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TReCCA Analyser User Manual

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1. Select which datasets you want to export as csv and give them filenames Separator Decimal separator Raw data export to file Medium data export to file Average medium data export to file Medium data standard deviation export to file medium sd csv OxoDish data export to file oxodish csv Export Files Status messages Graph output Data output EEE Z U lt BE Import export settings Import export R data Figure 35 Data output tab 5 5 3 Import export settings In order to make it easier to rerun the analysis it is possible to save all the settings chosen in the program Click on the Import export settings button at the bottom right of the screen and the pop up window displayed in Figure should appear The file will be saved in the working directory Type a file name ending with txt for example Settings _Experimentl txt or a path included in the working directory followed by the file name for example ResultsA Settings_Experimentl txt These settings will also be saved automatically when you click the Run analysis button under the name au tosave settings txt The Import export settings button can also be used to load previously saved set tings by clicking the Load button and selecting the settings file The layout of the program will then be changed accordingly Even after importing the saved settings it will be necessary to load the data and the template agai
2. a a 5 2 Data output wu 4 amp Ga eae Oo oe RA E E RSA GT Technical details 6 1 Average and standard deviation 22 2 2222 2 2 nn 6 2 Normalisation 2 2 Co m on 6 3 OxoDish sensor calibrationl 2 08005 2 6 3 1 Step 1 Homogenising the sensor read outs of each plate 6 3 2 Step 2 Setting the average read out to a defined target ee e a E 6 6 Data smoothing 4 2 0 2 0 0 4h aaa wo naar Lae kee ee E ee ee ee ee E O eo peteweeeeaeeereeaaeeeaaaeas ee ee ee ee en SA a Trouble shooting Tel Error messages abbiegen he L2 I COMSOIG oe we oe he eee ee bE RA a ER 5 5 3 Import export settings 2 2 2 2m nennen 5 5 4 Import export R data 2 2 aa Contents TReCCA Analyser user manual 1 Preface We thank you for choosing the TReCCA Analyser for your time resolved data The aim of this manual is to provide you with an overview of the functionalities of the program and a course of action in the case of errors This manual does not claim to be complete and we welcome any ideas for its improvement An article about the TReCCA Analyser has been published in the open access and peer reviewed journal PLOS ONE Lochead et al 10 6 e0131233 2015 Please refer to this publication for a more detailed explanation of the justification of some of the proposed analysis steps and for a quick illustration of the possibilities of the program For mor
3. S l 2 130 microL oxygen concentration for the linear fit of the kla value kla Std Error iuis pus Ranna The fully saturated oxygen concentration is 95 g 0 01691007 235 236 0 0 9924314 3 150 microL 1 l Std Error t value Pr gt t R squared 0 01316906 323 3689 0 0 9962652 2 130 microL 4 170 microL Std Error t value Pr gt t R squared 0 02149062 219 3059 0 0 9925498 4 1170 microl 5 200 microL Please enter the names for the different calibrations one per number in the template 3 150 microL 5 200 microL Std Error tvalue Pr gt t R squared 0 01457342 391 9502 0 0 9941371 The calibration was run successfully Figure 27 Oxygen calibration window Resulting values A Calibration of oxygen accessibility Use the oxygen values and their slope from the Raw data data The hillto be analysed lies approximately in the timeframe from 0 to 3 in the given output unit Use data from 40 to 90 oxygen concentration for the linear fit of the kla value The fully saturated oxygen concentration is 95 E Please enter the names for the different calibrations one per number in the template 1 1100 microl 130 microL 3 150 microL 4 1170 microl 5 200 microL The calibration was run successfully gt q E O Ln L 5 QO aS Se Ee BC LA Ss th uns RAY 40 60 Oxygen level Figure 28 Oxygen calibratio
4. Please check the console to see which settings are affected The settings you are trying to load are from TReCCA version 3 but you are only using TReCCA version 1 You won t be able to use part of the settings you are trying to load or to reproduce the analysis If you want to do so please download a higher version of the program The settings you are trying to load are from TReCCA version 1 but you are using the more powerful TReCCA version 3 The missing settings are replaced by default values The settings you are trying to load are from TReCCA version 1 but you are using TReCCA version 3 Since the settings or the computational design of these two versions differ you might not be able to reproduce certain analyses and you might encounter default settings instead of your personalised ones Error upon trying to import settings Either the file is corrupt or you are using settings from an earlier version of the TReCCA Anal yser Check the R console to see which set tings are affected or go through the different settings manually in the program Error upon importing settings Some set tings available in version 3 of the TReCCA Analyser were not available in the version 1 or 2 Download the most recent version of the TReCCA Analyser for the optimal func tionality or accept the missing settings and check through the settings manually Error upon importing settings Accept the default settings as they are or go through the
5. settings manually to check them all Error upon importing settings The compu tational design has changed from one ver sion to the next and therefore some analy ses might not be available Go through the settings manually to check them all TReCCA Analyser user manual Chapter 7 Trouble shooting Error upon importing an RData file Make The RData file could not be fully loaded Please check the console for errors sure that the file is saved as RData or check the console for further possible errors Table 6 Pop up errors and solutions Settings and R Data import TReCCA Analyser user manual 59 Chapter 7 Trouble shooting Labels and Colours errors Error upon trying to run the analysis A proper Labels amp colours template is not Please enter all labels and colours first wa ne nas Deen rare by loading settings Please go to the Labels amp colours tab and click the load template button to open a file chooser dialogue or create a new template using the autofill buttons Error upon trying to import data The names of the columns are not unique This Wellnumbers in imported data are not unique can happen easily by merging the data man ually Please change the names accordingly or use the automated file merging to prevent this error There are colours in the template which are not recognised by the program Please only use colour names and not numbers check The template could not be loaded
6. John Verzani gWidgetsRGtk2 Toolkit implementation of gWidgets for RGtk2 2012 R package version 0 0 81 Michael Lawrence and Duncan Temple Lang Rgtk2 A graphical user interface toolkit for R Journal of Statistical Software 37 8 1 52 12 2010 TReCCA Analyser user manual 11 Chapter 3 Installation guide amp R File Edit Format Workspace ZOSTO DEE Misc Window Help Package Manager Package Installer 3 Data Manager eoo R Package Installer Packages Repository CRAN binaries Get List V Binary Format Pac kages Q Package Search Installed Version Repository Version 1 2 1 0 3 2 1 0 2 1 5 5 Install Location At System Level in R framework Install Selected At User Level Install Dependencies In Other Location Will Be Asked Upon Installation As defined by libPathsQ Update All Figure 4 Package installation guide on Mac Need GTK 7 Restart R after i Need GTK Restart R after inst ltem Install GTK Install GTK Do not install GTK E ee j Do not install GTK Cancel Figure 5 Automatic GTK download 3 2 Running the TReCCA Analyser As soon as R all the necessary packages and GIK are installed run the program as follows All the files and graphs produced by the TReCCA Analyser will be saved in a defined working directory which is a folder placed in any convenient place of the com puter The main folder of the program has to be inserted into th
7. Mac users the package installation guide can be reached as seen in Figure 4 by clicking on Packages and Data and then Package Installer Pick which packages to download as seen for the package cairoDevice in Figure 4 and click Install selected 2 12 0 51 1 cairoDevice ackage details dre ackage details g Widgets ackage details gWidgetsRGtk2 ackage details RGtk2 Package details GTK combined package cairographics download Table 1 Detailed list of all system and software requirements Michael Lawrence cairoDevice Cairo based cross platform antialiased graphics device driver 2011 R package version 2 19 10 TReCCA Analyser user manual Chapter 3 Installation guide A IR RGui 64 bit Fichier Edition Voir Misc Fen tres Aide Charger le package Choisir le site miroir de CRAN R R Console Choisir les entrep ts ee 5 4 i ei Installer le s packagels Copyright C 2014 T Mettre jour les packages Platform x86 64 w64 Ea E Installer le s package s depuis des fichiers zip R est un logiciel libre livre sans AUCUNE GARANTIE Vous pouvez le redistribuer sous certaines conditions Tapez license ou licence pour plus de d tails R est un projet collaboratif avec de nombreux contributeurs Tapez contributors pour plus d information et citation pour la fa on de le citer dans les publications Tapez demo pour des d monstrations help pour l aide en
8. amp X Data input Labels amp colours Analysis options Graph options Run analysis Help Graph options Please fill in all the settings for the graphs can be refreshed after analysis General Title for all graphs Show title Celline Medium Date Subtitles for specific graphs W Show subtitles Raw data Raw data OxoDish calibration Sensor corrected data Medium normalisation Medium normalised data Average medium normalisation Average medium normalised data Axes X axis label Time day X axis limits min 0 max 10000 enter 10000 for automatic limits Y axis label Oxygen level air saturation Y axis limits min 10000 max 10000 enter 10000 for automatic limits Status messages ES Import export settings Import export R data Graph output Data output Figure 31 Graph options tab above every single graph The subtitles are displayed between the title and the plot on the corresponding graph 5 4 2 Axes Choose the label and limits of each axis To display limits optimised by the program choose 10 000 as minimum and maximum limit These settings will be applied to all the graphs unless mentioned otherwise in the specific Analysis options tab 5 5 Run analysis Once all the settings are set in the Data input Labels amp colours Analysis options and Graph options tabs click on the Run analysis button All the settings imported data and the templa
9. because l purple is not a supported colour Printallvalid there are no white spaces after the colour ones by typing colors in the console f name or spelling mistakes All the valid colours can be found by typing colours in the R console Error upon trying to load a template This means that the first row in the template does The template could not be loaded because not contain the right names It might help to its column titles don t fit the requirements remove all the quotation marks from the csv file Another reason might be the separator chosen for loading the template Table 7 Pop up errors and solutions Labels and Colours Graph output Error upon visualising data after using the Please be aware that the basis for rerun button for the data analysis Data the displayed data set changed sets used to calculate the data shown on this graph have been modified a posteriori Table 8 Pop up errors and solutions Graph output 60 TReCCA Analyser user manual List of Figures E REAR PERSAS AR 9 ee SE ee dad 10 ES EEE BER ae e 11 een 12 5 Automatic GTK download a a a a a a CE En nn 12 6 Folder and file content of the working directory 13 13 aro 13 amam de Ade EA E a e k 14 10 Buttons of the main tabs of the TReCCA Analyser 2 222220 17 11 Data input tab ooo aa a ee 18 12 Required input file format 2 2 a a a 000000008 18 13 Pop up windows after clickin
10. button refreshes the visualisation of the selected calibra tion area chosen that is delimited by four red lines Once everything is set press the Run 34 TReCCA Analyser user manual Chapter 5 Detailed user guide calibration button Two new tabs will appear next to the Data preview Resulting values and Plot result as seen in Figures 27 and 28 respectively In the last tab the diffusion flux d O2 dt is depicted against the oxygen level The line that fits this data has a slope that is equal to kLa The numerical value of the kr a for each condition as well as the standard error and the R squared of the linear fit are presented in the resulting values tab The calibration data can be saved by pressing the Save calibration button This file cannot be opened by traditional spreadsheet applications but can be loaded directly into the TReCCA Analyser as described in part By clicking the Save plot button the displayed graph will be saved in the folder currently being used for saving 5 3 11 ICs determination The TReCCA Analyser can calculate the IC or ECso of a drug automatically and in a time resolved manner thanks to the IC59 tab displayed in Figures 29 and First select the data set that should be used for the ICs calculation as seen in Fig ure Then choose the time points in the input unit between which the Cs 9 should be calculated For the log logistic fit of the data to be possible at every ti
11. calibration Figure 25 Pop up before oxygen calibration First fill in the data that should be used for the oxygen calibration Then decide which part of the curves are linear and should be used for the calibration by filling in the corresponding time frame and Y axis limits of the data Indicate the value of the oxygen in the fully saturated environment The names that appear in this section are determined by the numbers chosen in the Labels amp colours window under the columns Number of the template Each condition that has the same number will be analysed together and plotted under the same chosen name entered in the following boxes Fe Calibration of oxygen accessibility Use the oxygen values and their slope from the Raw data lt data The hillto be analysed lies approximately in the timeframe from 0 to 3 in the given output unit Use data from 40 to 90 oxygen concentration for the linear fit of the kla value The fully saturated oxygen concentration is 95 Please enter the names for the different calibrations one per number in the template 1 100 microL 2 130 microL 3 150 microL 4 170 microL 5 200 microL The calibration was run successfully Data preview Resulting values Plot result 1 5 2 0 2 5 3 0 Annuler Refresh data preview Run calibration Save calibration Save plot Figure 26 Oxygen calibration window Data preview The Refresh data preview
12. converted according to the average of those conditions The conversion will be done per imported plate so for all the data to be converted according to the overall average conditions of all the plates merge the data in one file Select the desired oxygen unit for the output data according to the ambient temper ature and pressure If the assay conditions differ from the given options please choose other for either of the conditions and enter the unit conversion factor manually in the last slot To calculate the unit conversion factor an excel sheet is provided under Tools and utilities on the PreSens homepage 5 3 6 HydroPlate pH conversion This option is only available once PreSens HydroPlate has been selected in the Data input window in the Data layout part It is displayed in Figure Please enter the pH values of the cal bration wells by name and 1 for all the others 100uM4 1 200uM 25uM 50uM uk Media II E f E IS E E f KA E nei e ee NT pH4 pHs pH pH pHs pH Figure 21 Analysis options HydroPlate oxygen conversion TReCCA Analyser user manual 29 Chapter 5 Detailed user guide When using a HydroPlate PreSens Precision Sensing GmbH with a classical spec trophotometer the emission intensity of a luminophore that is quenched differently de pending on the pH is measured in comparison to the emission intensity of a reference luminophore See the HydroPlate instru
13. details An overview of the possible analyses and their relations is displayed in Figure TReCCA Analyser user manual 23 Chapter 5 Detailed user guide 7 Fe TReCCA Analyser for time resolved cell culture assays V4 0 o B x Data input Labels amp colours Analysis options Graph options Run analysis Help Analysis options Please select the calculations you want to run and the corresponding options Analysis selection Basic calculations Also calculate Additional functions Print timepoints Raw data calculate average data smoothing slope S z Print dataset dimensions Sensor correction calculate average consumed oxygen IC50 Calibrate oxygen consumption Medium normalisation calculate average Basic data formatting Average Medium normalisation Sensor correction Please select according to which attribute the average should be calculated O name number colour calculate standard deviation Status messages Graph output Data output Import export settings Import export R data Figure 15 Analysis options tab with the basic data formatting option Each analysis tab has a Rerun xxx only button which starts the analysis of only the actual analysis in order to save time and prevent having to run the whole analyses repeatedly By pressing a Rerun xxx only button a new window will appear asking if the anal
14. independent read outs using an empty well sensor read out from the beginning of the measurement as reference also usually 24 reference read outs The calibration curve for the conversion of the phase to the oxygen level is a com plex equation which is the property of PreSens Precision Sensing GmbH which can be modelled by the exponential Equation 6 4 quite accurately where P is the Plateau of the model and S the Span The values of P and S vary from lot to lot as presented in Table 2 see part OxoDish sensor calibration r P S e 6 4 In order to make the calibration curve linear and thereby allow a correction by mul tiplication and division all the data is first converted to logarithmic values as described in Equation u 6 5 p In E 6 5 Then instead of using a discrete point as empty well reference value all the values for each sensor included in a certain time frame are averaged and taken into account This time frame is chosen by the user and ranges from the time points t to tn where t is the last time point at the end of the sensor calibration and n the number of points averaged for calibration The mean read out over the time frame of each sensor s is then described by Equation n 1 1 Ms a H ZL 4 8 6 6 1 0 The mean read out value for the total plate m is determined by averaging the average read outs m for each sensor as described in Equation where w is the number o
15. number of smoothed points on either side of it Select the number of points to be TReCCA Analyser user manual 31 Chapter 5 Detailed user guide used for linear fitting which will determine how precisely the slope should be determined for each data point It is important to note that the total number of time points in each data set will be reduced after slope calculation if n is the number of points on either side then n time points will be missing from the beginning and the end of the data set Finally select the data set s for which the slope should be calculated by the TReCCA Analyser by ticking the corresponding boxes For more details about the formulas used for the slope calculations please refer to part Numerical slope 5 3 9 Oxygen consumption The TReCCA Analyser will convert measured oxygen values to actual oxygen consump tion values for all experimental set ups that fit the model used The diffusion of oxygen into the liquid should be approximated linear with the sensor being at the bottom of the well and the lateral diffusion of oxygen being negligible The calculations implemented here are based on previous publications where the assumptions of the model are described in more precisely ITISa For more details about the formulas used for the oxygen consump y 8 tion please refer to part Oxygen consumption The fully saturated oxygen concentration ts 100 use sensor correction target value use medium nor
16. occurring sample of a specific group will be used to represent the average condition For example if Well 1 has the name Medium and the colour red and Well 2 has the name Medium and the colour blue then when representing the Medium average the line will be red The Colour must be a character string referring to one of the 657 R colours A list of the available colours can be found online by searching R colours or by typing colours in the R console to get the available list The Line type can be solid dashed dotted dotdash longdash twodash or blank not be visible 5 2 2 Filling the template automatically When creating a new template using the auto fill options will make sure the template has the right format The Autofill labels button will detect all the column names in the input data and place them in the Well column This requires the data to be loaded already Make sure that all the column names are different inside one imported file When importing data from two separate files all the Well names of the second file will be mod ified to display 1 at their end In this way it is possible to import files with identical column names without having to change these names manually In the case of importing three files which all have Al as first column name these will appear as Al Al l and Al 2 in the Well column of the template The Autofill labels button will automatically fill the Colour co
17. of the saturated liquid phase and kra is the oxygen mass transfer coefficient The change in oxygen over time is equal to the diffusion of oxygen into the well minus the amount of oxygen consumed by the cells yeast bacteria or chemical reaction d Os dt kra 05 02 OUR 6 17 To perform the oxygen consumption calibration it is necessary to work under condi tions where the OUR 0 which means by working with wells without any cells yeast bacteria enzymes etc Equation can then be simplified to Equation 6 18 where kpa 05 is a constant d O2 dt kpa 05 ER kra O3 6 18 The oxygen concentration Os is set to O by using either nitrogen or chemicals that react with oxygen as described in part 5 3 10 Oxygen consumption calibration and then the diffusion of oxygen back into the solutions is measured over time until it reaches the level of the ambient saturation The rate of oxygen change over time d Oa dt can be calculated using the slope function of the TReCCA Analyser Plotting this slope against the actual oxygen value 05 and fitting a linear model then yields kra and the corresponding residual e for the fit Using the plots that are displayed in the oxygen consumption calibration interface Figure 26 TReCCA Analyser user manual 49 Chapter 6 Technical details and 28 it is possible to determine if the model for the oxygen balance in the experimental set up is valid and if so to set
18. previous publications Eyer et al 1995 Hermann et al 2003 John et al 2003 and can only be used if the experimental conditions fit the prerequisites of the model described in these publications For example in our experimental set ups using 96 well plates this is the case if the solutions are mixed 48 TReCCA Analyser user manual Chapter 6 Technical details over time but not the case if they are not shaken The first step in knowing if the model applies or not is to see whether for at least certain oxygen saturation values the oxygen saturation value is proportional to the rate of change of the oxygen saturation value when the oxygen consumption rate is 0 This is described in more detail in parts 5 3 10 gen consumption calibration and 6 9 Oxygen consumption calibration When the model applies the oxygen uptake rate OUR is then described by the Equation where O gt is the measured oxygen concentration 05 the concentration of the saturated liquid phase and kya is the oxygen mass transfer coefficient For the OUR to be correct it is essential that the kra and the d O2 dt have the same time unit d O gt dt OUR kra 0 3 02 6 16 6 9 Oxygen consumption calibration The calculations implemented in the TReCCA Analyser are based on the assumption that the general formula of the oxygen balance is as described in Equation 6 17 where O2 is the measured oxygen concentration 05 the concentration
19. separator Sensor Make sure the exact time point for the start of the calculation interval given in the input unit is included in the data file Enter a number with as decimal separator Enter a number with as decimal separator Enter a number with as decimal separator Enter a whole number superior or equal 1 Enter a number with as decimal separator 1 for no conversion OxoPlate conversion Data Enter a whole odd number superior or equal to 1 Sensor Data input Enter a number with as decimal separator for the unit conversion of the luminescence data to the target unit Enter a number of time points for sensor correction superior to 1 the last time point is included and one point is insufficient for the correction If you do not have a header in your file s select No so the number will be ignored Otherwise enter a positive whole number 59 Please enter a positive number for last cali bration value Please enter a positive number for number of plates Please enter a posi tive number for skip ping points in Cs9 cal culation Please enter numbers for X axis limits Please enter numbers for Y axis limits Please enter numerical values gt 0 or 1 only for the ICs doses Please enter numerical values gt 0 or 1 only for the pH values Please enter positive numbers for wells Please enter positive num
20. the data in one file 5 3 7 Data smoothing The TReCCA Analyser can be used to smoothen the data which is especially necessary to reduce the data noise before calculating the slope of the data see part slope For smoothing each data point will be replaced by the average of the actual data point and of its surrounding neighbourhood The Data smoothing tab can be seen in Figure First choose the number of points to be selected in each neighbourhood This should always be an odd number so as to include the actual data point and the same number of time points on either side of the actual data point It is important to note that the total number of time points in each data set will be reduced after smoothing if n is the neighbourhood number then n 1 2 time points will be missing from the beginning and the end of the data set Also the bigger the neighbourhood the smoother and less 30 TReCCA Analyser user manual Chapter 5 Detailed user guide Smooth the selected datasets by averaging a neighbourhood of 3 points Needs to be an odd number Calculate smoothing for raw data averaged raw data Y converted data average converted data Figure 22 Analysis options Data smoothing precise the data will get Select the data set s that should be smoothed by the TReCCA Analyser by ticking the corresponding boxes For more details about the formulas used for the smoothing calculations please refer to par
21. the range for the linear fit in order to exclude the initial or final phases of the measurement 6 10 ICs determination For the ICs9 calculation the TReCCA Analyser uses concentrations assigned to the labels of the data by the user It is also possible to select how many time points to skip in order to reduce the run time of the calculation From this a data set is created where the concentrations replace the well names fewer time points are included and those wells which do not belong to the IC calculation are excluded The ICs is calculated for each line of the data set which represents one time point The possible models used for determining the IC are included in the drc package and are the two three four and five parameter log logistic models as depicted in Equations 6 19 6 20 6 21 respectively The error is reduced by a non linear least squares method 1 1 Tr exp Blog Toe 6 d 1 exp b log x log e en d c ee 1 exp b log x log e en fa ee 6 22 O DD E 1 exp b log x log e The curve of the Cs 9 fit for each time point as well as a curve of the IC value over time are shown 50 TReCCA Analyser user manual 7 Trouble shooting This section is designed to help find a fast solution to any error message or problem that may be encountered while using the TReCCA Analyser 7 1 Error messages Error messages regarding the settings are show
22. the right separators and file path s The file should contain the time points in the first column and the rest of the data in the following columns each headed by a unique column name 4 Click the Import Files button and solve status messages if necessary bottom middle of the screen 2 Click Labels amp colours 1 Load a previous template with Load template or follow the next points 2 Auto fill the template by clicking Autofill labels Autofill numbers All black and All solid 3 Export the template by clicking on Save template and edit it with any spreadsheet application without changing the first row 4 Import the modified template with Load template 3 Click Analysis options 1 Select the analysis you want to run on your data using the tick boxes 2 Fill out the settings for each chosen analysis bottom half of the screen 15 Chapter 4 Quick guide 4 Click Graph options 1 2 Choose all the titles for the graphs Enter the axis labels and limits All the graph options can be changed after the analysis is run once the graphs are made 5 Click Run analysis 1 2 Chose the name of the results folder in which the results will be saved Wait for the analysis to be run The analysis time should be under 15 minutes 6 Customise the graphs 1 2 On the right you can switch through the different graphs If you wish to visually exclude some lines click on their tick b
23. time f x 6 12 lian d nau ye lne 1 dp pHo 6 13 L Past 6 6 Data smoothing The data smoothing uses the built in mean function but in this case an average of several points over time is calculated in order to reduce fluctuations according to the formula given in Equation where n is the number of time points to average Every time point is thereby replaced by the average of the time point and n 1 2 neighbours on either side This causes the loss of n 1 2 data points in the beginning and the end of the measurement as these points do not have the necessary neighbours 6 7 Numerical slope For the numerical slope calculation it is usually necessary to smoothen the data first to get rid of the noise The TReCCA Analyser always uses a smoothed data set for the calculation but the number of points used or the smoothing can be set to 1 in which case no smoothing will actually occur For the slope calculation a certain number of points n on either side of the currently processed time point x are used for a linear model fit as described by Equation 6 15 The linear model fit is a built in function of R and yields the slope m and the corresponding residual e which is displayed as the standard deviation on the graphs Nens u m izn ee 6 15 6 8 Oxygen consumption As already described in the part Oxygen consumption the calculations imple mented in this part of the TReCCA Analyser are based on
24. 78 94 79 22 79 08 81 18 80 2 81 33 124 88 02 89 01 88 89 90 77 88 74 89 76 184 87 88 90 19 90 05 90 77 89 18 90 63 245 88 02 90 63 89 32 90 77 88 46 90 77 306 84 13 90 63 89 03 90 05 88 6 90 34 367 88 02 89 61 88 6 90 34 89 03 91 36 428 88 31 90 63 89 47 88 89 87 88 90 05 Figure 12 Required input file format 5 1 2 Data layout SJUSTB INSEIATA The first parameter defining the data layout is the number of plates which also regulates the number of input lines in the File import to the right of the screen The maximum number of plates which can be analysed at once is 10 18 If there are several plates to be analysed it is possible to either have all the plates TReCCA Analyser user manual Chapter 5 Detailed user guide in one single document with one time column for all or have the plates in multiple doc uments with individual time columns In this last case it is important that each file contains exactly the same time points Select multiple documents and this option will change the number of paths that can be given in the File import Since non numerical values in the files such as error NA or NAN will disturb the analysis they all have to be removed This can either be done manually in a spread sheet application or the program can replace all the non numerical values automatically with O For this select no for the previous accomplishment of non numerical data cleaning and when importing the
25. Analyser user manual List of Tables 1 Detailed list of all system and software requirements 10 2 Span and Plateau values for different SDR lots 2 222222 28 3 Error messages shown in the status messages bar in alphabetical order 55 4 Pop up errors and solutions General errors 048 56 5 Pop up errors and solutions Data input 2 2 a a a a nen 58 6 Pop up errors and solutions Settings and R Data import 59 T Pop up errors and solutions Labels and Coloursl 2 60 8 Pop up errors and solutions Graph output 60 63
26. CA Analyser will take the value of the time point given and for example the 9 time points before this one to calculate the basis for moving the whole dataset to a target starting value which must also be given Span and Plateau are two values which determine the exact form of the calibration curve for each OxoDish lot Some Span and Plateau values are presented in Table 2 As a rough estimation it is also possible to work with the default settings 5 3 5 OxoPlate oxygen conversion This option is only available once PreSens OxoPlate has been selected in the Data input window in the Data layout part It is displayed in Figure TReCCA Analyser user manual 27 Chapter 5 Detailed user guide OxoDish lot number OD 1437 01 2168 201 9 OD 1407 01 OD 1319 01 OD 1308 01 OD 1228 01 Table 2 Span and Plateau values for different SDR lots What is the name of the wells containing 0 oxygen Cal What is the name of the wells containing 100 oxygen Call 00 Please choose the target unit pressure and temperature or choose other for any of them and enter the conversion factor Target unit 6 oxygen saturation Pressure 1013 hPa Temperature 20 C 0 209 Figure 20 Analysis options OxoPlate oxygen conversion When using an OxoPlate PreSens Precision Sensing GmbH with a classical spec trophotometer the emission intensity of a luminophore that is quenched by oxygen is measured in comparison t
27. Desktop TRelCA Analyser gt getwd 1 C Users Julia Desktop TReCCA Analyser gt source Program mainApplication R Figure 7 Commands to set the working directory and launch the TReCCA Analyser To make the launching of the TReCCA Analyser easier and faster it is possible to save the two commands needed to change the directory and run the program to a text file and then copy paste them to the console when it is started as shown in Figure setwd C Users Julia Desktop TReccA Analyser source Program mainapplication R Figure 8 Text file for launching the TReCCA Analyser from the R console After starting the program a GTK application should start on your computer and by clicking on the GTK icon on your tool bar the Welcome screen of the program should appear as shown in Figure 9 indicating where the current working directory is If the directory is not changed beforehand the program will not be loaded Always restart the program if the working directory is changed If the TReCCA Analyser is not launched check that your pathways do not contain TReCCA Analyser user manual 13 Chapter 3 Installation guide any special characters for example ti Japanese Arabic characters fe TReCCA Analyser for time resolved cell culture assays V4 0 Data input Labels amp colours Analysis options Graph options Welcome E start modify a project Welcome to the TReCCA Analyser the analysis program f
28. User manual TReCCA Analyser Version 4 0 Time Resolved Cell Culture Assay Analyser Julia Lochead Julia Schessner T Universit t Heidelberg Institut f r Pharmazie und molekulare Biotechnologie INF364 69120 Heidelberg Germany 2 Hochschule Mannheim Institut f r analytische Chemie Paul Wittsack Stra e 10 68163 Mannheim Germany June 30 2015 Contents 2 Intended use 3 Installation guide 3 1 Downloading R and the complementary packages 2 2 222 200 3 2 Running the TReCCA Analyser 0 20 0000 ea 4 Quick guide 5 Detailed user guide Ook Daa Ples eee co pesa ee ME oh Eds E 5 1 1 Required format a oe ER a ee Dulac dl Ol s sza eb ee a oh PIS ARRASAR a eh eS 5 1 3 Cutting borders 22 2 Co oo oo ee a 9 1 4 Fileimport 22 2 oo Kon 5 2 Labels amp colours 2 2 oo oo nn 5 2 1 Template layo tl x amp zen a5 an u as E va 9 2 2 Filling the template automatically 2 22 2222 2222 5 2 3 Saving and loading 2 2 2 Cu na rn 5 2 4 Excluding data from the analysis 22 22 2 Em m nn nee 5 3 Analysis options Bae cee toe Bee eee eB Re 5 3 1 Basic data formatting 22 Co oo one en C er ER eee ee ee re yee sa ao gue eusseeuesseta en a a a 6 T 5 4 Graph options lt 4 is sis Sd RA 5 4 1 General serpe ee 5 4 2 Axes aoaaa aa a ee do R nanalysisjs s 4 as Gea me whee ewe REE REAR SES 9 5 1 Graph output
29. and 10 included Please enter a number Make sure the exact time point for the end of the ICso determi for end of Cs 9 calcu calculation interval given in the input unit is in 7 lation cluded in the data file Please enter a number oo Enter a target value for normalisation without any for normalisation tar Normalisation l l measurement unit and as a decimal separator get Please enter a number l l Sensor Enter a number with as decimal separator for Plateau 92 TReCCA Analyser user manual Chapter 7 Trouble shooting Please enter a number for sensor calibration target Please enter a number for Span Please enter a number for start of ICs calcu lation Please enter a number for the error of the kya value Please enter a number for the K a value Please enter a number for the saturated oxy gen concentration Please enter a num ber for the slope fit ting points Please enter a num ber for the unit con version factor used for the oxygen consump tion Please enter a number for the unit conversion factor Please enter an odd number for the curve smoothing Please enter a number gt 1 for sensor calibra tion range Please enter a positive number for Header length TReCCA Analyser user manual Sensor Enter a number with as decimal separator Enter a number with as decimal
30. bers or U for spare columns Please enter positive numbers or O for spare rows Please enter proper for all the data sets to be ex filenames ported Please only enter numerical values the parameters for formatting 54 Sensor Data input lC 0 determi HydroPlate Data input Data input Data input Data output formatting Chapter 7 Trouble shooting The time point specified for the last calibration point must be a positive number found in the time column of the data set in the input unit The number of plates has to be a whole number between 1 and 10 included Enter a whole positive number for the frequency of IC o calculation For all time points to be taken into account choose 1 Enter any number with as decimal separator and 10 000 if for automatic axis limits Enter any number with as decimal separator and 10 000 if for automatic axis limits Enter the concentrations as positive numbers with as a decimal separator or 1 to exclude a condi tion from the IC gt n calculation Enter the pH values as positive numbers with as a decimal separator or 1 to exclude a condition from the pH conversion There has to be a positive whole number in every field next to the rows for each imported plate For several plates just one number in the first field is not enough Enter a positive whole number for the data for matting or U to not delete any columns E
31. ce again not forgetting to take the empty rows into account 5 1 4 File import First fill in the cell separator and decimal separator The cells have to be separated by some character string that is not a white space and the decimal separator for numbers can be any single character To choose the files either browse the system by clicking on the button next to the input line or type the path to a file lying within the current working directory Fill in the number of wells per plate which will determine how many columns are expected from the imported data The number of wells per plate is important in order to perform the sensor correction or to normalise the plates independently to one condition present in each plate To normalise several plates to a condition only measured in one of the plates copy paste them together into one big file and enter the sum of the wells as the well number to be analysed 20 TReCCA Analyser user manual Chapter 5 Detailed user guide 5 2 Labels amp colours Click on the Data input button in the upper menu bar to see the tab displayed in Fig ure In this tab the labels and colours for each column of data or well of the plate are determined and defined into a template This information will be used during the data analysis to determine which conditions should be averaged which ones should be used for normalisation which ones for lC 0 determination or in the case of the OxoDish for s
32. ction manual on the PreSens homepage for more precise information The ratio of the indicator luminophore over the reference luminophore then has to be converted by the user to actual pH values using six calibration solutions at pH values between 4 0 and 9 0 The calibration can be done once for each lot of Hy droPlates or once for each plate and during the whole course of the experiment The time resolved calibration of each plate separately leads to more precise and less noisy results Whatever the calibration method the TReCCA Analyser will convert the relative emission intensity data automatically and if available in a time resolved manner accord ing to the formulas described in the HydroPlate user manual For more calculation details please refer to part HydroPlate pH conversion For each name of the template that appears in the HydroPlate conversion tab enter the corresponding pH value and 1 for the wells that are irrelevant to the HydroPlate pH conversion as seen in Figure If you pre calibrated the lot of plates add columns to your data where each column contains a pH relative intensity average repeated for each time point of the data If there are several columns with the calibration solutions the data will be converted according the average of those conditions Tho conversion will be done per imported plate so for all the data to be converted according to the overall average conditions of all the plates merge
33. data the first pop up window in Figure 13 will appear It might be the case that certain character strings force the program to replace whole columns with 0 When the data import is finished there will be a message indicating whether there were no replacements discrete replacements or column replacements and how many as seen in the rest of Figure If there are whole columns replaced the first five rows of the data will be printed into the R console so that it is easy to identify the columns containing character strings Allnonnumerical data will be replaced with 07 There were no single values replaced L DS 1 whole column s had to be replaced If you want them included clean nonumerical data manually The first five rows are printed to the R Console 1 single value s had to be replaced Figure 13 Pop up windows after clicking Import Files Finally select which kind of measurement has been done in order to have access to the specific analysis options further on in the program The default option is other select one of the other options in the case of a PreSens OxoDish PreSens OxoPlate or PreSens HydroPlate measurement 5 1 3 Cutting borders Since most of the documents written by the measurement software will not only contain the actual data but also additional information date of the measurement wavelength chosen identification number it is possible to cut off the extra fi
34. e specific information on the actual code of the program or for its further implementation please refer to the code of the TReCCA Analyser which is freely accessible on the website of our research institute in Heidelberg http www uni heidelberg de fakultaeten biowissenschaften ipmb biologie woelfl Research html If you wish to discover the use of the program through a practical example you may also refer to our tutorials also available on our website They will guide you through different analysing exercises that will exemplify the use of the program We wish you an enjoyable reading 2 Intended use The TReCCA Analyser is conceived to facilitate speed up and intensify the analysis and representation of your time resolved data more specifically in the case of cell cul ture assays Without having to type any formula it will perform at wish the following calculations Control condition normalisation Technical replicate averaging and standard deviation calculation Smoothing and slope calculation of the data in order to obtain the rate of change IC EC determination of a substance in a time resolved fashion In the particular case of an oxygen measurement over time where a model of linear diffusion of oxygen into cell culture well plates applies this program can convert the oxygen values measured to the actual oxygen consumption of the cell culture In the even more particular case of using the co
35. ensor correction The information from the template is also used for plotting as the colour and line type of each well are determined and to set the legend of the graphs The template can be modified directly in the interface of the TReCCA Analyser but for making major changes in a template it is probably faster to export the template to a spreadsheet application and modify it there r A TReCCA Analyser for time resolved cell culture assays V4 0 gt x Data input Labels amp colours Analysis options Graph options Run analysis Help Labels amp Colours Please fill in the template for each well This will determine the graph design If you want a well to be excluded completely name it Exclude It will also be removed from the raw data set Well Name Number Colour Linetype Ai Medium 1 purple solid Bi Medium 2 purple solid C1 Medium 3 purple solid L Di Medium 4 purple solid A2 Plate 5 purple solid B2 Plate 6 purple solid C2 Plate 7 purple solid D2 Plate 8 purple solid A3 Exclude 9 purple solid B3 Plate 10 purple solid c3 Plate 11 purple solid D3 Plate 12 purple solid E Autofill labels Autofill numbers Allblack All solid Save or load labels The required format of the template is a txt or csv file with a title for every column but the first Save template Load template Separator Status messages Graph output Data output EEE Imp
36. es the point size of all the text on the graphs title subtitle label descriptions legend ranging from 5 0 to 25 0 Legend columns Number of columns used to display the legend if appropriate ranging from 1 to 10 Legend position Determines the presence of a legend or not and its position The possible options are no legend below the plot area to the right of the plot area top left top middle top right middle right bottom right bottom middle bottom left middle left Line width Sets the width of the lines on the graph grid lines and frame included ranging from 0 5 to 4 0 Grid colour intensity Picks the intensity of the colour of the grid in percentage so ranging from 0 no grid to 100 dark grey grid Error colour intensity For the graphs where the standard deviation is calculated average graphs slope calculation and time resolved IC changes the intensity of the error display in percentage so ranging from 0 no error to 100 black error White space Varies the amount of white space around the graph and how compact the representation is ranging from 0 00 to 1 00 This option is present so that the exported graphs can be used directly for power point presentations as well as for graphs that are part of a figure in a publication Line selection The line selection determines which lines are displayed on the graph When selecting the lines to be displayed also for the select all none bu
37. et used for the reformatting is selected Any data set that is calculated on the basis of a formatted data set will also be formatted in the same manner see Figure 16 for a representation of the dependencies between analyses For example shifting the time scale of the raw data will imply that the time scale of the normalised or averaged raw data will also be shifted To remove the data before or after a certain time point fill in the corresponding boxes The time has to be given in the output unit chosen in the first part of the Basic data formatting tab If the times are only available in the input unit use the Print time points button at the top right of the program window This way all the time points of the data set will be printed in the R console in two columns with the input and output unit automatically It is important to note at this point that the data will simply be removed before or after the time points but the actual value of the time points will stay the same To actually transform the time scale fill in the next lines once again in output units The given m value will be multiplied to all the points of the time scale for example to have the time scale in years and with day as output unit choose m 1 365 2425 and the given b value will be added to all the points of the time scale The latter is useful if deleting the first part of the data for example In contrary to the shifting possibility of the second
38. f wells per plate L O Ma E m 6 7 The corrected values are then calculated according to Equation whereby the data which is still in a logarithmic scale is then also reconverted back to the actual data values through the exponential function PHS em tu 6 8 46 TReCCA Analyser user manual Chapter 6 Technical details 6 3 2 Step 2 Setting the average read out to a defined target In a second step the corrected values x are linearly normalised to a target value T chosen by the user according to the experimental conditions The read outs of the correction time frame for each sensor Mes are averaged according to the Equation n 1 1 Mes S a 2 Ut pis 6 9 The resulting corrected oxygen values at the time point t and for the sensor s are then Y s Equation 6 10 Dis T Yt s 0 6 10 ies 6 4 OxoPlate oxygen conversion Prior to the data processing by the TReCCA Analyser the user has to divide the intensity measurement of the indicator luminophore by that of the reference measurement for each time point in a classical spreadsheet application The resulting data called IR can then be loaded to the TReCCA Analyser to be converted to oxygen values The OxoPlate oxygen conversion is performed per plate using the two calibration conditions that expose the sensors to 0 and 100 oxygen in percentage of air saturation The TReCCA Analyser first calculates the average of all 0 and 100 oxygen wells for eac
39. g Import Files 2 2 2 2 22222 19 14 Interface of the Labels amp colours tab sample template loaded 21 15 Analysis options tab with the basic data formatting option 24 16 Analyses available in the TReCCA Analyser and their relationship 25 17 Analysis options Average tab ooa aa a a nn 26 18 Analysis options Normalisation tabi o 22 a a nn a 2004 26 19 Analysis options OxoDish sensor calibration tab 27 20 Analysis options OxoPlate oxygen conversion 28 21 Analysis options HydroPlate oxygen conversion 29 22 Analysis options Data smoothing 2 2 CE Em nn nn 31 23 Analysis options Numerical slope 0 2 0048 31 24 Analysis options Oxygen consumption 2 222222 2 m 2 nn 32 25 Pop up before oxygen calibration CK Em nn 34 26 Oxygen calibration window Data preview 22 2 2 2 2 2 nme 34 C 36 28 Oxygen calibration window Plot result 36 29 Analysis options First part of the ICsg tab 2 22 2 2 nn nn 37 30 Analysis options Second part of the ICs tab 2 a 37 31 Graph options tab oaoa aa 38 62 List of Figures 32 Pop up window to run the analysis 2 2 CL EL Een 39 33 Window seen while the analysis is running 40 34 Graph outputtab cccccll ee 40 do Padon PU Tab u van a 0 Aara rA aN 43 36 Pop up for saving loading settings ooa a a a 44 TReCCA
40. ge installation guide included in the standard R program IR Core Team R A Language and Environment for Statistical Computing R Foundation for Statistical Computing Vienna Austria 2013 Chapter 3 Installation guide amp R File Edit Format Workspace Packages amp Data Misc Window Help 0009 R Console A RMS EQI INE E a ae R version 3 1 0 2014 04 10 Spring Dance Copyright C 2014 The R Foundation for Statistical Computing Platform x86 64 apple darwinl0 8 0 64 bit R is free software and comes with ABSOLUTELY NO WARRANTY You are welcome to redistribute it under certain conditions Type license or licence for distribution details Natural language support but running in an English locale R is a collaborative project with many contributors Type contributors for more information and ceitation on how to cite R or R packages in publications Type demo for some demos help for on line help or help start for an HTML browser interface to help Type qQ to quit R R app GUI 1 63 6734 x86_64 apple darwin19 8 0 gt Figure 2 R Console just after launch on Mac For Windows users the package installation guide can be reached as seen in Fig ure BA by going to Packages and then Install package s You will be asked to choose a CRAN mirror for download Figure 3B and then you can pick which packages to download as seen for the package cairoDevice Plin Figure 3k For
41. ghtly over time slight fluctuations of oxygen in the incubator drift of the sensors normalisation can reduce the fluctuations This analysis step can also be used to normalise all the conditions of an experiment to a non treated control The Normalisation tab is displayed in Figure Please enter the name of the wells only containing medium Medium What is the target value for the medium normalisation after optional rescaling of the data 90 Figure 18 Analysis options Normalisation tab Enter the exact name with capital letters or not assigned to the normalisation con dition in the Labels amp colours template and give the target value for the normalisation this value could be 100 to get percentages in the case of viability studies and a non treated control normalisation The normalisation is performed once per plate which means that each condition in a plate is normalised to the normalisation condition present in the same plate To normalise to the overall average normalisation condition merge the 26 TReCCA Analyser user manual Chapter 5 Detailed user guide data first in a spreadsheet application and import them as one file 5 3 4 OxoDish sensor calibration This option is only available once PreSens OxoDish has been selected in the Data input window in the Data layout part It is displayed in Figure Please adjust the time frame for the sensor correction Use 10 time points where the last included one
42. h time point again using Equation 6 1 thereby determining the values over time of k0 or k100 respectively Every data point is then converted according to the Equation 6 11 where F is the unit conversion factor and x the value Ip over time KO ye F 100 E 6 11 k100 6 5 HydroPlate pH conversion Just as in the case of the OxoPlate prior to the data processing by the TReCCA Analyser the user has to divide the intensity measurement of the indicator luminophore by that of the reference measurement for each time point in a classical spreadsheet application The resulting data called Ir can then be loaded to the TReCCA Analyser to be converted to pH values The HydroPlate pH conversion is performed per plate using 6 calibration conditions that expose the sensors to different pH conditions covering the range of pH 4 0 to pH 9 0 The TReCCA Analyser first calculates the average of the calibration pH wells for each time point again using Equation 6 1 These values are fitted using a four parameter TReCCA Analyser user manual 47 Chapter 6 Technical details logistic function curve over time the function L4 of the drc package as described in Equation 6 12 d c CH m u 1 exp b log x log e From this fit the calibration constants Imin d Imar c dpH 1 b pHo log e are determined Every data point is then converted according to the Equation 6 13 where t is the value Ip over
43. he actual code of the program is freely available 6 1 Average and standard deviation The average and standard deviation are calculated according to standard statistical formu las using the predefined functions of R The function for the average is mean numerical vector see Equation 6 1 where n is the number of averaged points and x the ini tial value of the replicate 2 The function for the standard deviation is sd numerical vector and calculates the sample standard variation see Equation 6 2 where is the average for each time point 6 1 6 2 6 2 Normalisation The normalisation first calculates the average m of all normalisation wells on one plate at each time point see Equation 6 1 Every data point from this plate is then normalised according to Equation 6 3 where M is the target value for the normalisation wells x the initial value for each time point and y the value after normalisation for each time point x M TTL Yi 6 3 6 3 OxoDish sensor calibration The OxoDish sensor calibration can be divided into two distinct steps First all the sen sor read outs inside one plate are brought to a common value and in a second step the sensor read outs are homogenised from plate to plate 45 Chapter 6 Technical details 6 3 1 Step 1 Homogenising the sensor read outs of each plate The first part of the sensor correction corrects each sensor read out from an OxoDish so usually 24
44. ircles Exporting graphs To export graphs click on the Export displayed graph button at the bottom right of the plot area and give the file name and size of the desired output size in inches The choice of the size of the file will not influence the size of the text on the graph so make sure that the text on the graphs still fits properly after export To change the output file type change the ending to either pdf Portable Document Format ps PostScript format svg Scalable Vector Graphics or png Portable Network Graphics The files will automatically be saved to the folder specified when running the analysis included in the working directory 5 5 2 Data output The Data output tab on the bottom of the screen contains one line for every data set that was created during the analysis as seen in Figure First fill in the appropriate column separator and decimal separator Then select which data sets to export by click ing the corresponding tick boxes and give them file names ending with csv Click the Export Files button at the bottom right of the screen and the files will all be written into the specified result folder A pop up window will confirm the success of the export 42 TReCCA Analyser user manual Chapter 5 Detailed user guide f TReCCA Analyser for time resolved cell culture assays V4 0 x Data input manan Analysis options Graph options analysis Help Data output
45. is att lt 8169 inthe given input unit What ts the target value for the sensor correction in air saturation 90 Span 2406 Plateau 2725 2 Figure 19 Analysis options OxoDish sensor calibration tab When measuring each sensor of an empty 24 well OxoDish PreSens Precision Sens ing GmbH more information is available in the SensorDishes amp SensorVials instruction manual a read out difference of between 2 and 8 can be noticed between each sensor although when being empty the value should be exactly the same This slight difference can be reduced thanks to the TReCCA Analyser To do so first measure the empty sensor plate under the experimental conditions temperature humidity on the SDR Reader that will be used later for the experiment until the readout is stable for at least 5 time points This will give information about the average offset value for each well which can then be set to a theoretical target value for example 100 of air saturation if working in the lab or 95 of air saturation if working in an incubator with 5 CO After the sensor calibration all the wells will start at a much more similar value For more calculation details please refer to part OxoDish sensor calibration First fill in how many time points should be used for the sensor correction once the read out is stable 10 points is a good number and enter the time point of the last of these time points in the input unit The TReC
46. is working directory and it should also contain the SampleProject folder as well as the default settings txt and the qualitycontrol txt files as shown in Figure 6 In the R console the path to the actual working directory is given by the command getwd To set the working directory type setwd path and define the path leading to the working directory as exemplified in Figure The working directory folder does 2 TReCCA Analyser user manual Chapter 3 Installation guide di Program SampleProject default settings bt qualitycontrol bt Figure 6 Folder and file content of the working directory not have to be in the same place on the hard drive every time the program is run but it should always contain the main folder of the program and the accompanying files Once the working directory is set type source Program mainApplication R to launch the TReCCA analyser as shown in Figure 7 In the example of this figure typing getwd gives back the localisation of the actual working directory in this case the Docu ments file By typing setwd C Users Julia Desktop TReCCA Analyser the work ing directory is set to a folder called TReCCA Analyser placed on the desktop of the computer The change of working directory is confirmed by the command getwd By typing source Program mainApplication R the program is then launched gt getwd 1 C Users Julia Documents gt setwd C Users Julia
47. le it is possible to type Exclude as its name thus bypassing the more time consuming step of actually deleting the column from the original data set and then from the template Naming a condition s Exclude will automatically remove the column s from the data set before any analysis is run so it will not be taken into account for the average calculation ICs determination oxygen consumption determination In the graphs of the raw data though the conditions may be represented in the wrong colour as the template is not automatically modified If it is important for the individual condi tions to be represented in the right colour then it will be necessary to delete the condition from the csv file and the template Either way it is possible to access the reduced data set as Raw data after the analysis is run 5 3 Analysis options In the Analysis options tab displayed in Figure 15 the analysis to be performed on the data are selected and the corresponding settings are filled in First fill in the Analy sis selection top part of the screen As the boxes are ticked new fields to fill in will appear in the bottom part of the screen corresponding to each possible analysis and the respective settings that have to be filled in These settings are described in the following paragraphs and for a more precise description of the mathematical calculations that take place please refer to chapter 6 Technical
48. le lines and columns automatically using the program This way it will not be necessary to delete the extra TReCCA Analyser user manual 19 Chapter 5 Detailed user guide data using classical spreadsheet applications If there is a header in the file s not taking into account the first line of the file which belongs to the data see Figure 12 select yes and whether it should be saved to a separate file or just be removed Either way the original file will not be changed just the imported version If you chose to save the header a csv file called Header exported csv will be saved after the analysis is run in the result folder that you will name The number of rows contained in the header should be equal to the number of rows in the spreadsheet program used In the exceptional case of Excel files containing Excel reports it will be necessary to count the rows in a text file as their number will increase with the report Empty lines between the header and the data also count as headers and have to be taken into account In a similar way if there are columns in the beginning or in the end of the files error messages time stamps time columns in other units they can also be removed auto matically by the program To do this enter the right numbers in the respective fields without forgetting to take the empty columns into account The same thing can be done also with rows at the end of the document on
49. ligne ou help start pour obtenir l aide au format HTML Tapez q pour quitter R gt B CRAN mirror C Packages France Lyon 2 a CAGExploreR a France Montpellier Cairo France Paris 1 France Paris 2 CALIBERrfimpute Germany Berlin calibrate Germany Bonn calibrator CALINES Greece calmate Hungary CAMAN India camel Indonesia Jakarta Indonesia Jember i cancerTiming candisc OK Annuler OK Annuler Figure 3 Package installation guide on Windows You will need to install the packages dref gWidgets gWidgetsRGtk2PJand RGtk2 f in the same manner as listed in Table As soon as RGtk2 is installed it can be loaded by typing library RGtk2 in the R console You will automatically be asked to install GTK if it is not installed already as seen in Figure The automatic download will install the whole GTK framework together with all the required packages from the all in one bundle listed in Table 1 that can also be found on the GTK website www gtk org Restart R after installing GTK C Ritz and J C Streibig Bioassay analysis using r Journal of Statistical Software 12 2005 4John Verzani gWidgets gWidgets API for building toolkit independent interactive GUIs 2012 Based on the iwidgets code of Simon Urbanek and suggestions by Simon Urbanek and Philippe Grosjean and Michael Lawrence R package version 0 0 52 Michael Lawrence and
50. lumn with black and the rest of the template with 0 so this button should be the first used and be sure to save the template displayed beforehand if necessary 22 TReCCA Analyser user manual Chapter 5 Detailed user guide The Autofill numbers button will give a unique number to all the wells All black and All solid will set all the colours to black and all the line types to solid respectively 5 2 3 Saving and loading As mentioned before even if it is possible to modify the template directly in the TReCCA Analyser interface it is probably easier to use a classical spreadsheet program for major changes as for example after using the auto fill options To save the template click the Save template button and type a file name ending with txt or csv It is also possible to type a path to a different folder within the current working directory for example Templates NewTemplate csv The template can then be changed at will and loaded again once it is completely filled To load a previously saved template click on the Load template button which will open the file chooser When importing a template be sure to specify the right cell separator After importing saved settings the template will have to be loaded again from the file system for the program to run smoothly 5 2 4 Excluding data from the analysis In order to exclude one well from the data analysis if you realise that it is an outlier condition for examp
51. m the Sample Project folder lead to an existing file colours E is missing This is fine but the error will always occur upon start up Use the button showing a folder to open a file chooser dialogue and select the data typing the Entered paths don t file name path can lead to typing errors avoidin 9 l Data input l p ae l S lead to existing files exotic characters in the pathways When import ing Settings make sure the input data is still in the same place on the computer Given last value for Make sure the time point is included in the data sensor calibration l no file A rough time point is not enough the exact can t be found im soa date time point has to be given in the input unit imported data l The name for the normalisation condition has to Given name for nor Eana l l be in the Labels amp colours template in exactly malisation is not in l l l l the same spelling and capital small letter combi the labels list l nation Given name for 0 The name has to be in the Labels amp colours oxygen wells is not in template in exactly the same spelling and capi the labels list tal small letter combination Given name for 100 The name has to be in the Labels amp colours oxygen wells is not in template in exactly the same spelling and capi the labels list tal small letter combination Max number of The number of plates has to be a whole number Data input E l plates is 10 between 1
52. malisation target value Load calibration data from file D Work Programs R WORKIN 2 RDATA 1 A kla value of 8 8 with an error of 1 and a unit conversion factor of 1 will be used for the analysis Please enter the Y axis label for the converted oxygen consumption Oxygen consumption 5 air saturation h Calculate consumed oxyqen for Figure 24 Analysis options Oxygen consumption The Oxygen consumption tab can be seen in Figure First fill in the oxygen concentration of the fully saturated environment this would typically be 100 of air sat uration for a measurement in the lab or 95 of air saturation for a measurement in the incubator with 5 CO If this is the same value as the one used for sensor correction for the OxoDish users or for medium normalisation click the corresponding use sensor correction target value and use medium normalisation target value buttons IK Eyer A Oeggerli and E Heinzle On line gas analysis in animal cell cultivation II methods for oxygen uptake rate estimation and its application to controlled feeding of glutamine Biotechnology and Bioengineering 45 1 54 62 1995 2R Hermann M Lehmann and J B chs Characterization of gas liquid mass transfer phenomena in microtiter plates Biotechnology and Bioengineering 81 2 178 186 2003 3G John I Klimant C Wittmann and E Heinzle Integrated optical sensing of dissolved oxygen in microtiter plates a novel tool for microbial c
53. many columns as desired each containing the measured data from one condition typically from one well The first line after the file header must contain unique names for each column An example of 17 Chapter 5 Detailed user guide EA TReCCA Analyser for time resolved cell culture assays V4 0 Data Input Status messages Graph output mw Import export settings Import export R data Please choose the format and source of your data input and import the files Data layout File import How many plates do you want to analyse 1 Insert Document Path to csv files starting within the current working directory Separator Decimal separator Do you have one document with all the data or one for each plate Plate Document path Wells single document multiple documents 1 SampleProject Data csv 24 E Import Files Did you already clean all non numerical data yes no What did you measure PreSens OxoDish PreSens OxoPlate PreSens HydroPlate other Cutting borders Is a header included in the file s yes no Are there any columns in the document s that are irrelevant for the analysis 0 columns in the beginning and 0 columns in the end have to be removed How many rows have to be deleted in the end of the document s 0 Figure 11 Data input tab input file format can be seen in Figure Time p oints Text Umique wellnames E Ti Time sec Al Bl cl D1 A2 B2 63
54. me cases the analysis should not last more than 15 minutes Each time an analysis step is completed it will appear in the analysis window followed by done as seen in Figure and the first ten rows of each data set will be printed in the R console Under Windows the messages and data sets will appear progressively as the analysis is performed under Mac all the information will appear at once as soon as all the analysis steps are finished Once the program has finished running press the Close button and the raw data graph in the Graph output tab should be visible 5 5 1 Graph output After running the analysis the Graph output tab should be opened automatically as seen in Figure In this tab it is possible to customise many more items on the graphs and export them by using the buttons and drop down menu at the bottom of the screen To the right the different buttons select which calculated data sets are displayed in the graphical area To the left a list of checkboxes are visible which select which lines are displayed on the graphs TReCCA Analyser user manual 39 Chapter 5 Detailed user guide Analysis running This can take some time Emptying data sets done Calculating raw data done Correcting OxoDish sensors done Normalising data to medium condition done Calculating average medium normalised data done Calculating 1050 done The analysis is finished and took 60 880890369415 seconds Your settings we
55. me point it is important to select the time frame where the conditions are sufficiently different from each other If the data cannot be fitted at one time point then all the IC fits will not appear in the graph output It is then necessary to change the selected time points In order to speed up the analysis time of the data it is possible to calculate the Cx for only some of the data for example for every third time point Fill in the frequency of the calculation accordingly Select which function to use to fit the data For ICs9 determination the 4 parameter log logistic curve is the most commonly used An exact description of each fitting for mula can be found in IC o determination Enter the X axis label that should be displayed in the IC graphs Finally enter the concentration of each condition used for ICs 9 determination without any unit in front of the corresponding condition The wells that are irrelevant to the IOso determination should be filled in with 1 as seen in Figure TReCCA Analyser user manual 39 Chapter 5 Detailed user guide Calibration of oxygen accessibility Use the oxygen values and their slope from the Raw data E data Data preview Resulting values The hill to be analysed lies approximately in the timeframe from 1 100 microL 0 to 3 in the given output unit kLa Std Error t value Pr gt t R squared 3 507472 0 01481849 236 6957 0 0 9915752 Use data from 40 to 90
56. mmercially available 24 well oxygen sensor plate the OxoDish PreSens Precision Sensing GmbH Germany this program will recalibrate the 24 oxygen sensors at the beginning of the experiment making the read out more homogeneous For the users of the commercially available 96 well pH or oxygen sensor plates respec tively the HydroPlate or OxoPlate PreSens Precision Sensing GmbH Germany this program will convert the relative fluorescence intensity data to respective pH or oxygen values The results of all these calculations will be automatically plotted using a simple tem plate and allowing an easy fast and reproducible visualisation of the data The graphs produced are highly customisable titles axis description legend content as well as sizes and colours exportation format can all be modified as wished The TReCCA Analyser is of course not restricted to the analysis of the results of cell culture assays and can be used for any time resolved data that need to be averaged normalised derivated and plotted 3 Installation guide The TReCCA Analyser runs on every system which is compatible with the freely acces sible statistical analysis software REI as long as the packages described hereafter are also available on the computer Some details of the appearance of the program may vary from one system to the other In order to use the program R and the corresponding packages have to be downloaded to the computer as well as
57. n for the analysis to run It is possible to customise the default settings of the TReCCA Analyser once it is first opened To do this set all the settings as wished and then save them using the Im port Export settings button under the name default _settings txt thereby replacing the existing file The next time the TReCCA Analyser is opened the settings should be the way they were last left TReCCA Analyser user manual 43 Chapter 5 Detailed user guide Please enter the path Tilename bet starting in your current working directory or choose a file to be loaded D Work Programs R Working directory 4 Cancel Figure 36 Pop up for saving loading settings 5 5 4 Import export R data To save all the data that has been imported and analysed in order to possibly replot graphs without rerunning the analysis for example press the Import export R data button The corresponding file should end with RData and will be saved in the work ing directory as described by the pop up window To load previously saved R Data click on the Load button and select the correct file The analysed data sets will once more be available in the R console and for plotting 44 TReCCA Analyser user manual 6 Technical details In this section more details concerning the mathematical analysis of the data are pre sented sorted for each analysis option available in the tab Analysis options For more precise information t
58. n in the status messages bar in the bottom of the program window This bar is a scrollable list In Table 3 all possible error mes sages are listed in alphabetical order A short explanation and solution to each message is presented and each error message is linked to the section in the manual it is related to for more details Errors that prevent an action from being taken will appear as a pop up window The Tables 6 and 7 give suggestions to solve each problem 7 2 R console Some error messages and warnings can appear in the R console and be ignored If after solving all the error messages in the scrollable list status messages bar and all the pop up errors something still is not functioning correctly double check whether the data is cut properly whether there is non numerical data left in the data set this can be checked eas ily using the program whether the template contains exotic characters that might affect plotting and whether all the paths and folders given are valid If the problem persists it is possible to check the R console for additional complications Many problems can then be resolved by following the instructions in the R console by searching the error message on line or by asking the questions on an online forum ol Chapter 7 Trouble shooting Use the file chooser properly to select the template If this error occurs when starting the program the Entered path doesn t Labels amp g 9 e default template fro
59. n window Plot result 36 TReCCA Analyser user manual Chapter 5 Detailed user guide Please select the data set to be used for the IC50 calculation Raw data 5 Calculate IC50 from time 112324 to time 1343 in green input unit Calculate IC50 only for every 3 timepoint Please select which function to f t to your data 4 parameter log logistic function Please enter a name for the X axis on the IC50 graphics Concentration of substance Figure 29 Analysis options First part of the ICs5o tab Please enter the concentration without units for each well name To exclude wellnames from the IC50 calculation put 1 100 uM 100 200uM 200 25ulv 25 SU UKM 50 DuM 75 Media 4 NT n pH4 1 pH5 4 pHo 1 Figure 30 Analysis options Second part of the ICso tab 5 4 Graph options In the Graph options tab as seen in Figure fill in the settings to determine the appearance of the graphs All the options that are filled in this part can be changed after the graphs are visible Many other settings can be modified after the analysis is run in the Graph output tab 5 4 1 General In the top part of the window choose if the graphs should have titles and subtitles and if so what these titles should be The title will be displayed in the exact same form TReCCA Analyser user manual 37 Chapter 5 Detailed user guide EA TReCCA Analyser for time resolved cell culture assays V4 0 o
60. nter a positive whole number for the data for matting or U to not delete any rows Enter file names for all the chosen files to be ex ported They should all end with csv Enter numbers with as decimal separator in the output unit for each formatting parameter TReCCA Analyser user manual Chapter 7 Trouble shooting Starting time point of o l l ICs9 determi The ICso calculation interval time points in input unit are not in the right order IC calculation has to be before end point The given time point Make sure the exact time point for the end of the for end of ICs9 calcula calculation interval given in the input unit is in tion can t be found in nation l men cluded in the data file imported data The given time point Make sure the exact time point for the start of for start of ICs 9 calcu C59 determi the calculation interval given in the input unit is lation can t be found nation o a included in the data file in imported data There are not as man 9 There are not enough time points prior to the time points for cali l starting point specified Enter a smaller number bration as you want to l l l l l l l l clude calibration of time points or set a higher starting time point include You are about to re l i By proceeding with this basic data formatting move the stop time step the time point used for OxoDish sensor cali point of the sensor f
61. o the emission intensity of a reference luminophore see the OxoPlate instruction manual on the PreSens homepage for more precise information The ratio of the indicator luminophore over the reference luminophore then has to be converted by the user to actual oxygen values using two calibration solutions one con taining a 100 of oxygen Call00 and one containing a chemical which depletes all the oxygen present by reacting with it Cal0 This calibration step can be done once for each lot of OxoPlates or once for each plate and during the whole course of the experiment The time resolved calibration of each plate separately leads to more precise and less noisy results Whatever the calibration method the TReCCA Analyser will convert the relative emission intensity data automatically and if available in a time resolved manner accord ing to the formulas described in the OxoPlate user manual For more calculation details 28 TReCCA Analyser user manual Chapter 5 Detailed user guide please refer to part OxoPlate oxygen conversion First enter the exact names assigned in the template in Label amp colours for the 0 and 100 oxygen calibration solutions often Cal0 and Call00 If the lot of plates is pre calibrated add two columns to the data where each one contains the Cal0 and Call UU average value for conversion for each time point of the data If there are several columns with the calibration solutions the data will be
62. or time resolved cell culture assays The current working directory ts D Work Programs R Working directory 5 If you change the working directory please restart the application view and export results save load a project Status messages S Graph output Data output EE Import export settings Import export R data Figure 9 TreCCA Analyser welcome screen In the console there will be a message indicating that all R objects were deleted This is to make sure that there are no conflicts when running the program several times within the same R session This also means that any progress made in R before starting the program will be lost 14 TReCCA Analyser user manual 4 Quick guide This quick guide will give the key steps to follow to use the TReCCA Analyser Please refer to the more detailed descriptions following in chapter 5 Detailed user guide for any extra information Throughout the use of the program whenever something is entered in an entry box it is necessary to press return to process the setting directly otherwise it will only be processed as soon as it is needed by the program If you already have a personalised settings file load it by clicking on Import Export settings import the input files and the template and go directly to step 5 1 Click Data input 1 Fill in the Data layout top left of the screen 2 Define the Cutting borders bottom left of the screen 3 In File import select
63. ormatting l bration will be removed correction Your imported data doesn t contain Reduce the number of points set for smoothing or enough time points prepare a data set with more time points for smoothing Your imported data doesn t contain Reduce the number of points set for the slope cal enough time points culation or prepare a data set with more time slope for the slope calcula points tion Table 3 Error messages shown in the status messages bar in alphabetical order TReCCA Analyser user manual 55 Chapter 7 Trouble shooting General errors The script for the TReCCA Analyser that We hereby inform you that the TReCCA Analyser version you use has been modified Any technical details or you are using has been mo dified Download guidelines in the user manual might be invalid and you are ss 5 prohibited to refer to this program unless you use the the original files again to work with the COT original version rect version Error upon data import or when trying to run the analysis Some of the settings are in Please solve all status messages and try again valid and there are errors in the status mes sages bar Please look up solutions in Ta Table 4 Pop up errors and solutions General errors Data input Error upon trying to automatically fill the template s well number column or trying to run the analysis Either the data is not im Please import data first ported yet or a seve
64. ort export settings Import export R data Figure 14 Interface of the Labels amp colours tab sample template loaded 5 2 1 Template layout The template is a table containing 5 columns which are Well number Name Num ber Colour and Line type The Well numbers must be the same as the names of the columns of the data sets in the imported data from example Al to D6 or 1 to 20 TReCCA Analyser user manual 21 Chapter 5 Detailed user guide These can be detected automatically and added to the template by clicking the Autofill labels button The Name can be any type of character string but many special characters will not be recognised or will cause problems depending on the system running This name will be used for the legend labels and to determine groups of wells for averaging for example Instead of using the Name it is also possible to use the Number column to define groups of columns wells The oxygen calibration pop up also determines the groups of wells using this number If all the numbers in the template are the same the legends of the graphs will be sorted alphabetically By assigning each condition a number it is possible to determine in which order each label will appear in the legend the name assigned the smallest number will be displayed first and so on As a general rule in the case of the legend for average conditions the Number Colour and Line type of the first
65. oxes to the left of the screen and then on Refresh lines If you wish to exclude some conditions from the analysis go back to the template and name them Exclude You will have to rerun the analysis by clicking Run analysis for the changes to be taken into account Customise the graphs by using the options displayed at their bottom point size error and grid intensity the legend position and format You can also change the settings in Graph options menu and apply them by clicking Refresh options 7 Export graphs and data 1 16 Export each graph by clicking on the Export displayed diagram button and enter a file name and size in inches It will be saved in the result folder To save the data as csv files click on Data output and select which data sets to export their name and click on Export Files By clicking on Import export R data you can save the R data so that you will not have to rerun the analysis to change the graph customisation By clicking on Import export settings you can save the settings for the next analysis TReCCA Analyser user manual 5 Detailed user guide In this part of the manual the TReCCA Analyser is described in full detail with an overview of all the modifiable options and the consequences of their selection The buttons at the top of the screen displayed in Figure 10 should be filled in one after the other and will be successively desc
66. re error message occurred upon trying to import it The data input is also cleared whenever settings are loaded Please go to the data input interface and press import data and if necessary solve the other error messages Error upon trying to import data without EN oF getting rid of non numerical data Your data mpored data Is NOT AUMEFIC contains non numerical data Either change your tick for the data cleaning or clean the data manually Error upon trying to import data from sev eral plates This means that the time Imported Files don t have the same columns in your files are not identical This timepoints Please solve the problem and try again can be due to different line numbers in the files Otherwise it is recommended to analyse the plates independently 56 TReCCA Analyser user manual Chapter 7 Trouble shooting Error upon trying to import data The sum of the well numbers you specified 1 for the Number of columns doesn t match total number of wells time column doesn t match with the total number of columns in the merged data you tried to import Please check the columns again and change the numbers accordingly Warning pop up that all the data that is not All nonnumerical data will be replaced with 0 recognised by the program as a number will be replaced by 0 This will not affect your original data files Information pop up The files imported con There were no single value
67. re saved automatically to the results folder Figure 33 Window seen while the analysis is running EA TReCCA Analyser for time resolved cell culture assays V4 0 Experiment 1 18 06 2014 Raw data SI S S x Before formatting lt S S S SSB Relative intensity ratio SI iS SI lt Time day Sl 4 0 51 L Status White space Figure 34 Graph output tab To change options that were set previously in the Graph options tab change them 40 TReCCA Analyser user manual Chapter 5 Detailed user guide in the corresponding tab and then press the Refresh options button for the changes to be applied To change colours line types or names change the template without running the analysis again as long as the changes do not influence the calculations many of the analysis options use the names or numbers in the template as a basis for calculation see the corresponding analysis details Graph customisation The drop down menu at the bottom of the screen allows the customisation of different graphical parameters Select the parameter to be modified and then use the scroll bar or the second drop down menu that appears to modify it The graphs will only be modified once the slider 19 set and it is also possible to click on the bar directly to skip through bigger steps instead of pulling the slider The different possible customisations are as follows Point size Chang
68. ribed in this part of the handbook Figure 10 Buttons of the main tabs of the TReCCA Analyser 5 1 Data input When starting the program the first step consists of importing the data Click on the Data input button in the upper menu bar to see the tab displayed in Figure It consists of three subunits Data layout Cutting borders and File import described more precisely hereafter Io have a second look at data previously imported analysed and saved as R data click Import export R data and load the corresponding file If the settings from a previous analysis or from a similar experiment were saved it is also possible to import them by clicking Import export settings and loading the corre sponding file Clicking through the different settings is still possible to check that they are set as wished and they can be modified if necessary Even after importing the saved settings it will be necessary to load the template again When importing R data it is also essential to first import the settings so that the interface is correctly set for the imported data to be shown 5 1 1 Required format The format required for the input files is csv or txt It is possible to convert files to these formats with usual spreadsheet applications for example excel files xls or xlsx using the save as function In each document the first column has to be the time column after cutting off the edges Following this column there can be as
69. row of the advanced data formatting here the time points will be taken away and the beginning of the time scale will be set to 0 TReCCA Analyser user manual 25 Chapter 5 Detailed user guide The last box will multiply all the data by a given number allowing a conversion of the measurement unit In this way it is possible for example to convert data from milimolars to molars by entering 1000 5 3 2 Average and standard deviation The Average option tab can be seen in Figure 17 Select the criteria from the template in Labels amp colours to use for the average calculation The TReCCA Analyser can ei ther average conditions having the same Name Number or Colour The arithmetic mean and the standard deviation will be calculated accordingly for every time point If only one replicate is available then the TReCCA Analyser will just take the actual value of that replicate the standard deviation in this case will be set to 0 Please select according to which attribute the average should be calculated name number colour calculate standard deviation Figure 17 Analysis options Average tab 5 3 3 Normalisation The TReCCA Analyser can be used to normalise all the measurements at each time point to a specific condition For example in the case of an oxygen measurement over time a well containing only medium and placed in an incubator should have a stable read out throughout the measurement As this can differ sli
70. s replaced pop up P tained data that was all recognised as numer ical no data point was replaced by 0 5 discrete value s are not recognised as nu merical Accept that they will have the value 5 single value s had to be replaced 0 or check out the original files and modify them The files could contain text such as Error gt 9 0 pH or numbers with the wrong decimal separator 2 whole column s are not recognised as nu merical Accept that they will have the value 0 or check out the original files and mod 2 whole column s had to be replaced If you want them included clean nonumerical data manually ify them The columns could contain values The first five rows are printed to the R Console recognised as text such as Error gt 9 0 pH or numbers with the wrong decimal sep arator TReCCA Analyser user manual 57 98 Wellnumbers in template and imported data don t match please correct the template Chapter 7 Trouble shooting Error upon trying to import data load template The well numbers in your data and in the currently loaded template do not match If you haven t filled in the Labels and columns tab yet this error can be ig nored Otherwise you should check all the files again or import the data and use the auto fill button for well names in the tem plate Table 5 Pop up errors and solutions Data input Settings and R Data import The settings could not be fully loaded
71. t Data smoothing 5 3 8 Numerical slope Calculating the slope of time resolved data can provide valuable information as it will highlight the changes in the speed of the observed phenomenons rather than their actual value It is important to note that the unit of the calculated slope will be the measure ment unit divided by the output time unit The y axis label of the slope graphs will have to be changed manually by filling in the Y axis label in the Graph options tab please refer to part 5 4 Graph options In many cases it can be useful to smoothen the data before using it for slope calculation as noise could hide the actual data trends The Slope tab can be seen in Figure 23 The unit of the slope will be converted measurement unit target time unit When exporting graphs remember to change the Y axis label accordingly Smooth the curves prior to calculation by averaging a neighbourhood of 3 points needs to be an odd number Lise 3 smoothed points on either side of the centre for a linear fit to determine the slope Calculate slope for raw data averaged raw data W converted data average converted data Figure 23 Analysis options Numerical slope Fill in the number of points to be included in each neighbourhood as explained in part Data smoothing To use non smoothed data fill in the number 1 The slope of each data point will be determined by performing a linear fit of this point and n
72. te are checked for validity A pop up window will appear asking the name of the results folder to be created as seen in Figure This folder will automatically be created in the current working directory and within this folder the current settings are also saved automatically It is possible to enter a path leading inside different folders included in the working directory as long as the upper directories are already created 38 TReCCA Analyser user manual Chapter 5 Detailed user guide gt Please enter the name of the results folder to be created If you want to enter a path the upper directories have to exist already Results experiment 1 Continue Cancel Figure 32 Pop up window to run the analysis If this pop up window does not show up then another pop up window with a sugges tion about what has to be corrected will probably appear If this is not the case make sure all the Status messages at the bottom middle of the program are clear and check the error messages in the R console In extreme cases restarting the program can also be an option After the name of the result folder is given the analysis will start running This will take a different amount of time depending on the size of the data sets the variety and complexity of the analyses that have to be performed and the speed of the computer that is running As a rough estimate most basic analyses are usually run in under a minute and even in extre
73. the GTK widget toolkit The pro gram is then unpacked to the computer according to the following instructions 3 1 Downloading R and the complementary packages The TReCCA Analyser requires R version 2 12 0 or higher which can be downloaded from the site R project org www r project org index html Choose a CRAN mirror from the country that you are in and follow the instructions for installation At the end of this step it should be possible to launch R as seen in Figure l for Windows and in Figure 2 for Mac IR RGui 64 bit a B X Fichier Edition Voir Misc Packages Fen tres Aide R R Console ollel z R version 3 1 0 2014 04 10 Spring Dance Copyright C 2014 The R Foundation for Statistical Computing Platform x86_64 w64 mingw32 x64 64 bit R est un logiciel libre livr sans AUCUNE GARANTIE Vous pouvez le redistribuer sous certaines conditions Tapez license ou licence pour plus de d tails R est un projet collaboratif avec de nombreux contributeurs Tapez contributors pour plus d information et citation pour la fa on de le citer dans les publications Tapez demo pour des d monstrations help pour l aide en ligne ou help start pour obtenir l aide au format HTML Tapez q pour quitter R gt Figure 1 R Console just after launch on Windows In addition to R the TReCCA Analyser also requires special packages to run They can be installed via the packa
74. to deplete it such as sodium dithionite Na 5 0 or sodium sulphite Na SO or by using a nitrogen gas chamber Note that the cited chemicals while being easier to use might react with the composition of the media Once each well has a read out of near to 0 oxygen measure oxygen diffusing back into the system by either waiting for all the chemical substance to be consumed or by placing the plate in an oxygen saturated environment again The speed at which the oxygen rises in each well will determine the oxygen mass transfer coefficient kya First import the calibration data into the TReCCA Analyser fill in the template and perform the wanted analysis including probably averaging and definitely slope calcula tion It is important to run a slope calculation as the data will be needed for the oxygen consumption calibration To reach the calibrating platform click on the button Calibrate oxygen consump tion at the top right of the Analysis options tab The TReCCA Analyser will ask for confirmation that the slope calculation has been done as seen in Figure and once Continue with the calibration is clicked a new pop up window will appear as seen in Figure TReCCA Analyser user manual 33 The calibration needs a dataset of timeresolved oxygen concentrations and its slope If you didn t run Chapter 5 Detailed user guide this analysis yet please do this before you continue Go back to run required analysis Continue with the
75. ttons click the Refresh lines button to show the selected lines on the actual graph TReCCA Analyser user manual 41 Chapter 5 Detailed user guide Graph selection The buttons on the right of the screen determine which graph is represented in the plot area Only graphs of the analyses that have been run can be represented In the case of the average graphs where the standard deviation was calculated an extra tick box appears next to the graph customisation drop down menu Selecting it will display the standard deviations of the averages of the curves as a grey shade behind the averaged line For the slope calculation the standard deviation tick box is also present and enables showing the error of the linear fit on the graphs For the time resolved IC data the error of the fit can also be displayed In the case of the display of the sigmoidal fits for the IC calculation extra tick boxes appear above the customisation drop down menu allowing for a further customisation of the graphs The first line allows to differentiate IC fittings by varying Colour Each fit is displayed in a different colour the concentration points are represented as empty coloured circles Line type symbols All the lines and symbols are black the line types and sym bols vary from time point to time point Nothing All the lines and symbols are black the lines are all solid and the symbols are all represented as empty c
76. ultivation Biotechnology and Bioengineering 81 7 829 836 2003 32 TReCCA Analyser user manual Chapter 5 Detailed user guide The experimental set up first has to be calibrated in order to determine the diffusion constants This can either be done using the TReCCA Analyser See part 5 3 10 gen consumption calibration of the manual and loading a calibration file which will automatically fill in the following fields or by filling in the fields manually for the oxygen mass transfer coefficient kra value and error It is essential that the time unit used for the kra value and for calculating the slope of the data be the same To change the unit of the oxygen measurement fill in the corresponding unit con version factor To calculate the unit conversion factor the excel sheet provided by the company PreSens GmbH under on the PreSens homepage can be useful Enter the Y axis label that should be used for the converted oxygen consumption and choose the data that should be used for the oxygen consumption calculation 5 3 10 Oxygen consumption calibration In order to convert the oxygen measurement data to actual oxygen consumption data it is necessary to first calibrate the system and determine its oxygen mass transfer coefficient kya The use of this constant is further detailed in part Oxygen consumption Under the experimental conditions deplete the oxygen in each well by using either a chemical that will react with oxygen
77. ysis based on this calculation should also be updated refer to Figure 16 for an overview of the dependencies For example if the normalisation target value is changed and the Rerun normalisation only button is pressed then the TReCCA Analyser will ask whether the average normalisation calculation which is based on the normalised data should also be accordingly recalculated 5 3 1 Basic data formatting The Basic data formatting tab is always displayed in the Analysis options window as seen in Figure The first half of the tab has to be filled in accordance with the time resolved experiment the second half is optional In the first row tick the time unit of the input data The time unit used for all the output graphs can be chosen in the second row and the TReCCA Analyser will automati cally perform the corresponding unit conversions if necessary The output time scale unit will also have an influence on the slope calculation of the data see part 24 TReCCA Analyser user manual Chapter 5 Detailed user guide Data input Time scaling data exclusion Raw data Basic data formatting Average sd Smoothing IC50 HydroPlate calibration Slope OxoPlate calibration kLa calibration Oxygen consumption Medium normalisation Figure 16 Analyses available in the TReCCA Analyser and their relationship The second half of the tab is for performing more advanced data formatting First the data s

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