed using its own trace colour colours are displayed darker to make them easier to read the baseline has been subtracted each trace is in a separate lane as opposed to have all traces from a file displayed in a single lane a calibrated axis BP is used and peaks are marked 32 Chapter 5 Trace Types in DAx Trace types are used in DAx for a number of purposes Their principal use is to distinguish between up to five traces that can be contained in a single measurement file where obviously the file name alone would be insufficient A secondary use is to limit certain operations to specific trace types These operations include e Automatic Trace Calibrations The trace type is used to indicate which trace contains the sizing calibration cf Chapter 2 1 e Normalise using the total peak area of named peaks of a specified trace type cf Chapter 4 4 e Identification Database and Marker Peak items The trace type is used to limit the detection of certain components to a specified trace t
4. are lalebelled with their fragment sizes This is because ATC has named each calibration peak with its fragment size and peaks are being labelled with their names 21 To verify that the calibration is well formed click on the data tag of the ROX trace and select the Calibration Curve menu item from the popup menu that appears Ladder_E10 ROX DNA Calibration Ladder_E10 ROX Linear Local Southern BP C faa Base pairs 400 e T T T T T T T T T 3000 Peak Migration Coordinate For a quick verification of multiple calibrations try the following e Use the File Open command to once again display the File Open dialog Select multiple data files Check the Stacked checkbox at the bottom of the dialog Click Open e In the TAP dialog click Finish All without changing any settings 22 The result might look like this eS a B Semple_A04 ROX pem B04 ROX 5 Sample_B04 ROX Bi sample_cos ROX B Sample_Cco4 ROXx Sampie D04 ROX B Sample_D04 ROXx Ladder E10 ROX B Lsdder_ 10 ROX Despite running at different times all 5 calibrations are well recognised 23 Chapter 4 Analysis of the Unknowns Invoke the File Open command Select Files of Type ABI Genescan files or one of the other trace file types SCF files MegaBACE files CEQ files Select the file or files you want to analyse then click the Open button ia Open g ne Look in J w_example6 X fe
5. dialog contains a further 7 pages dealing with peak find signal and slope thresholds skipping an initial part of the measurement detecting shoulder peaks normalising peak widths to a set number of base pairs removing bleed through peaks All these settings can be left at factory defaults in almost all analyses To finish filling out the TAP dialog click the Finish button If more than one data file was selected in the File Open dialog click Finish All to load all data without showing the TAP dialog again 28 Chapter 4 2 Results The FAM and ROX traces will now be loaded and analysed The results might look like this E4 D w_example6 Sample_B04 fsa CE D w_example6 Sample_C04 fsa CE D w_example6 Sample_D04 fsa CE D w_exemple6 Sample_A04 fsa CE gt faa amp B Ssmple_804 ROX Bisemple_Bo4 FAM B Semple_B04 FAM Ef Sample_C04 ROX al B Sample_co4 ROx Semple_C04 FAM B Ssmple_CO4 FAM ElSample_D04 ROX S Bse 4 ROX Ej Sample_D04 FAM B Sample_D04 FAM Ej Sample_A04 ROX a El Sample_A04 FAM Sample_A04 FAM B Sample _A04 ROX Y 200000 4 150000 4 O E se 100000 4 a Pr W100 i75 200 P225 pe par m 310 p325 p350 pars pe 400 Pr 425 Paso Par P500 P525 p550 g s Q o o 1 oi 0 75 q4 100 Bizs Eiso pir 200 225 250 Par pe 310 32 350 P375 400 Pas Paso az soo P52 P
6. loaded Since this is the calibration trace it will have a baseline constructed and peaks found even if the baseline construction and peak find items are not checked here Maxed out signal correction Parameters Choose if you want to mark and correct intervals where the trace Panor piocessed data signal exceeds a specified limit Trace names Maxed out traces Mark intervals Trace selection Trace drawing colours I Correct signals Colour separation method Colour separation matrix Corect spikes Sizing calibration derivation ere Calibration trace selection Signal limit fer 30 A Horizontal axis TON Signal normalisation Marking signals adds tacks at the begin and end of intervals Extra data and headers where the signal is out of range Correcting signals uses lower intensity signals to predict the values of out of range signals Removing spikes interpolates between 2 out of range signal j points if a single intermediate point drops below 1 of the signal limit Finish Cancel Finish All Cancel Al Tabbed Trace Parameters Load Save Sometimes the signal values in a trace file will exceed what the data format can handle DAx can detect such occurrences and annotate them in the loaded data Shown here is a comparison of current DAx analysis parameters and pertinent qualities of the analysis present in the file Trace selection Calibration trace selected ROX used byATC ROX Adjust Tra
7. the data are to be loaded can be set up The TAP dialog contains a list of parameters at left which you should fill out in order34 Raw or processed data Parameters Choose if you want to import raw data or data that have already been colour separated and processed Raw data r Colour separation matrix This ABI file contains no processed data Sizing calibration derivation Horizontal axis Signal normalisation IF you choose raw data you will have to indicate how E the traces are to be colour separated Maxed out signal correction Some functionality such as importing peaks or base calls requires processed data to be imported E Next gt Cancel Finish All Cancel Al Tabbed Trace Parameters Load Save The example file contains no processed data so raw data must be loaded Click Next to display the next page of parameters 3 The list changes in accordance with the types of parameters that are needed 4 if your TAP dialog does not look like the examples you should click the Wizard button third from bottom left 12 Trace names Trace analysis parameters for D w_ Parameters Choose or adjust the names of the traces in the file Each trace should preferably F ad use one of the defined trace names Some functionality such as Automatic Trace aw of processed data Calibration requires the use of a defined trace name Trace name in file 1 FAM lt Dyet HEX lt
8. 550 o N o4 S D o4 S DNA BP The following has happened in succession e For each file the ROX trace is loaded It was analysed using the current settings Since the Automatic Trace Calibration parameters define ROX as the calibration trace the sizing standard was recognised and a calibration relating time coordinates to fragment sizes was derived from the ROX trace e the FAM traces were loaded and analysed using the current settings e The axis was converted to base pairs using the calibration derived from the ROX trace You can now click any of the peak list buttons at the left side of the data tags for the ROX or FAM traces You will note that the correct fragment sizes are listed in the Base Pairs column It is also possible to display the fragment sizes of the peaks that were found in the graphics window To do this invoke the File Customise menu option Select the Plotting Peaks tab Under Peak Labeling select Component Name as the first peak label 29 Select Base Pairs as the second peak label it is near the bottom of the list of possible peak labels Click the OK button The graphic window will now display base pair counts for all the peaks You can click and drag the mouse on the graph to zoom in on the peaks and see more detail Chapter 4 3 Converting to a Base Pair axis In the example above the horizontal axis was immediately converted to a BP axis by checking Convert to BP axis in th
9. Chapter 1 Chapter 2 Chapter 2 1 Chapter 2 2 Chapter 3 Chapter 3 1 Chapter 3 2 Chapter 4 Chapter 4 1 Chapter 4 2 Chapter 4 3 Chapter 4 4 Chapter 4 5 Chapter 5 Chapter 5 1 Chapter 6 Contents Introduction Defining the Standard Defining the ATC parameters Troubleshooting ATC Analysis of the Calibration Ladder Trace Analysis Parameters TAP dialog Results Analysis of the Unknowns Trace Analysis Parameters TAP dialog Results Converting to a Base Pair axis Normalising the data Displaying data as a colour view Trace Types in DAx Assigning trace types Conclusion 5 10 11 12 21 24 25 29 30 31 32 33 33 35 Chapter 1 Introduction DAx can be used to read and analyse trace files such as the files that are created by the Amersham MegaBACE and ABI Genescan systems The MegaBACE and ABI systems like other DNA analysis systems can analyse four or five traces in a single run using four five different coloured labels One way to use this feature is to include a standard sample with known DNA fragment sizes as one of the traces and apply this standard to the other traces The standard is used to ascertain the fragment sizes of the components in the unknown samples Chapter 2 Defining the Standard This chapter describes the steps needed to set up the analysis of the sizing standard contained in trace files Typically you will go through this process once for a particular siz
10. atch files All the data on an entire hard disk can be analysed with a single command while highly flexible report files are created These features exceed the scope of this manual Refer to the DAx User s Manual for details 35
11. aw the traces Raw or processed data Colour separation method 3961 data FAM __ Colour separation matrix a Sizing calibration derivation 3961 data HEX m Horizontal axis 9961 data TAMRA MM 9961 data ROX ME Maxed out signal correction Use standard colours Traces that will be drawn using default DAx colours Opa are shown with a white hatched button here Finish Cancel Finish All Cancel Al Tabbed Trace Parameters Load Save The Use standard colours button is disabled here because the colours are already the standards for the trace names shown 14 Colour separation method Trace analysis parameters for D w_ gt l Parameters Choose the method to be used for colour separation Raw or processed data C No colour separation Use specified colour matrix Use matrix reported in file Optimum colour separation Config Maxed out signal correction lt Prey Finish Cancel Finish All Cancel Al Tabbed Trace Parameters Load Save The signals measured for each of the traces are made up of contributions of not just the trace itself but also contain bleedthrough from the other traces To correct for that a colour separation should ideally be used The best way to do this is to specify a colour separation matrix that has been ascertained by measuring a standard mixture of samples Colour separation matrix Trace analysis parameters for D w_exe Parameters Specify the col
12. ce drawing colours Colour separation method Coordinate of 60 00 BP calibration peak in file lt unknown gt Colour separation matrix Peak find skipped start interval 30 00 Adust Sizing calibration derivation 3 3 Calibration trace selection ATC sl start interval lt not used gt Horizontal axis ae yi Signal normalisation Relative area of smallest calibration peak in file lt unknown gt Extra data and headers Peak find minimum relative area lt not relative gt Adis Baselines peaks ATC minimum relative area 0 20 Maxed out signal correction ee Compare analysis settings Comparison of sizes in file and ATC sizes Copy sizes from file to ATC lt Prev Ney File contains no sizes Number of ATC sizes found in file 0 E STA z er Adjust HER A Minimum number of ATC calibration points 22 Finish All Cancel Al Tabbed Trace Parameters Load Save DAx will compare and verify a number of analysis settings and flag any that do not match In such instances clicking the Adjust button will remove the conflict 20 To finish filling out the TAP dialog click the Finish button Chapter 3 2 Results The ROX trace will now be loaded and analysed The result is shown below EX D w_example6 Ladder_E10 fsa CE fo laj S 3 10000 4 i ry Ss gt 4 175 5000 a IEEE z T z T T 0 1000 2000 3000 Time s Note that the calibration peaks
13. e ROX trace Note that the list of parameters at left has changed because the selection on this page was changed from Do not derive calibration to Derive size calibration from a trace The warning symbol displayed next to Compare analysis settings is there because DAx has detected an inconsistency This will be resolved momentarily 16 Calibration trace selection Parameters Choose what trace to use to derive the sizing calibration Raw or processed data C 9961 data FAM f 9961 data HEX Trace drawing colours C 9961 data TAMRA Colour separation method Colour separation matrix Sizing calibration derivation Choose if you want to use private baseline construction and peak find settings for Signal normalisation the calibration trace Turning on private settings first copies the current settings Extra data and headers Baselines peaks S Maxed out signal correction Compare analysis settings v Automatic Trace Calibration ATC is a method of deriving a sizing calibration lt Prev Next gt M Use ATC Config In the ATC setup you can choose from a number of predefined sizing calibrations mance import the sizes reported in the file if available or enter a sizing calibration manually Finish All Cancel Al Tabbed Trace Parameters Load Save By changing the calibration trace to ROX the warning icon next to Compare analysis settings is removed Make sure Use ATC is checked so that the ET 550 R calibration tha
14. e E A Name Date modified _ Ladder_E10 fsa 07 02 2013 11 22 Sample _A04 fsa 07 02 2013 11 22 _ Sample _B04 fsa 07 02 2013 11 22 _ Sample_C04 fsa 07 02 2013 11 22 07 02 2013 11 22 Files of type ABI Genescan files abi fsa z Cancel E M Sorted Reduce memory use F Multiple windows E F Automatic analysis F Minimised MV Stacked Config Check Multiple windows if you want each file to be put in its own window not typically used for trace analysis If you are opening hundreds or thousands of files you can check Minimised The window into which the files are loaded will be created minimized so that the traces are not drawn saving time Check Sorted and use the Config button to indicate how the data will be sorted after loading For instance the data can be sorted by well or plate name Opening files as a Colour view creates a gel type view of the data immediately upon loading You can immediately display the data Stacked and use its Config button to indicate how to stack Automatic analysis is used to create baselines and find peaks immediately upon loading For trace files using the TAP dialog see below is the preferred method so do not check this option Click the Open button to start opening all selected sample files 24 Chapter 4 1 Trace Analysis Parameters TAP dialog As was mentioned in the previous chapter when trace files are opened the Trace Analysis Parameter
15. e TAP dialog If this option had not been checked the axis can still be converted using the Calibration Axis Conversion menu option This displays a dialog box D gt Convert to BP Axis s a fa il Ois D w_example6 Sample_B04 fsa CE AE Sample_B04 ROX VIII Sample_B04 ROX Low DNA 0 0 yA Sample_B04 FAM 2 aae r High DNA e423 DA M Sample_C04 ROX Points per unit DNA 10 pJ II Sample_C04 ROX VIM Sample_Cc04 FAM i EN Sample_C04 FAM m Sampie_D04 ROX II Sample_D04 ROX vill Sample_D04 FAM I Sample_D04 FAM i MIB Sample_A04 ROX m Sample_A04 ROX vill Sample_A04 FAM I Sample_A04 FAM Cancel Help Make sure to check the New Window check box and then make sure that all data are selected Note that the dialog box was resized to make all data items visible by dragging its bottom right corner 30 Chapter 4 4 Normalising the data When loading trace data it can be desirable to normalise the data after they have been loaded One common form of normalisation stretches the data vertically in such a way that the total area of the size calibration peaks is equal across several measurements Normalisation can be achieved after data have been loaded using the Data Overlay menu option A more convenient way to normalise is to check the Normalise data check box on the Signal normalization page in the Trace Analysis Parameters dialog box Click
16. ing standard DAx uses a heuristic method called Automatic Trace Calibration to recognise the sizing standard s fragments Chapter 2 1 Defining the ATC parameters Use the Analysis Edit ATC menu option to invoke the Automatic Trace Calibration or ATC dialog box The dialog box looks like this A EO O Trace type amp fragment sizes Import sizes Restrictions Calibration Advanced Calibration trace X Horizontal axis type Not DNA BP Calibration sizes lL Size Annotation Paste Add line No calibration sizes have been entered yet It is possible to enter these sizes manually Taking the MegaBACE ET550 R size standard as an example you would 1 Earlier versions of DAx used a process that recognised the sizing standard s peaks essentially by their positions refer to daxtrace pdf for an explanation of this procedure e Pick ROX as the Calibration trace e Keep the Horizontal axis type as Not DNA BP This means that whenever some data have already been converted to a BP axis the ATC will not be applied All other axis types such as time axes will have ATC applied converting them to a BP axis e Enter the Calibration sizes 60 75 100 525 and 550 As an alternative to manually entering the sizing calibration click on the Import sizes tab Automatic Trace Calibration Trace type amp fragment sizes Import sizes Restrictions Calibration Advanced
17. ll be used to find the standard peaks in a ROX trace This is normally a very straightforward process However should the procedure run into a snag there are some steps that should be followed The first step is to make sure that the calibration trace being looked at does indeed contain all or some expected calibration peaks Here are some reasons why this might not be the case e the most common reason is that too large an interval at the start of the trace is being excluded from peak finding causing one or more initial calibration peaks to be skipped Make sure not to skip too large an interval at the start of the trace when finding peaks e peak find parameters might have too high a detection limit e the trace may actually not contain the calibration peaks or not all of them If 10 calibration peaks are expected but only 8 peaks are present ATC will fail if the require minimum number of calibration points parameter was set higher than 8 If the calibration peaks are present and found correctly but ATC still fails some parameters may have been given values that are too limiting It is important to understand that most ATC parameters are used to speed up the ATC algorithm without being strictly necessary for the algorithm to work Therefore if the ATC fails to find a calibration these parameters can be relaxed advisably in this order e if some early sizes are being missed skip a smaller interval e if you are sure all size
18. nal normalisation Trace analysis parameters for D w_example6 Ladder_E10 fs Parameters Raw or processed data Trace names Trace selection Trace drawing colours Colour separation method Colour separation matrix Sizing calibration derivation Calibration trace selection Signal normalisation Maxed out signal correction Compare analysis settings lt Prev i Finish Cancel Finish All Cancel Al Tabbed Choose if the traces should be normalised 7 Normalise data Config Choose if traces should be converted to base 2 log values Baseline subtraction is implied in this I Convert to 2Log Trace Parameters Load Save The data will not be normalized 18 Extra data and headers Trace analysis parameters for D w_exai Parameters Choose the extra data items that you wish to load from Raw or processed data nee Trace names Trace selection F Voltage 10 V Trace drawing colours Colour separation method T Current m Colour separation matrix Sizing calibration derivation Electrophoretic Power W Calibration trace selection Horizontal axis Temperature C Signal normalisation Extra data and headers Choose if you want the file s headers and structure to be copied to the logfile I Copy file headers to logfile Finish Cancel Finish All Cancel Al Tabbed Trace Parameters Load Save No extra data will be loaded Baselines peaks Trace analysis parameter
19. nas pair size This requires that a calibration is derived from the data or loaded from the file lt Prey Next gt Vv Cancel Finish All Cancel Al Tabbed Trace Parameters Load Save This time the horizontal axis will immediately be converted to a BP axis Baselines peaks Trace analysis parameters for Diw Parameters Choose if you want to construct a baseline for each imported trace this is recommended when loading raw data If no baseline is constructed processed data are considered to have had a baseline subtracted Raw or processed data Trace names Trace selection V Construct baseline Subtract amp remove Trace drawing colours s i Colour neka method You can choose to find peaks in each imported trace Colour separation matrix IV Find peaks Sizing calibration derivation er 5 Calibration trace selection Finding peaks requires constructing 4 baseline or loading processed data Horizontal axis Signal normalisation Choose if you want peaks to be imported from the file This requires loading processed data F Import peaks from file F Unless you choose to recalculate peaks the values for height area and base pair t A 7 i Compare analisis s iireis count will be imported from the file and the data will be fixated lt Prev i i _ Recalculate peaks If a calibration is derived from a trace its imported peaks will be used unchanged Finish Cancel u
20. nless they are recalculated Finish All Cancel Al Tabbed Trace Parameters Load Save For both the ROX and FAM traces a baseline will be constructed and peaks will be found Let s look at the baseline construction and peak find parameters 26 Click on the blue underlined Construct baseline to display the baseline construction parameters dialog m Baseline type Parameters Drifting Auto width 5 00 7 Percent Time s C Points C Derim Fourier Moving Average C Just below trace IV Auto Max Passes 3 Z C Moving Median 1 MV Auto Percentile 75 A IV Auto Median 50 Al Straight under peaks m Trend removal Horizontal doh Remove polynomial trend C Ave Below Ave Use HPLC Gradient Modus Close Help The baseline type has been changed to Moving Median but elsewhere all settings have been left at factory defaults When items are checked to use Auto values DAx analyses the data to find values that fit the data being analysed 27 Click on the blue underlined Find peaks to display the peak find dialog box Indicate if positive and or negative peaks should be found V Positive Peaks l Negative Peaks Indicate the minimum area for valid peaks Minimum area ol WA C Absolute f s Percentage Select all I Deselect Trace files should not contain negative peaks so they are not being found The peak find
21. our separation matrix Raw or processed data FAM HEX TAMRA ROX Trace drawing colours FAM 1 0000 cJ 0 0000 WA 0 0000 WA 0 0000 WA Colour separation method Colour separation mate Hex o o000 24 fr co00 4 o o000 74 aoo00 74 izing Calbration denyation Heron ans TAMRA 0 0000 77 fo oo00 4 1 0000 WA 0 0000 WA ignal normalisation ft Rox 0 0000 Z 0 0000 WA 0 0000 WA 1 0000 Al Maxed out signal correction Load matrix Paste Copy lt Prev IE Invert Transpose Unity diagonal Find matrix Finish Cancel Finish All Cancel Al Tabbed Trace Parameters Load Save Sizing calibration derivation Trace analysis parameters for D w_s le6 Ladder_E10 eee s Parameters Choose if a size calibration should be derived from the data or imported from the file Raw or processed data Trace names Trace selection Trace drawing colours Derive size calibration from a trace rae Seran pek At a minimum using this option causes automatic analysis baseline construction and peak finding unless peaks are imported from the file Do not derive calibration C Use re ported calibration from file For base call files this option requires processed not raw data to be loaded It also requires the file to contain a calibration r Finish Cancel Finish All Cancel Al Tabbed Trace Parameters Load Save In the example we want to derive the sizing calibration from th
22. peaks Maximum allowed discontinuities 10 pJ Low and high allowed direction ratio 10 60 A 1 80 p Start looking for highest size runs the ATC algorithm in reverse which can sometimes be helpful Allow missing points at both start and end allows for low end as well as high end calibration points to be missing Reward higher numbers of calibration points tells the ATC algorithm that searches that turn up more calibration points are to be considered better Reward higher total calibration peak area percentage tells the ATC algorithm that calibration points are likely to have peaks with significant sizes Reward calibration peak areas being similar instructs the ATC algorithm to make use of the fact that calibration peaks probably have similar sizes Maximum allowed discontinuities can be used if the measurement is known to contain a discontinuity in the horizontal axis It is rarely necessary to allow discontinuities The low and high allowed direction ratios determine how forgiving the ATC algorithm will be More forgiving settings further away from 1 0 will result in more inclusive searches which may take longer to run Generally values of 0 7 and 1 5 give excellent results Once all fields have been filled click the Close button to close the ATC dialog You should now use File Save Analysis Procedure to store this setup on disk Chapter 2 2 Troubleshooting ATC In the next chapter Automatic Trace Calibrations wi
23. s TAP dialog is displayed so that the way in which the data are to be loaded can be set up Since we are now analyzing the data some changes will be made Trace selection Parameters Choose the traces that you want to load from the file Raw or processed data Trace name in file MV 9961 data FAM Dyel Sizing calibration derivation 7 9961 data HEX Dye2 Calibration trace selection i Horizontal axis A Dye3 Signal normalisation V 9961 data ROX Dyed Maxed out signal correction Compare analysis settings lt Prev Next gt Cancel Finish All Cancel Al Tabbed Trace Parameters Load Save The example files only contain data in the FAM trace so only FAM and ROX are checked vr 7 It is even possible to check only FAM The ROX trace will temporarily be loaded to set up the calibration and will then be discarded 25 Horizontal axis Parameters Traces can have a horizontal axis that is index based or time frequency based Raw or processed data Trace names Horizontal axis Time s Trace selection Trace drawing colours Colour separation method Data frequency Hz WA sain N Hed am Leave the frequency field blank to use the frequency in the file Calibration trace selection Signal normalisation Extra data and headers Baselines peaks palahi ea al correction Choose if the horizontal axis of the traces should be converted to base Compare analysis setti
24. s a sizing standard peak A similar effect could be achieved by limiting ATC to a number of the largest peaks expressed in the ROX trace If n standard peaks are present a value of 2n generally works well Some measurements will have very tall peaks early in the measurement which may bleed through into the ROX trace When that happens it can be helpful to exclude an initial time range2 2 Under these circumstances it is sometimes also necessary to leave out two or three of the initial sizes in the standard from the list of sizes because those low sizes 60 75 would have been expressed in the initial time range Click on the Calibration tab to indicate the type of calibration that should be set up Trace type amp fragment sizes Import sizes Restrictions Calibration Advanced Select the type of calibration curve that should be derived from the data r Calibration Curve Type Multilinear Cubic Spline Polynomial Degree 2 Al Global Southem Click on the Advanced tab to set up some advanced option avonateTace coon E Trace type amp fragment sizes Import sizes Restrictions Calibration Advanced l Start looking for highest size Allow missing points at both start amp end V Reward higher numbers of calibration points V Reward higher total calibration peak area percentage Reward calibration peak areas being similar F Allow mixture of positive amp negative calibration
25. s are present and early sizes sometimes get called wrong use the advanced option Start looking for highest size e require a smaller peak area e use more peaks e require fewer points to be present 10 Chapter 3 Analysis of the Calibration Ladder Invoke the File Open command Select Files of Type ABI Genescan files or one of the other trace file types SCF files MegaBACE files CEQ files select a file containing the calibration then click the Open button T Open Look in Jo w_example mc e A Name Date modified Ty _ Ladder_E10 fsa 07 02 2013 11 22 ES _ Sample_A04 fsa 07 02 2013 11 22 FS _ Sample_B04 fsa 07 02 2013 11 22 FS _ Sample_C04 fsa 07 02 2013 11 22 FS _ Sample_D04 fsa 07 02 2013 11 22 FS 4 w r File name Ladder E10fsa Files of type ABI Genescan files abi fsa Cancel M Add to win l Sorted Reduce memory use Multiple windows Colour view F Automatic analysis Minimised Stacked Soni The options at the bottom of the File Open dialog are discussed in the next chapter Note that typically each sample file will contain the calibration ladder so any sample file can be selected to set up the calibration Here one file contains only the calibration so this file is used Chapter 3 1 Trace Analysis Parameters TAP dialog When trace files are opened the Trace Analysis Parameters TAP dialog is displayed so that the way in which
26. s for D w_example6 Ladder_E10 fsz Parameters Choose if you want to construct a baseline for each imported trace this is recommended when loading raw data If no baseline is constructed processed data are considered to have had a baseline subtracted Raw or processed data Trace names Trace selection V Construct baseline Subtract amp remove Trace drawing colours Colour separation method You can choose to find peaks in each imported trace Colour separation matrix IV Find peaks Sizing calibration derivation ee Calton trace selection Finding peaks requires constructing a baseline or loading processed data Horizontal axis Signal normalisation Extra data and headers Choose if you want peaks to be imported from the file This requires loading processed data F Import peaks from file Maxed out signal ti A E aE Unless you choose to recalculate peaks the values for height area and base pair count will be imported from the file and the data will be fixated lt Prev i 7 Recalculate peaks If a calibration is derived from a trace its imported peaks will be used unchanged Finish Cancel unless they are recalculated Finish All Cancel Al Tabbed Trace Parameters Load Save After the data are loaded baselines will be constructed and peaks found Click on the blue underlined items to set up baseline construction and peak find parameters 6 In this example only the ROX trace is
27. t was set up in Chapter 2 will be applied Sometimes it is desirable to analyse the calibration trace with different baseline construction and peak find parameters than the other traces To do that check Private baseline settings and Private peak find settings When these options are checked the texts become blue and underlined and can be clicked to set up parameters Private baseline settings Private peak find settings Horizontal axis Parameters Raw or processed data Trace names Trace selection Trace drawing colours Colour separation method Colour separation matrix Sizing calibration derivation Calibration trace selection Signal normalisation Maxed out signal correction Compare analysis settings lt Prev i Finish Cancel Finish All Cancel All Tabbed exam m aaaeei ple6 Ladder_E10 fs Traces can have a horizontal axis that is index based or time frequency based Horizontal axis Time s Data frequency Hz WA Leave the frequency field blank to use the frequency in the file Choose if the horizontal axis of the traces should be converted to base pair size This requires that a calibration is derived from the data or loaded from the file I Convert to BP axis Trace Parameters Load Save The horizontal axis can be a time axis or can be converted to a BP axis When setting up and verifying a calibration leave the axis as a time axis Sig
28. the Config button to set up normalisation Data normalisation parameters 2 x m Horizontal C Move data left right Stretch data Method None m Vertical Stretching Method Equalise named peaks x In trace ROX Target total named peak area fi o000 WA Derive from first data set encountered i Cancel Help In this example the horizontal axis is not affected The vertical axis will be stretched in such a way that the total area of all named peaks in the ROX trace will become 10000 Named peaks are used because the named peaks in the ROX trace will be the calibration standard as called by ATC The other trace FAM will be stretched an equal amount as the ROX data 31 Chapter 4 5 Displaying data as a colour view Use the View Colour view menu option to display the data as a colour gel view E D w_example6 Sample_B04 fsa CE D w_example6 Sample_C04 fsa CE D w_example6 Sample_D04 fsa CE D w_example6 Sample_A04 fsa CE fon Se Ej Sample _B04 ROX B Sample _B04 ROX E Sample _B04 FAM B sampie_B04 Fam il Sampie_co4 Rox B sample_co4 ROx Hl Sample _c04 FAM B se ROX E Ssmple_D04 FAM B Semple_D04 FAM E Sample _A04 ROX igj Sample_A04 FAM B Sampie _aos Fam Data set Y Sample_A04 FAM 4 kss s J s ti 1 30000 Sample_A04 ROX 4 aeo g I Sample_D04 FAM 4 las a i i 20000 Sample_D04 ROX 4
29. ype Chapter 5 1 Assigning trace types DAx attempts to retrieve the trace type of each trace from the measurement file which often but not always contains trace names The trace names are displayed in the TAP dialog cf Chapter 3 1 The standard trace types that DAx recognises are FAM HEX JOE NED ROX TAMRA TET VIC PAT LIZ GRODY and PET Mainly for use with SCF files there are also A C G and T trace types 33 The Config trace names button in the TAP dialog allows additional trace types to be added A dialog box like this one is displayed r Enter trace names m Standard trace names are FAM HEX JOE NED ROX TAMRA TET VIC PAT LIZ GRODY PET 4 C G T these names cannot be modified Name Default colour won moe Ww wee 34 Chapter 6 Conclusion DAx can be used to perform highly complex analyses of trace files with relatively little effort There are a large number of additional features that have not been discussed in this manual Examples are e peak width standardisation If a sample is known to contain mostly peaks that have a width of 1 fragment DAx can be told to preferentially assign peak widths of 1 fragment If a peak is clearly wider its width will not be affected e command line analyses Typically trace analysers will be used to perform dozens to hundreds of analyses per day To make it possible to analyse such large quantities of data DAx can be run from b
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