USER GUIDE
Contents
1. Nanopore Analysis USER GUIDE 14th July 2014 Contents 1 Introduction 2 Get start to Nanopore Analysis 3 GUI of Nanopore Analysis 4 Manage your data 5 Routine for data analysis 6 Preview the events 7 Statistical Tool box 8 Reference Nanopore Analysis Nanopore Analysis 1 Introduction Nanopore Analysis is free software built for people who struggles in data analysis of nanopore study It provides an automatic and accurate process for measuring the dwell time and current amplitude of every individual blockades With the help of this program management of data detection of events and statistical analysis could be achieved easily and conveniently Following this guide you will certainly have a clear idea on how to analyze your nanopore data through this software Methods based on different strategies are applied to recover measure or evaluate the dwell time and amplitude of events Considering the difference of criterions or purposes for data analysis all of the methods in this software are optional In addition three user parameters offered by the software which can be used to adjust the sensitivity of blockades locations and accuracy of measurements If you find this software cannot meet your needs in data analysis or encounter an error in usage please feel free to inform us by E mail yitaolong gmail com The more you can tell us about the circumstances leading up to the bug the better for us to improve2. the software Nanopore Analysis 2 Get start to Nanopore Analysis A Matlab platform is necessary to open this software To ensure the software runs properly we strongly recommended you to using a version higher than Matlab R2008a So far the software can only run in Windows OS but the next version will come with compatibility for Mac OS The software was packaged before uploaded After downloading from our website Nanopore Analysis should be extracted by specific tools such as Winrar WinZip If you do not have one please Click here to get one After extraction please do not delete any files keep all the files in a same folder Then you can open the software in Matlab by either of following two ways 1 Open Matlab gt set Current Folder to the folder of the software gt type in main in the Command Window gt press Enter Current Folder DE Command Window Name fx gt gt main El Scripts I temps E cOVerJpg fe dataReflesh m fe eventListRefresh m fe eventView m main fig H main m StatisticalTool fi g fe StatisticalTool m 2 Double click main m in the folder gt the Matlab platform will automatically gt launch gt click this icon in the script editor of main m Nanopore Analysis 3 GUI of Nanopore Analysis Two GUls exist in the current version including a main GUI for data process figure 1 and a StatisticalTool GUI for stat
3. ct the start and end point of the events Risetime correction is based on an investigation which reveals the influence of filter to the shape of events Through this method the dwell time will be modified by reset the end point of event see reference 1 and 2 Unselecting the check box could be used to turn off the methods in data analysis Nanopore Analysis The integration method is also developed by our group which can evaluate the current amplitude of events Note that the integration method should be used together with DBC method and risetime correction to ensure the result is accurate The cutoff frequency of filter should be given correctly Alternatively you can also choose the plateau averaging method to measure the current amplitude Click the button Start the software will automatically analyze the data selected in the data list and a message box will show after finishing the analysis Preview the events After data analysis all the detected events are listed in the Results panel figure 5 You can select one of the events and then it will be figured in the preview window As shown in figure 6 the corrected event pulse is depicted as red solid line the baseline is shown in black fitting result of event is a green dashed line and the uncorrected event is red dashed line reference 1 All of the elements are optional through the check box in the left of panel In the list box the length of event number of data poi
4. he right column is dwell time ms Statistical Tool DataList E scripts matlab data Available data Scatter X Y Reverse Plot Histogram Dwell time kai Bin width ms x Limit auto Pot 362 events Figure 7 GUI of the StatisticalTool Dwell time and amplitude of events in the selected data can be used to make a scatter diagram by click the Plot button in the Scatter panel If the checkbox X Y Reverse is unselected x axis will be the amplitude and vice versa The pop up menu in the Histogram panel is used to select which will be distributed by histogram Then you can set the bin width of histogram If the checkbox before auto is selected the x limit will be set automatically and otherwise you can customize the x limit in the textboxs Nanopore Analysis 8 Reference 1 A Data Process for Accurate and Automatic Analysis of Blockades in Nanopore Study unpublished 2 Pedone D Firnkes M Rant U Anal Chem 2009 87 9689
5. istical analysis figure 2 The main GUI is divided into four parts Upper half of the interface is a window for data preview When an event is depicted in the axis you can tune the Y scale and X scale to adjust the limit of the axis In the Data List panel you can load rename and remove your data or select a data for further analysis In Analysis panel method options and user parameters is available The Results panel lists all the detected events you can choose one to see its detailed information a BB Nanopore Analysis 2014 Lo e jes file Tools i ih O 32 N h Ye Wanopore Analysis Version 1 3 Data List Analysis window width 200 Y scale m X scale mn ns Load data u0 20 Results M l Dwell time Measurement F Event puise Events list 7 DBC method Baseine V Risetime correction a Previous Remove data ooo Fitting Uncorrected Renae a Amplitude Evaluation Integration method Dwell time O een Plateau averaging ms Low pass filter 7 used in experiment 10 kHz Amplitude a pA Figure 1 main GUI for data process After detection of events you can use the StatisticalTool GUI to distribute the dwell time or amplitude of events Data in the selected data list could be chosen to make a scatter or hist
6. lick the button Save List and then a dialog will appear to ask you type in the name of list If the name is valid a folder will be created in the path of the software Data as well as the detected events are saved as an individual file in the folder Note that you can shift the directory of the folder but not to delete files in it especially the file info mat Saved data list could be loaded into the software by click the button Load list and selecting the corresponding folder Be aware that the loading process will delete the unsaved data list Routine for data analysis The first step in the data process is detection of events A moving window method is used to evaluate baseline of the current Default window width is 200 data points while the user could adjust it according the noise level of the signal The parameter Up is used to tune the sensitivity of detection and its default value is 20 pA uo of 20 pA means events with amplitude lower than 20 pA from the baseline will not be detected Analysis window width 200 ud Fall Dwell time WWleasurement v DBC method V Risetime correction Amplitude Evaluation Integration method Plateau averaging Low pass filter used in experiment 10 KHz Figure 4 the Analysis panel in the main GUI To achieve accurate and robust analsis a 2nd order differential based calibration DBC method was developed by our group This method helps to corre
7. nts is demonstrated Results W Event pulse Events list SF penn 1 length E Baseine 2 length re Fitting 3 length 12 4 length 14 Uncorrected 5 length 15 ae 6 length 18 Dwell time 7 length 10 0 12 ms 16 length 16 Amplitude jopa 10 length 12 40 5579 pA 11 length 13 Figure 5 the Results panel in the main GUI 3 519 3 5195 3 52 3 5205 3 521 3 5215 3 522 Figure 6 event depicted in the axis Nanopore Analysis 7 Statistical Tool box The statistical tool is a standalone program which can be run without the main GUI You can open it in menu of Tools in the main GUI or just type in StatisticalTool in the commend window and press Enter when the current folder of Matlab is set to the path of the software By using this tool you should first load a data list by click the button Select Data in the selected folder will be listed in the left list box You can use the gt lt and select all button to select or unselect data The number of events is labeled on the left of the name of data linked by a hyphen The total number of events in selected data is calculated and shown under the right listbox The information of dwell time and amplitude of events in selected data can be export to a excel file for analysis in other software by click the button Export The left column in the excel file is the current amplitude pA and t
8. ogram Nanopore Analysis EJ Statistical Tool 23 DataList Select Export Available data Selected data Histogram Dwell time Bin wridth ms x Limit auto Total 0 Figure 2 GUI for statistical analysis 4 Manage your data Firstly click the button Load data main GUI to select the file of your data and then it will be imported into the data list Note that the data file should be format of txt with one column of time and another column of current The unit of time is ms and the unit of current is pA A sample data file is available on our website Click here to download Similarly you can also remove a selected data by click the button Remove data To rename your data click the button Rename data and type in a new name on the pop up dialog Do not to use a name which already exists in the data list Data List Reflesh oad Lt Save Let Figure 3 the Data List panel in the main GUI All of the data in the list and their events information are stored in a temporary folder in the same directory of the software If your computer or Matlab was crashed you can try to restart the software and click the button Refresh to recover your data Note that Nanopore Analysis if the software was closed in a proper way the temporary folder will be deleted so it is strongly recommended to save the data list after every analysis You can just c
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