Manual for Demo Data. SEQUENCE Pilot module MLPA

Transcription

1 Manual for Demo Data SEQUENCE Pilot module MLPA Version (July 31st 2018) developed by JSI medical systems GmbH Tullastr Ettenheim GERMANY phone: / fax: / web: (for research use only)

2 Table of Contents 1 Introduction to SEQUENCE Pilot MLPA Installation SEQUENCE Pilot Installation of demo data The first login SEQUENCE Pilot screen Create your own user Import MLPA mixes MLPA screen What is shown? Order/Protocol/Family or Control Files File Information Tab Electropherogram Tab Analysis Functions Analysis of the Demo Data Copy Number Variation Control result files Load MLPA control result files Controllist Operation MLPA - Analysis of the control result files First control result file Second control result file Third control result file Fourth control result file Patient result files Load MLPA patient result files Joining Worklist Operation MLPA Analysis of orders First order Control assignments Result file 7_P034_M_P 1111.fsa Result file 7_P035_M_P 1111.fsa Final result and printing the report Second order Control assignments Result file 8_P034_M_P 1111.fsa Result file 8_P035_M_P 1111.fsa Final result and printing the report Third order Control assignments Result file 9_P034_F_P 1111.fsa Result file 9_P035_F_P 1111.fsa Final result and printing of a report How to make modifications Manual peak assignments Remove or ignore copy number changes How to analyse the order again Archiving of processed orders Analysis of Demo Data methylation Import of Mix ME Controls Load MLPA control result files Controllist Operation MLPA - Analysis of the control result files Control result files with the DNA number

3 Control result file with the DNA number Orders Load MLPA patient result files Joining Operation MLPA Analysis of order Order with DNA-number Final result and printing of a report

4 1 Introduction to SEQUENCE Pilot MLPA The module MLPA is made for bioinformatical analysis of your MLPA data and is compatible with all sequencers. On our homepage pre-configured SALSA MLPA kit descriptions are available which can be easily imported. For your analysis you can select different analysis modes for each MLPA kit. Moreover MLPA offers configurable control settings and relative peak area limits. For calculation it uses automated peak assignment and a data correction for probes with tailing-off-effect. If desired manual peak assignments are easy to do afterwards. Moreover warnings are displayed for undersized and oversized peak areas and heights, too little DNA quantity, incomplete denaturation and hybridisation and in case the wrong gender is set. After the analysis, a diagnostic report for each sample is received. MLPA is a simple, easy to use desktop application including a variety of functionalities to fit the program to many different specific needs. 2 Installation In case you have done the Installation of SeqPilot already, please proceed with chapter SEQUENCE Pilot For the installation of SEQUENCE Pilot, please do the following: Go to our website to obtain a free trial license. Please use the instructions you will receive via to download and install our latest version of SeqPilot. 2.2 Installation of demo data The demo data package MLPA_DemoData.exe includes the complete configuration and example files for the mixes P034, P035 and ME028 and shows you step by step how to load and analyse the data. Please note, that this package shouldn't be installed to systems already including data for mixes P034, P035 and ME028. This might overwrite your local configuration. In this case, please contact our support associates (support@jsi-medisys.de) for a separated installation. The demo data package MLPA_DemoData.exe includes three MLPA patient result files for copy number variation analysis and one MLPA patient result file for methylation analysis. Moreover corresponding control result files are included. For the installation of the demo data, do the following: Stop SEQUENCE Pilot. The program should not run during installation!!! Go to our website Download MLPA_DemoData.exe and open it. Press [Execute] in the dialogue file download security warning. Press [Execute] again in case of another security warning. Press [Next] in the dialogue Setup MLPA Demo Data. Check the box I accept the agreement after reading the Software License Agreement and press [Next]. Press [Next] after reading the Info and Installation Instructions for Sequence Pilot demo data. Select the destination folder, where the SEQUENCE Pilot demo data should be installed. By default this is c:\seqpilot. Then press [Next]. 3

5 Press [Install]. Press [Finish] when the SEQUENCE Pilot installation is completed. 3 The first login Once SEQUENCE Pilot is installed and licensed, you can login with the Name jsi (leave the Password field empty). 4 SEQUENCE Pilot screen In SEQUENCE Pilot a menu is available which consists out of the items System, MLPA and Help. Furthermore several categories and operations are shown on the left side of the screen. Open a category or an operation by clicking on the corresponding icon. menu operations categories 5 Create your own user How to get there: Category Administration Operation Users [master file] After the first login, create a new user. After the new user has been saved, the user jsi is deleted automatically. 4

6 Click on the category Administration on the left side of the screen and then click on the operation Users. Fill out the field User (max. 4 letters). Check active and all options user is authorized to... Press [Save]. Select the operation Logout and then login with the new user. 5

7 6 Import MLPA mixes How to get there: Category MLPA Operation Mixes [master file] Go to the category MLPA and open the operation Mixes [master file]. To import the master files for the MLPA mixes P034 and P035, do the following: Press [Import] and the dialogue Import file... is opened. Open the folder MLPA-Mixes, which is located in the Demo folder of your installation (by default C:\SeqPilot\Demo\MLPA-Mixes). Select the files P034.mme and P035.mme and press [Open]. All data belonging to these mixes (data for the operation Mixes [master file] and the corresponding data for the operations Mix Sequencer [master file] and Analysis Mode [master file]) are imported. If the import was successful, you will get a corresponding message. In case a mix with the same name already exists, you will be asked, if you want to overwrite it. 6

8 7 MLPA screen What is shown? How to get there: Category MLPA Operation MLPA Before we start analysing our demo data we first explain the most important features of the operation MLPA. The operation MLPA is the main operation for your analysis. If you switch to this operation you won't see any entries because we have no result files loaded yet! 7.1 Order/Protocol/Family or Control This dialogue part differs depending on the analysed file: patient or control. For orders this dialogue part is divided into three tabs. The tab Order shows all important information about the order, like order number, patient, DNA number, date etc. The tab Protocol shows a list of all changes for the currently selected result file. The tab Family shows a list of all orders belonging to the same family ID. For controls only DNA number and rundate are shown. 7

9 7.2 Files This dialogue part shows all patient result files belonging to an order or the control result file to analyse. The selected file is displayed in all tables of the operation MLPA and in the tabs Electropherogram or Analysis. 7.3 File Information This dialogue part shows all important information about a selected patient or control result file in the dialogue part Files, like the used mix, gender, total peak area (TPA) and Date as well as the joined control result files. 7.4 Tab Electropherogram The tab Electropherogram is divided into two main parts, the Mix/Result file Peak Table and the Electropherogram. The Mix/Result file Peak Table shows the defined peaks for the used mix in relation to the peaks of the selected result file. If the program does not find a peak in the selected result file, the value of this peak in the column Rf. Length is 0.00, the cells in the columns Mix Length and Rf. Length of this peak are marked red. If you select a peak, by clicking on the corresponding entry in the table, the assigned result file peak in the Electropherogram will be selected, too. The Electropherogram shows the raw data curves. Peaks assigned to a peak of the used mix and peaks, which are not assigned, are highlighted differently. By default assigned peaks are highlighted blue for ctrl DNA peaks (peak types: DNA ctrl, ctrl fragment, ctrl DNA quantity, ctrl DNA denaturation, ctrl DNA ligation, ctrl gender), orange for control probes (peak type ctrl probe) or yellow for gene probe peaks (no peak type or peak type mutation). Not assigned peaks are highlighted grey. 8

10 Electropherogram Mix / Result file peak table peak height dye buttons warnings peak length horizontal and vertical compression Below the electropherogram you find warnings and dye buttons (here: [Joe], [Rox]). The names of the dye buttons depend on the dyes, that are present in the result file. If the program cannot find any dye entries, the dyes are named with consecutive numbers. To switch the dye data curves on and off, press the corresponding dye button. Moreover there are several warnings. If there is a warning state the dot will be red, otherwise it will be green for the analyse state and grey for all other warnings: analyse state: shows, if peak assignment was successful. peak not found: shows, if all peaks defined in the operation Mixes [master file] for the used mix are found. DNA Quantity: shows in case the DNA quantity is too low. DNA Denaturation: shows in case of incomplete denaturation. 9

11 DNA Hybridisation: shows in case of incomplete hybridisation. Gender: shows if the wrong gender is set. Calculations based on the peak types ctrl gender. > max peak height: shows, if there are peaks that exceed the max. peak height defined in the operation Mix Sequencer [master file]. > max peak area: shows, if there are peak areas of the assigned peaks which exceed the max peak area defined in the operation Mix Sequencer [master file]. < TPA (total peak area): shows, if the total peak area of all assigned peaks of a result file is higher than the min total peak area defined in the operation Mix Sequencer [master file]. 7.5 Tab Analysis The tab Analysis is divided into two main parts, the Result table and the Analyse. Result table analyse modes peak length warnings relative peak area of the control result files with standard deviation limits regression relative peak area of the patient result file ratio RPA (relative peak area) Analyse horizontal and vertical compression The Result table shows all peaks, defined in the selected analysis mode of the used mix. You will get a corresponding entry in the column Result if there is a deletion, duplication or multiple copies at a 10

12 peak position. Moreover the result is mutation present or mutation absent if a MLPA kit that includes mutation analysis was used (peak type mutation defined in Mixes [master file]). Result calculation: The ratio relative peak area is the relative peak area of the patient result file divided by the relative peak area of the control result files. If this value is around 100 % the relative peak area of the patient is about the relative peak area of the control and there is probably no copy number change. Depending on your settings there will be a copy number change shown if the ratio relative peak area exceeds or falls below a certain limit. The default settings for this limit are 75 % (indicates a deletion) and 125 % (indicates an insertion). A value above 175 % indicates multiple copies. Moreover there is the result mutation present or mutation absent possible in the column Result if the peak type mutation was defined in Mixes [master file]. For the peak type mutation no copy number variation analysis is performed. If the peak is present (peak must exceed the upper limit) the mutation is present, otherwise it is absent. If you select an entry of the table, the corresponding position in the Analyse is selected, too. The Analyse shows the results in the form of a histogram: The upper histogram shows the relative control peak area as blue bars and the relative patient peak area as green bars. The lower histogram shows the ratio relative peak area as blue bars if it is within the limits and as dark blue bars if it exceeds or falls below the limits. The limits are indicated as red lines. Below the Analyse warnings are shown. If there is a warning state the dot will be red, otherwise it will be green for analyse state and grey for <> std. deviation: analyse state: shows, if peak assignment was successful. <> std. Deviation: shows, if the relative peak area (RPA) of the patient result files is between the standard deviation of the corresponding control result files multiplied by the Std. deviation Factor defined in the operation Analysis Mode [master file]. Moreover the buttons [Regression On/Off] and [show Regression] are available. The regression is used to compensate the tailing off effect (decreasing peak height with an increasing time ( = fragment length)). By default the regression is activated, but it is switched off automatically in case: no or only one ctrl probe peak(s) is/are present. it was not successful (red warning dot). The warning dot for the regression can be differently colored: grey: The regression is switched off. green: The regression was successful. Red: The regression was not successful. Below buttons are shown for all activated analysis modes which are defined in the for the used mix. You can switch to another analysis mode by pressing on the corresponding button. 11

13 7.6 Functions This dialogue part comprises different functions to edit the orders or control result files. [Previous] and [Next]: to jump to the previous or next file in your Worklist or Controllist. [T.V.] and [M.V.]: to technical and medical validate your order or control result file. [print...]: to print a report. [Extras ->]: you can enter a comment or change the order state to repeat or cancelled. [Protocol]: to open the protocol that shows all edits done in the current order. 12

14 8 Analysis of the Demo Data Copy Number Variation For the analysis it is best to use the following instructions: First load all control result files. Check the control result files and technically validate them. This way all technically validated controls of the same run date are joined to patient orders automatically during loading of patient result files. Then load all patient result files. Check if all control result files are joined to the orders. As we will see you can join other control result files manually (Files / editing / control settings...). 8.1 Control result files Before analysing our demo data, we first load, analyse and validate our controls Load MLPA control result files How to get there: Menu MLPA Item Load MLPA Resultfiles... To load the MLPA control result files of the demo data for the mixes P034 and P035, do the following: Select the menu item MLPA Load MLPA Resultfiles... Open the folder MLPA-Files\Controls, which is located in the demo folder of your installation (by default C:\SeqPilot\Demo). Select all result files. Press [Open], to load the selected result files. You can follow the process by having a look at the status bar shown at the lower right of the SEQUENCE Pilot screen: The dot will be red, if the loaded files still import. The number of remaining files to import is shown behind then. The dot will be green, if all loaded files are imported. The number behind is 0 then. Depending on the used hardware the loading of all demo result files needs approximately some seconds Controllist How to get there: Category MLPA Operation Controllist Control result files are independent from all orders and are therefore listed separately in the Controllist. If you deactivate the field Date and click on [Search], all controls are listed in this table. 13

15 For automatic assignment of the DNA number, corresponding mix, gender and type of probe, SEQUENCE Pilot needs the following information for each result file: DNA number, e.g. 4. name of the used mix, e.g. P035 (is defined in the operation Mixes [master file]) gender of the sample (F or f = female, M or m = male, U or u = unknown gender) type of result file (C or c = control result file, P or p = patient result file) digestion (D or d = digested, U, u or empty = undigested) in case of methylation mixes only family ID (optional, for patient result files only) This information is included in the sample name and has the following formate: mix name type of probe family ID (4_P035_M_C_D_1023) DNA number gender digestion All of the information is enclosed in parentheses. Within the parentheses the six sectors are separated by underscores. Any text can be placed before and after the parentheses. This text is not regarded by SEQUENCE Pilot. For our control resultfiles this information is listed in the file name (see column Filename). We do not have an entry for digestion (no methylation mix) and family ID here. Because of correct sample naming of our controls the DNA number, mix, gender and type of probe were assigned automatically. 14

16 8.1.3 Operation MLPA - Analysis of the control result files How to get there: Category MLPA Operation MLPA First, we have to edit and technically validate the control result files. A selected control of the Controllist is displayed in the operation MLPA. Therefore select the first control in the Controllist and switch to the operation MLPA First control result file (result file 4_P035_M_C.fsa) If you have a look at the Mix/Result file peak table within the tab Electropherogram you can see, that mix peaks 64, 70 and 76 are not assigned with a control result file peak. They are therefore marked red and have the entry 0.00 in the column Rf. Length. But this doesn't matter for the analysis, because these are DNA control peaks. All other mix peaks are assigned correctly to a control result file peak. 15

17 So we can technically validate this control result file, by pressing [T.V.] in the dialogue part Functions or Files: The state of the file then changes to compl. TV (see dialogue part Files, column State) Second control result file (result file 2_P035_M_C.fsa) To show the second control result file press [Next] in the dialogue part Functions. 16

18 The Mix/Result file peak table shows, that mix peaks 64, 70 and 76 are not assigned with a control result file peak. But those are DNA control peaks again. All other mix peaks are assigned. So we can technically validate this control result file, by pressing [T.V.] as done for the first control Third control result file (result file 3_P034_F_C.fsa) To show the next control result file press [Next] in the dialogue part Functions. The Mix/Result file peak table shows, that again only DNA control peaks (64, 70, 76 and 82) are not assigned with a control result file peak. So we can technically validate this control result file, by pressing [T.V.] in the dialogue part Functions or Files. 17

19 Fourth control result file (result file 1_P034_F_C.fsa) To show the last control result file press [Next]. Here again only DNA control peaks (64, 70, 76 and 82) were not assigned with a control result file peak. So we can technically validate this control result file, by pressing [T.V.] in the dialogue part Functions or Files. 8.2 Patient result files Because all control result files are technically validated now, we can load the patient result files Load MLPA patient result files How to get there: Menu MLPA Item Load MLPA Resultfiles... To load the MLPA patient result files of the demo data for the mixes P034 and P035, do the following: Select the menu item MLPA Load MLPA Resultfiles... 18

20 Open the folder MLPA-Files/Patients, which is located in the Demo-folder of your SEQUENCE Pilot installation (by default this is C:\SeqPilot\Demo). Select all result files. Press [Open], to load the selected result files Joining How to get there: Category MLPA Operation Joining All loaded patient result files are joined to an order. An order therefore includes all result files having the same DNA number. By default the operation Joining lists all result files or orders loaded at the current date. (To show orders from other dates, deactivate Date/Select Orders and press [Search]). Patient result files that could not be joined to an order or control result files that were not detected as control are shown in the Upper Table. Patient result files that could be joined to orders and are ready for analysis are shown in the Lower Table. Upper Table Select Orders Lower Table For automatic generation of orders the sample naming conventions have to be used, to ensure that the program gets all necessary data (see chapter 9.1.2). Only the DNA number is obligatory for automatic joining of result files to an order. All files with the same DNA-number are automatically joined to the same order. This is what happened to our patient result files of mixes the P034 and P035. They are ready for analysis. The file name of our patient resultfiles is shown in the column Filename (e.g. 7_P034_F_P 1111). This file name is conform to the needed sample naming conventions and gives information about DNA number (7), the used mix (P034), the gender (F=female), the type of probe (P=patient) and the family ID (1111). The entry for digestion is empty (no methylation mix) Worklist How to get there: Category MLPA Operation Worklist This operation lists all existing orders and their joined result files. The dialogue part Select Orders and the Worklist Table are the same than Select Orders and the Lower Table in Joining. 19

21 8.2.4 Operation MLPA Analysis of orders How to get there: Category MLPA Operation MLPA A selected order in the Lower Table of the operation Joining or in the Worklist is displayed in the operation MLPA. Therefore select the first order in the Worklist and switch to the operation MLPA First order (DNA number 7; result files 7_P034_M_P 1111.fsa and 7_P035_M_P 1111.fsa) Control assignments First of all we have to check, if we have to assign the control result files of the same mix to the patient result files. Technically validated control result files of the same date (and mix) are joined to orders automatically during loading of patient result files. That is why we loaded and validated our controls before loading our patient result files. In our demo data, some controls of the same mix are not of the same date as the patient result files. We have to assign those manually. Therefore select the first patient result file in the dialogue part Files. The dialogue part File Information shows that two control result files are assigned. File information Control Settings Now we check if there are more control result files which can be assigned (of the same mix but not of the same date). Therefore right-click the first patient result file in the dialogue part Files and select the item editing>control settings... from the context menu. 20

22 The dialogue Control settings consists of three tabs Date, Default and Selection. Click on the tab Selection. Now you can see all control result files of the used mix. There are only the two controls available that are already assigned. All assigned controls are marked in the column Select. Therefore just press [OK] or [Cancel]. Now switch to the second patient result file in the dialogue part Files. Here no control result files are assigned since all technically validated controls of the same mix are from another date. Therefore open the dialogue Control settings/tab Selection for the second result file. There are two controls available. To select them, check the boxes of the column Select and press [OK]. At that moment SEQUENCE Pilot reanalyses the complete result data. The control result files are now listed in the dialogue part File Information Result file 7_P034_M_P 1111.fsa Now select the first patient result file (7_P034_M_P 1111.fsa) in the dialogue Files. 21

23 The Mix/Result file peak table within the tab Electropherogram shows, that only the mix peaks 64, 70, 76 and 82 are not assigned with a patient result file peak. They are therefore marked red and have the entry 0.00 in the column Rf. Length. But this doesn't matter for the analysis, because these are DNA control peaks. So we can change to the tab Analysis. The first shown analysis mode is Female, because of the gender and the settings in the operation Analysis mode [master file] for the used mix. If the picture below looks different click once on the column header Location to sort the Result table. Moreover please press the button [Regression On/Off] in case the regression is switched off (dot behind is grey!). The dot behind should be green then as shown in the picture below. 22

24 The Result table shows deletion of exon 8 to 10. If we compare the values of the control relative peak area (RPA) with the patient relative peak area (RPA) of exon 8, 9 and 10, we can see, that the patient RPA is much smaller and outside the standard deviation of the control RPA. limits The lower histogram shows that the ratio relative peak area (patient RPA divided by control RPA) falls below the limit of 75 %. These points argue for real deletions. So we can technically validate this patient result file, by clicking [T.V.] in the dialogue part Files. 23

25 Result file 7_P035_M_P 1111.fsa Now select the next patient result file in the dialogue part File. The Mix/Result file peak table within the tab Electropherogram shows, that all mix peaks except for the DNA control peaks (64, 70, 76 and 82) are assigned correctly to a patient result file peak. 24

26 So we can change to the tab Analysis. The first shown analysis mode is Female. The Result table shows a deletion of exon 11 to 13. If we compare the values of the control RPA with the patient RPA for exon 11 to 13, we can see that the patient RPA is much smaller and outside the standard deviation of the control RPA. The ratio relative peak area (patient RPA divided by control RPA) falls below the limit of 75 % by far. This argues for real deletions. So we can technically validate this patient result file, by pressing [T.V.] in the dialogue part Files Final result and printing the report The final result for our first order is a deletion of exon 8 to 13. Because all patient result files are edited and technical validated, we can medically validate the order, by pressing [M.V.] in the dialogue part Functions. 25

27 The state of the order then changes to compl. MV and the order is in read only mode. Then we can print a report. To do this press [print...] in the dialogue part Functions. Within the following dialogue Print/Preview the diagram as well as the results of the corresponding analysis mode are already preselected. If desired you can additionally print other information. After all settings are done press the corresponding button to show a preview or to print the report. 26

28 Report: 27

29 Second order (DNA number 8; result files 8_P034_M_P 1111.fsa and 8_P035_M_P 1111.fsa ) To show the second order press [Next] in the dialogue part Functions Control assignments First of all we have to check, if the corresponding control result files are assigned. For the first patient result file no controls are assigned. Therefore right-click the file in the dialogue part Files and select editing>control settings... Now click on the tab Selection to see all technical validated control result files of the used mix. Select these control result files, by checking the column Select and press [OK]. For the second patient result file the controls are assigned already Result file 8_P034_M_P 1111.fsa If we select this patient result file and have a look at the Mix/Result file peak table within the tab Electropherogram we can see, that all mix peaks except for the DNA control peaks (64, 70, 76 and 82) are assigned correctly to a patient result file peak. So we can change to the tab Analysis. The first shown analysis mode is Male. The Result table shows a duplication of exon

30 The grey dot behind the button [Regression On/Off] shows, that the regression is switched off automatically. This is the case because the regression was not successful. Comparing the control RPA with the patient RPA for the duplication, we can see that the patient RPA is much smaller and outside the standard deviation of the control RPA. The ratio relative peak area (patient RPA divided by control RPA) exceeds the limit of 150 % by far. These points argue for a real duplication. So we technically validate this result file, by pressing [T.V.] in the dialogue part Files Result file 8_P035_M_P 1111.fsa Select the second patient result file in the dialogue part Files. The Mix/Result file peak table within the tab Electropherogram shows, that only the DNA control peaks (64, 70, 76 and 82) are not assigned with a patient result file peak. 29

31 So we can change to the tab Analysis. The first shown analysis mode is Male. The Result table shows duplications in exons 52 to

32 Here again the regression was switched off automatically because it was not successful. Comparing the values of the control RPA with the patient RPA for all duplications, we can see that the patient RPA is much smaller and outside the standard deviation of the control RPA. The ratio relative peak area (patient RPA divided by control RPA) exceeds the limit of 150 % by far. This argues for real duplications. So we can technically validate this result file, by pressing [T.V.] in the dialogue part Files Final result and printing the report The final result for our second order is a duplication from exon 52 to exon 61. Because all patient result files are edited and technically validated, we can medically validate the order, by pressing [M.V.] in the dialogue part Functions. Now we can print a report as done for our first order (chapter ). For this press [print...] in the dialogue part Functions. 31

33 Third order (DNA number 01743; result files 9_P034_F_P 1111.fsa and 9_P035_F_P 1111.fsa) To show the third order press [Next] in the dialogue part Functions Control assignments First of all we have to check if the corresponding control result files are assigned as done before. The controls for the first result file are already assigned. The controls for the second patient result file have to be assigned manually. Therefore right-click the patient result file in the dialogue part Files and select editing>control settings... Then switch to the tab Selection and select both control result files, by checking the column Select. Press [OK] Result file 9_P034_F_P 1111.fsa If we select this patient result file we can see, that all result file peaks, except for the DNA control peaks are assigned to the mix peaks correctly. 32

34 Therefore select the tab Analysis. The first shown analysis mode is Female. The Result table shows no copy number changes. So we can technically validate this result file, by pressing [T.V.] in the dialogue part Files and then have a look at the second patient result file of this order Result file 9_P035_F_P 1111.fsa Here again only the DNA control peaks 64, 70, 76 and 82 are not assigned with a patient result file peak. All other mix peaks in the Mix/Resultfile table are assigned. 33

35 So we can change to the tab Analysis. The first shown analysis mode is female. The Result table shows no copy number changes. 34

36 So we can technically validate this result file, by pressing [T.V.] in the dialogue part Files Final result and printing of a report The final result for our third order is no copy number changes. Because all patient result files are edited and technically validated, we can medically validate the order, by pressing [M.V.] in the dialogue part Functions Then we can print a report as done before (see chapter ). 35

37 9 How to make modifications To make modifications in our demo data you have to remove the state compl MV by clicking [MV] in the dialogue part Functions again. 9.1 Manual peak assignments In our demo data all peaks were assigned correctly. If this is not the case you have the possibility to assign peaks or to delete peak assignments manually in the tab Electropherogram. If you right-click a selected result file peak within the electropherogram there are two different context menus that are opened, depending on if the result file peak already is assigned to a mix peak. result file peak assigned to a mix peak result file peak not assigned to a mix peak delete peak assignment: Deletes the peak assignment. After the assignment is deleted, the result file peak in the electropherogram is highlighted grey and the cells in the columns Mix Length and Rf. Length of the corresponding mix peak in the Mix/Result file peak table are highlighted red. Furthermore the program reanalyses the result data. assign peak: To assign the peak with a mix peak from the Mix/Result file peak table, select the corresponding mix peak in the Mix/Result file peak table first and then select this item. After the assignment the result file peak in the electropherogram is highlighted blue (ctrl DNA peak), orange (ctrl probe) or yellow (gene probe peak). Furthermore the program reanalyses the result data. If you want to move an assigned peak in the electropherogram to another not assigned peak, select the peak, keep the left mouse button pressed, move it to the other peak and then release the mouse button. Here the program reanalyses result data as well. 9.2 Remove or ignore copy number changes If you have copy number changes you do not want to print in your report, they can be removed or ignored in the tab Analysis. Therefore right-click a ratio RPA, to open the following context menu: 36

38 set limit value selected peak: Changes the limits of a selected peak. If you change this setting and a peak does not exceed the limit any more the result will be removed in the result table. set limit value all peaks: Changes the limits of all peaks. ignore peak (toggle) (peaks with a result only): Ignores a peak. The corresponding entry in the column Result of the Result Table is written in parenthesis with the remark ignore in front then. To remove the state ignore, select this item again. Ignored results are not printed in the report. 10 How to analyse the order again The same result files can not be loaded twice. To load patient result files again, the order has to be deleted. Therefore select the category LIS and the operation Orderlist. 37

39 Then right-click on the first order (DNA No 7) and select the context menu item delete order. Now you can load the patient result files again as described in chapter 9.2. Control result files can be deleted in the Controllist, but only if they are not joined to an order. Therefore right-click a control result file and select delete from the context menu. 38

40 11 Archiving of processed orders How to get there: category MLPA operation Archiving Processed orders with the state compl. MV (complete medical validation) are automatically listed in the operation Archiving. This operation serves to archive orders, that is to move the electropherogram data out of the database, write them to files and save this files in a for each month of a year generated folder. All other information is still stored in the database. The database is much smaller and faster, if you archive analysed orders regularly. The state of these orders is changed to archived. 39

41 12 Analysis of Demo Data methylation For the analysis later on it is best if you load the result files as explained in the following: First load all control result files. Check the control result files and technically validate them. This way all technically validated controls of the same run date are joined to orders automatically during loading of patient result files. Then load all undigested patient result files. Check if all control result files are joined to the orders. Only technically validated controls of the same date are joined automatically. You can join other control result files manually (Files / editing / control settings...) Then load all digested patient result files. Check if all control result files are joined to the orders. If you did not load the undigested result files first, you have to reanalyse all digested result files after loading the undigested ones! (Files / editing / reanalyse) Import of Mix ME028 Open the operation Mixes [master file]. To import the master files for the MLPA mix ME028, do the following: Press [Import] and the dialogue Import file... is opened. Open the folder MLPA-Mixes, which is located in the folder C:\SeqPilot\Demo\MLPAMixes. Select the file ME028.mme and press [Open]. All data belonging to this mix (including the corresponding data from the operations Mix Sequencer [master file] and Analysis Mode [master file] are imported. If the import was successful, you get a corresponding message Controls Load MLPA control result files How to get there: Menu MLPA Item Load MLPA Resultfiles... To load the MLPA control result files of the demo data for the mix ME028, do the following: Select the menu item MLPA Load MLPA Resultfiles... Open the folder MLPA-Methylation\Controls, which is located in the folder C:\ SeqPilot\Demo. Select all result files. Press [Open], to load the selected result files Controllist How to get there: Category MLPA Operation Controllist If you deactivate the field Date and click on [Search], the following controls for methylation mix ME028 are listed in the table: 40

42 Operation MLPA - Analysis of the control result files How to get there: Category MLPA Operation MLPA First, we have to edit and technically validate the control result files. A selected control of the Controllist is displayed in the operation MLPA. Therefore select the first control in the Controllist and switch to the operation MLPA Control result files with the DNA number 5 (result files 5_ME028_M_C.fsa and 5_ME028_M_C_D.fsa) For the calculations it is necessary to join the corresponding undigested control result file to each digested control result file. This is done automatically if the sample naming is correct. Therefore for methylation mixes the undigested as well as the digested control result file are listed in the dialogue Files. There is the entry yes in the column Digested for digested result files: If we have a look at the Mix/Result file peak table within the tab Electropherogram for the undigested control, we can see, that mix peaks 64, 70 and 82 are not assigned with a control result file peak (marked red). But this doesn't matter for the analysis, because these are DNA control peaks. All other mix peaks are assigned correctly to a control result file peak. So we can technically validate this control result file, by pressing [T.V.] in the dialogue part Files. The state of the file then changes to compl. TV (see dialogue part Files, column State). Then select the digested control in the dialogue part Files. The data for the digested control is now displayed: 41

43 Here several peaks were not found. The corresponding entries are marked red in the Mix/Result file peak table. For digested control result files it is expected that no peak is found if the estimated signal is 0 %. The estimated signal is displayed in the column estimated signal of the Mix/Result file peak table. If you look through the table entries you can see, that all peaks that were not found have an estimated signal of 0 %. So we can technically validate this control result file, by pressing [T.V.] in the dialogue part Files Control result file with the DNA number 6 Press [Next] in the dialogue part Functions to open the next control result files (DNA number 6). Like for the previous files all peaks of the undigested result file are assigned correctly (except for some DNA ctrls). So we can technically validate this control result file, by pressing [T.V.] in the dialogue part Files. For the digested result file all peaks that have an estimated signal of 0 % are missing as expected (see previous chapter). So we can technically validate this control result file, by pressing [T.V.] in the dialogue part Files. 42

44 12.3 Orders Load MLPA patient result files How to get there: Menu MLPA Item Load MLPA Resultfiles... It is important to load undigested MLPA patient result files first, before loading the digested patient result files. Therefore do the following: Select the menu item MLPA Load MLPA Resultfiles... Open the folder MLPA-Methylation\Patients\Undigested, which is located in the folder C:\SeqPilot\Demo. Select the result file. Press [Open], to load the selected result file. Then select the menu item MLPA Load MLPA Resultfiles... again. Open the folder MLPA-Methylation\Patients\Digested, which is located in the folder C:\SeqPilot\Demo. Select the result file. Press [Open], to load the selected result file Joining How to get there: Category MLPA Operation Joining In the Lower Table of Joining one order and its joined result files is listed. The same order is listed in the Worklist! Operation MLPA Analysis of order 10 How to get there: Category MLPA Operation MLPA A selected order in the Lower Table of the operation Joining or in the Worklist is displayed in the operation MLPA. Therefore select the order (DNA number 10) in Joining and switch to the operation MLPA. 43

45 Order with DNA-number 10 (DNA number 10; result files 10_ME28_U_P_U_1111.fsa and 10_ME28_U_P_D_1111.fsa) The analysis of the undigested result files (first result file listed in Files) is the same as the normal copy number analysis, which was shown in the previous chapters. As you can see in the dialogue File Information both undigested control result files (DNA number 5 and 6) are joined to the undigested patient result file already, so the control settings do not have to be changed. Moreover as you can see in the electropherogram and the Mix/Result file peak table, all peaks were assigned expect for the DNA ctrl peak 74. But this does not matter since it is a DNA ctrl. Change to the tab Analysis: 44

46 No copy number changes are present. Therefore we can technically and medically validate the undigested patient result file by pressing [T.V.] and [M.V.] in the dialogue part Files. Now select the digested patient result file (second file in the dialogue part Files) and switch to the tab Electropherogram. 45

47 As you can see in the dialogue part File Information the digested as well as the undigested control resultfiles were joined to this file automatically. The Mix/Resultfile Peak Table shows that some peaks were not found, but this is expected for digested result files. Switch to the tab Analysis now. As for non methylation mixes this table shows all peaks, defined in the selected analysis mode. The column estimated signal shows the estimated methylation signal. The column RPA P. (relative peak area patient) shows the methylation of the patient result file in % whereas the column RPA C. (relative peak area control) shows the methylation of the control result file in %. The ratio RPA is the RPA P. divided by the RPA C. times 100. In case the RPA C. is 0 % the ratio RPA field is empty. The column Result shows the methylation rate for the patient as a % value, which is the RPA P. divided by the RPA C. times 100 (= ratio RPA). In case the RPA C. is 0 % the Result is: 100 % (meth. absent) if the RPA P. is also 0 %. >100 % (meth. present) if the RPA P. is more than 0 %. All other entries and functions are the same as for non methylation mixes. As for non methylation mixes the results is also shown in the form of a histogram (sort the table by clicking on the table header Gene to get the sorting shown below). 46

48 The methylation of the control (RPA C) is shown as blue bars whereas the methylation of the patient (RPA P.) is shown as green bars in the upper histogram. The ratiorpa is shown as blue bars in the lower histogram. If the ratio RPA (or the Result) is around 100 % the methylation rate of the patient is about the methylation rate of the control. Values below 100 % show that the patient has a lower methylation rate than the control, values above 100 % indicate that the patient has a higher methylation rate than the control. For the analysis of methylation there are no limits and no context menu available in the lower histogram as for non methylation mixes! Final result and printing of a report In our example the sample has a lower methylation rate compared to the control for some locations. To finish the analysis you can technically and medically validate the second file and print a Report as described before. For a detailed description have a look at the Manual SEQUENCE Pilot module MLPA. 47