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PyroMark : Analysis Software Guide

1. Note The value cannot be lower than the required peak height for check quality value Required percentage The minimum signal value for a peak to achieve for check quality Check quality assessment for the variable positions The warning Uncertain due to low peak height is triggered during the analysis Note This rule is only used if the rule for Passed quality is not met The default value is 10 A signal value for a peak that is lower than the set value will result in a Failed quality assessment tor the variable positions The warning Failed due to low peak height is triggered during the analysis Note The value cannot be higher than the required peak height for passed quality value 22 PyroMark Q24 Analysis Software User Guide 01 2009 Stringency levels The stringency of the warnings for Pattern deviation in variable positions and Sum deviation in variable positions can be set to Low Normal default or High A high stringency level narrows the allowed deviation Pattern in deviation in The deviation between the measured peak pattern in variable positions the variable position and the theoretical peak pattern It the deviation is higher than the set stringency level allows the warning Uncertain Failed due to high pattern deviation in variable position is triggered during the analysis Whether the warning will yield a Check or Failed quality assessment for the analysis r
2. PyroMark Q24 Analysis Software User Guide 01 2009 25 Add assay files To add an assay to a well you can either E Right click the well and select Load Assay from the context menu E Select the assay in the shortcut browser and press and hold down the left mouse button while you drag the assay to the well Note If you wish to add an assay to several wells select the wells see Select Wells page 36 and drag the assay to the selection Note It is not possible to add an assay with no dispensation order or add two or more assays that share the same assay name but have different dispensation orders Plate Setup A well is colored according to the assay loaded to the well Enter sample IDs and notes E To enter a sample ID or note select the cell and enter the text E To edit a sample ID or note either select the cell the current contents will be selected or double click the cell E To import a sample and note layout defined in a text file tsv or csv right click a well and select Insert Sample Layout File trom the context menu For more information see Define Sample ID ad Note Externally page 28 E To paste a sample layout trom the clipboard right click a well and select Paste Sample Layout from the context menu For more information see Define Sample ID ad Note Externally page 28 A selected cell is highlighted with a blue background color 26 PyroMark Q24 Analysis Software User Guide 01 20
3. Select New Run from the context menu Enter the filename and press Enter To set up a run see Run Setup page 24 Copy a processed run file and rerun it by right clicking the run tile and selecting Copy and Rerun from the context menu The process and analysis data will not be copied PyroMark Q24 Analysis Software User Guide 01 2009 E Copy a tile using Windows Explorer by right clicking the folder containing the tile and selecting Explore from the context menu Windows Explorer opens For more information press the F1 key to open the online help Note To avoid losing data do note copy a tile that is open in PyroMark Q24 Software E Open a tile by double clicking it or right click the file and select Open trom the context menu fo open a processed run tile select Open with and then select the analysis mode AQ or CpG E The following icons are used to display information about the files AQ assay file CpG assay tile A run file that has not been processed A run tile that has been processed xado Broken shortcut This may be due to a network server thot is temporarily inaccessible or that the file or the folder has been moved renamed or deleted outside the software Main Menu and Toolbars File menu and toolbars Select AQ Assay or CpG Assay from the New faci pe ia Assay submenu or click in the toolbar and select New AQ Assay or New CpG A
4. Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 1544 7145 Fax 1544 7146 Technical 1544 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1122 330 Technical 01 800 7742 639 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 65 67775366 Fax 65 67785177 Technical 65 67775366 Spain Orders 91 630 7050 Fax 91 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 QIAGEN Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 P Sample amp Assay Technologies
5. SNP overview report 46 Manage Instrument Methods 47 Method parameters 47 Troubleshooting Guide 48 References 48 E 4 PyroMark Q24 Analysis Software User Guide 01 2009 Product Use Limitations Use the PyroMark Q24 Analysis Software only with the PyroMark Q24 Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product tail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or retund the purchase price We reserve the right to change alter or modity any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire tor more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specitications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www qgiagen com Technical Assistance At QIAGEN we pride ourselves on the quality and availability of o
6. and 2 Check quality assessment for the CpG sites The entered values are compared to the single peak height value that the analysis algorithm determines Allowed percentage for The highest acceptable percentages of unconverted passed quality sequence to achieve Passed quality assessment for the CpG sites The default value is 5 Note The value cannot be higher than the allowed percentage for check quality value PyroMark Q24 Analysis Software User Guide 01 2009 21 Allowed percentage for The highest acceptable percentages of unconverted check quality sequence to achieve Check quality assessment for the CpG sites The warning Uncertain bisulfite conversion at dispensation is triggered during the analysis Note This rule is only used if the rule for Passed quality is not met A higher percentage of unconverted sequence than the set value will result in a Failed quality assessment for all CpG sites The warning Failed bisulfite conversion at dispensation is triggered during the analysis The default value is 7 Note The value cannot be lower than the allowed percentage for passed quality value Peak height threshold These parameters set the lower intensity limit for the single peak level of Pyrogram Required peak height The minimum signal value tor a peak to achieve for passed quality Passed quality assessment for the variable positions The default value is 20
7. cell 2 To increment by row Position the mouse pointer over the lower right square of the selection Press and hold down the Ctrl key the left mouse button while moving the mouse to change the selection First release the left mouse button then the Ctrl key When the left mouse button is released the sample ID of the first selected cell is incremented and pasted into the selected cells PyroMark Q24 Analysis Software User Guide 01 2009 27 3 To increment by column Position the mouse pointer over the lower right square of the selection Press and hold down the Shift and Ctrl keys the left mouse button while moving the mouse to change the selection First release the left mouse button then the Shift and Ctrl keys When the left mouse button is released the sample ID of the first selected cell is incremented and pasted into the selected cells AG Assay 1 Sample 3 Tan Assay 1 Sample 4 The sample ID Sample 1 is copied and incremented by column Print or export plate setup as image The Plate Setup can be printed or copied as an image to the clipboard by right clicking the plate and selecting Print or Copy as Image from the context menu The image can be pasted into applications that support Enhanced Metafile EMF images To print a list of required volumes of reagents enzyme mix substrate mix and nucleotides and the plate setup select Pre Run In
8. computer Adobe Acrobat Reader is available on the PyroMark Q24 Software CD but can also be downloaded at www adobe com 46 PyroMark Q24 Analysis Software User Guide 01 2009 Manage Instrument Methods Window Instrument Methods Volume Information The instrument method shall be selected according to the reagents and reagent cartridge that will be used for the run see the instructions supplied with the products Note We recommend that only methods supplied by QIAGEN are used To import a new method 1 In the Instrument Methods dialog click Import The Find Instrument Method dialog appears 2 Locate and select the method that you wish to import and click Open To create a new method 1 In the Instrument Method dialog select an existing method and click Save As 2 Enter a name for the method and press Enter 3 Change the settings and click Save Method parameters In the Instrument Methods dialog the tollowing parameters are available Reagent pressure Pressure millibar during dispensation of the enzyme mix and substrate mix Enzyme pulse time Dispensation time milliseconds for the enzyme mix Substrate pulse time Dispensation time milliseconds for the substrate mix Nucleotide pressure Pressure millibar during the dispensation of nucleotides Nucleotide pulse time Dispensation time milliseconds for nucleotides Note Optional Note on the in
9. frequently the file is saved the more information is recovered if there is a power failure or similar problem while the file is open To save the Tile click lal in the toolbar Overview tab At the Overview tab in the analysis view you can analyze the run get an overview of the results compare Pyrograms of different wells and enter an analysis note Analyze There are two ways to perform the analysis Hifi Analyze all wells with a valid analysis setup for the selected analysis mode Om Analyze the selected wells see Select Wells page 36 Note It is also possible to right click the selection and select Analyze Selected from the context menu During the analysis a progress dialog is shown This dialog contains a progress bar a stop button and the name of the well that is being analyzed The analysis is stopped by clicking the Stop button PyroMark Q24 Analysis Software User Guide 01 2009 31 AQ assays are analyzed in the AQ mode and CpG assays are analyzed in the CpG mode To toggle between the analysis modes select AQ or CpG in the toolbar Note Since the CoG mode does not support automatic analysis of SNPs methylation percentages and quality assessments are only determined for the CpG sites SNPs in CpG assays can be analyzed in the AQ mode using the CpG setups select AQ in the toolbar select the wells and click G28 the Analyze Selected Wells button To exclude the Cp
10. mode AQ or CpG and analysis version results including any warnings date and time and the Windows user account used to perform the analysis see the General Hints and Tips section Text files tsv or csv can be imported into Microsoft Excel or other applications that can handle PyroMark Q24 Analysis Software User Guide 01 2009 Reports menu for CpG runs Window Reports Help CpG Analysis Statistics CpG nalysis Results Cp Pyrogram Report Cp Full Report Reports menu for AQ runs Window Reports Help AQ Analysis Statistics 40 Analysis Results 40 Pyrogram Report AQ Full Report SMP Analysis Results SNP Pyrogram Report SMP Full Report SNP Overview Report Window menu Window 1 CpG Run Analysis C119 Cp6 example run z CpG assay 0 1441561F5 3 40 Run Analysis 01 40 example run 4 Run Setup Untitled 5 AQ Assay Untitled Help menu 2 PyroMark Q24 Software Help PyroMark Q24 User Documentation About PyroMark G24 data that is separated by semicolons or tabs This is useful when doing turther calculations on the data The Analysis Statistics report includes analysis statistics for all or selected wells The Analysis Results report includes well intormation and analysis results tor all or selected wells The Pyrogram Report includes well information and Pyrogram for all or selected well
11. qiagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor 48 PyroMark Q24 Analysis Software User Guide 01 2009 Notes PyroMark Q24 Analysis Software User Guide 01 2009 49 Notes 50 PyroMark Q24 Analysis Software User Guide 01 2009 Trademarks QIAGEN Pyrogram Pyrosequencing QIAGEN Group Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the PyroMark Q24 to the following terms 1 The PyroMark Q24 Analysis Software may be used solely in accordance with the PyroMark Q24 Analysis Software User Guide and for use with components contained with the Software only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Software with any components not included within this Software except as described in the PyroMark Q24 User Guide and additional protocols available at www giagen com Other than expressly stated licenses QIAGEN makes no warranty that this Software and or its use s do not infringe the rights of third parties This Software and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated er r ee o The purchaser and user of the Software agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibite
12. the mouse pointer over the diamond and a tooltip will describe the consequence of a click Zoom histogram It is possible to zoom in on the histogram by selecting an aspect of it with the left mouse button Zoom out either by right clicking the histogram area and selecting Zoom Out from the context menu the zoom is set to the previous level or by double clicking the histogram area the zoom is set to 100 Adjust heights of histogram bars This feature is normally not used but can be used when previous experiences have shown a reproducible deviation in the measured pattern from the theoretical pattern 18 PyroMark Q24 Analysis Software User Guide 01 2009 1 Press and hold down the Cirl key while left clicking the top of the histogram bar left click when the pointer changes from a white arrow to 2 Either enter the height in the text box that appears or increase or decrease the height by using the arrows next to the text box 3 To apply the new height press Enter Note Instead of removing nonstandard methylation patterns from the sequence to analyze for example methylations of Cs that are not followed by Gs set the expected heights of the Gs to zero 0 Orange decreased height Blue increased height Export histogram as image The histogram can be copied as an image to the clipboard by right clicking the histogram and selecting Copy as Image from the context menu The image
13. with a blue gray background color E Reference peaks are marked with blue diamonds over the histogram bars E Bisulfite treatment controls are marked with orange diamonds over the histogram bars and highlighted with a yellow background color CpG assays only Add or remove bisulfite treatment controls Add Bisulfite Treatment Control Before Dispensation Add Bisulfite Treatment Control After Dispensation CpG assays should contain at least one internal control to assess successful bisulfite treatment preferably at the beginning of the sequence C bases that are not followed by G in the sequence are usually not methylated and should therefore be fully converted to T after bisulfite treatment and PCR As a result of successful bisulfite treatment all templates should show only Ts and no Cs in these positions For reverse assays all templates should show only As and no Gs in these positions The potential positions for bisulfite treatment controls are illustrated with a bold orange letter T in a forward assay and A in a reverse assay A bisulfite treatment control can be added by left clicking the bold orange T or A and selecting the desired option from the context menu A bisulfite treatment control can be removed by left clicking the control C in a forward assay or G in a reverse assay and selecting Remove Bisulfite Control from the context menu Note In the sequence before bisulfite treatment check whether the suggested bis
14. 09 Copy or delete contents from cells E To cut the contents of a cell to the clipboard right click the cell and select Cut trom the context menu E To copy the contents of a cell to the clipboard either right click the cell and select Copy Cell from the context menu or select the cell and press Ctrl C E To paste the clipboard to a cell or a selection of cells see Select Wells page 36 either right click the cell or the selection and select Paste from the context menu or select the cell s and press Ctrl V E To delete one or more assays sample IDs or notes either right click the cell or the selection and select Delete from the context menu or select the cell s and press Delete Drag copy To drag copy the contents of a cell to other wells 1 Select the cell that you wish to copy 2 Position the mouse pointer over the lower right square of the selection and press and hold down the left mouse button while you move the mouse to change the selection 3 When the left mouse button is released the contents of the first selected cell are pasted into the selected cells CpG Assay CpG Assay 2 CoG Assay CpG Assay 2 25 pl PCR prod i 25 ul PCR prod 25 pl PCR prod Drag copy of the note 25 ul PCR prod Drag copy and increment sample ID If the last part of an entered sample ID is a number the number can be incremented when drag copying the sample ID 1 Select the sample ID
15. ATT GA TC AGTE GT ATOT C GTA z i 15 20 25 30 The following information icons and colors are displayed and used in the Pyrogram area E The well name and the sequence to analyze are shown in the upper left corner E The analysis result the allele frequencies or the methylation percentage is displayed above each variable position tor example 26 The background color shows the quality assessment of the analysis result see Color Legend page 35 If the mouse pointer is positioned over the analysis result a tooltip displays the position number and any analysis warnings Note Deselected by the user 4 in white The software does not support analysis for example SNP when in the CoG mode 4 in red Not possible to analyze due to lack of data E Variable positions are highlighted with a blue gray background color E The theoretical histogram is gray and positioned over the peaks It is best viewed when zoomed in E Reference peaks are marked with blue diamonds over the peaks For more intormation see Histogram page 17 Bisulfite treatment controls are marked with orange diamonds over the peaks and highlighted with a light yellow background color For more information tor CpG assays only see Histogram page 17 View histogram and or reference peaks You can toggle between viewing and hiding the theoretical histogram or the reference peaks by right clicking the Pyrogram area and selecting Show Histogram
16. Analysis Statistics AQ Analysis Results AQ Pyrogram Report AQ Full Report SNP Analysis Results SNP Pyrogram Report SNP Full Report SNP Overview Report Reports Window Help AQ Analysis Statistics AQ Analysis Results 40 Pyrogram Report AQ Full Report SNP Analysis Results SNP Pyrogram Report SMP Full Report SMP Overview Report Reports Window Help CpG Anahsis Statistics Cp Analysis Results CpG Pyrogram Report CpG Full Report The Full report includes the following information for or all or selected wells see Select Wells page 36 E Run parameters run name run date and time instrument method operator plate ID barcode reagent ID and run note and a run log Information on whether the analysis settings have been changed by the user or not Wells with changed analysis settings are listed E Well information assay name Sample ID and well note analysis version and sequence to analyze Analysis results for all or selected wells E Pyrogram For information on icons and colors used in the Pyrogram area see Pyrogram Report page 44 E The allele frequencies AQ report genotypes SNP report or methylation percentages CpG report and the quality assessments for the variable positions E The mean methylation percentage of all CpG sites except tailed in the well CoG report E Any analysis warnings Report options In the Full Report dialog you c
17. G sites in the SNP reports turn off clear the Analyze option for these positions at the Analysis Setup tab see page 34 Well colors The color of the well indicates whether a well has been analyzed or not E Beige Not analyzed or not used Either the well has not been analyzed or the assay has no valid analysis setup for the selected analysis mode E Lightblue Analyzed View analysis results By selecting an analyzed well in the plate overview its Pyrogram and histogram are displayed in the lower part of the window Well information including any analysis warnings is listed in the Well Information area For more information see Well Information Area page 39 and Pyrogram Details page 37 The following well information can be viewed in the plate overview Select to show the assay name acer f Select to show the sample ID ee Select to show the well note Hean Select to show the quality bar The quality bar shows the quality assessment of all variable positions in the well For more information see Color Legend page 35 Ranges Select to show the mean methylation percentage of all CoG sites except for failed in the well Only available when in the CpG mode 32 PyroMark Q24 Analysis Software User Guide 01 2009 Select to show the methylation bar The methylation bar shows the methylation level for each CpG site in the well For more information see Color Legend page 35 Only available wh
18. January 2009 PyroMark Q24 Analysis Software User Guide For use with the PyroMark Q24 QIAGEN Sample amp Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 5 Introduction 6 About this user guide 6 PyroMark Q24 Analysis Software 7 Analysis Modes 7 Shortcut Browser 8 Main Menu and Toolbars 9 General Hints and Tips 12 Protection of files 12 Validation of assays 13 Assay Setup 13 Workflow 13 Sequence to analyze 14 Variable positions tab 19 Analysis parameters tab 21 Run setup 24 Workflow 24 Run parameters 24 Plate setup 25 Run 30 Workflow 30 Analysis 30 Workflow 30 Overview tab 31 Analysis setup tab 34 Color legend 35 Reports 40 Analysis statistics report 4 Analysis results report 42 PyroMark Q24 Analysis Software User Guide 01 2009 3 Pyrogram report 44 Full report A5
19. To add a shortcut to a folder or drive click Add Folder Shortcut Note An assay note can be displayed in the shortcut browser by positioning the mouse pointer over the assay file Note The more frequently the file is saved the more information is recovered if there is a power failure or similar problem while the file is open To save the file click ll in the toolbar If the file has never been saved select location and name of the file in the dialog that appears PyroMark Q24 Analysis Software User Guide 01 2009 13 Sequence to analyze Type or paste Ctrl V the sequence to analyze into the Sequence to Analyze text box If creating a CoG assay enter the sequence after the bisulfite treatment Variable positions can be entered using either IUPAC codes or a forward slash as a separator between the two potential bases for example C T If the sequence to analyze contains an error this is displayed by a red exclamation mark at the end of the text box Position the mouse pointer over the exclamation mark and a tooltip will display an explanation of the error The character or characters that caused the error will be red Sequence to 4nalze TYGT T nvalid sequence As T T is not a valid variable position it causes an Invalid sequence error Note If you are analyzing nonstandard methylation patterns for example methylations of Cs that are not followed by Gs these patterns can be analyzed in t
20. a column with the analysis version is included If this option is checked a column with the names of the variable positions is included It this option is checked a column with the original quality assessments for the variable positions is included It this option is checked a column with the current quality assessments for the variable positions is included If this option is checked a column with information on whether the current quality assessments for the variable positions have been edited by the user Yes or not is included If you wish to view the report before you save or print it click Preview PyroMark Q24 Analysis Software User Guide 01 2009 43 Pyrogram report Reports Window Help AQ Analysis Statistics AQ Analysis Results AQ Analysis Statistics AQ Analysis Results AQ Full Report AQ Full Report Reports Window Help SNF Analysis Results SNP Analysis Results CpG Analysis Statistics SNF Pyrogram Report CpG Analysis Results SNP Full Report SNP Full Report SNF Overview Report SNP Overview Report CpG Full Report The Pyrogram report includes well information assay name sample ID and well note and Pyrogram for all or selected wells see Select Wells page 36 The following information icons and colors are displayed and used in the Pyrogram area E The well name and the sequence to analyze are shown in the upper left corner E The analysis result allele frequencie
21. ade in the histogram at the Analysis Setup tab click Apply 3 In the Apply Analysis Setup dialog either apply the changes to All wells that share the same assay and dispensation order as the displayed well i e all the white wells in the Apply Analysis Setup or the selected wells i e the white wells that are selected in the Apply Analysis Setup dialog by clicking To Selected Note The changes can only be applied to wells that share the same assay and dispensation order as the displayed well During the analysis a progress dialog is shown This dialog contains a progress bar a stop button and the name of the well that is being analyzed The analysis is stopped by clicking Stop 38 PyroMark Q24 Analysis Software User Guide 01 2009 Note All saved changes are logged To view the analysis log tor a selected well select Analysis Log from the Tools menu Edit quality assessments Quality assessments can be edited by left clicking the analysis result the allele frequencies or the methylation percentage in Pyrogram and selecting Passed Check or Failed from the context menu If a quality assessment has been edited by the user this is displayed in Pyrogram by a border around the analysis result for example E and in the plate overview or navigator by a black square in the well for example Note All saved changes are logged To view the analysis log tor a sele
22. an select the wells to be included in the report All wells or Selected wells If you wish to view the report before you save or print it click Preview Note In order to view the report you must have Adobe Acrobat Reader installed on your computer Adobe Acrobat Reader is available on the PyroMark Q24 Software CD but can also be downloaded at www adobe com PyroMark Q24 Analysis Software User Guide 01 2009 45 SNP overview report Window Reports Help 40 Analysis Statistics 40 Analysis Results 40 Pyrogram Report 40 Full Report SNP Analysis Results SAP P rogram Report SMP Full Report SAP Overview Report The SNP Overview report includes genotypes and quality assessments for all SNPs The information is presented in plate overviews with one plate per position number The background color of the wells shows the quality assessment of the SNP see Color Legend page 35 Position 4 Position 5 l 2 3 4 5 6 7 5 A B c Extract from a report The Analyze option has been turned off for position 5 in well B1 Well B2 B3 and B7 B8 have no SNPs in position 5 Note Variable positions can be excluded from the report by turning off the Analyze option at the Analysis Setup tab see Analysis Setup Tab page 34 Note The SNP Overview report is only available when in the AQ mode In order to view the report you must have Adobe Acrobat Reader installed on your
23. and the lowest methylation percentage in a well CpG report only Optional The analysis version the names of the variable positions information on whether the quality assessments have been edited by the user or not the original and or the current quality assessments for the variable positions and any analysis warnings The report can be saved as a text file tsv or csv or an HTML file html The report can be imported into Microsoft Excel or other applications that can handle text files tsv or csv with data that is separated by semicolons or tabs This is useful when doing further calculations on the data The first line in the report states the name of the run The following two or three lines contain the column headings Each of the lines following the column headings contains detailed well information and statistics of a specitied well 42 PyroMark Q24 Analysis Software User Guide 01 2009 Report options In the Analysis Results Report dialog there are the following options All wells selected wells Row column Note column Warning columns Analysis version column Position name columns Original quality columns Quality columns Edited quality columns The results to be included in the report The sorting order of the wells If this option is checked a column with well notes is included It this option is checked a column with any analysis warnings is included It this option is checked
24. aoeks amp amp fy 0 C f amp Notes for well A3 Sample ID Note DNA 342 Notes for well Al DNA 360 Notes for well A3 l M 4 gt gt Sample Layout Example Ready An example of a sample and note layout file created in Microsoft Excel Plate Setup DNA 360 Hotes for well 41 Notes for well A3 The result when importing the sample and note layout tile above Using the paste sample layout feature You can tor example generate and copy layouts trom your LIMS Sample layouts can be copied from Microsoft Excel Word Notepad and similar applications In the source file each column of sample IDs must be delimited by a tab and each row of sample IDs must be delimited by a line break The sample layout can be pasted into an existing run file by copying all the intormation in the source file and right clicking a well in the Plate Setup and selecting Paste Sample Layout from the context menu The software will paste the sample IDs into the plate starting at well Al If any well notes have been entered into the wells these are kept P Paste Sample Layout Example txt Notepad Sel File Edit Format View Help 324 An example of a sample layout created in Microsoft Notepad PyroMark Q24 Analysis Software User Guide 01 2009 29 Plate Setup The result when copying and pasting the sample layout above Run Workflow When a run is set up and ready to run on the PyroMark Q24 per
25. are the same assay and dispensation order as the displayed well i e all the white wells in the Apply Analysis Setup dialog click To All 5 To apply the changes to the selected wells i e the white wells that are selected in the Apply Analysis Setup dialog click To Selected Note The changes can only be applied to wells that share the same assay and dispensation order as the displayed well During the analysis a progress dialog is shown The dialog contains a progress bar a stop button and the name of the well that is being analyzed The analysis can be stopped by clicking Stop 6 To save the changes click H Note All saved changes are logged To view the analysis log tor a selected well select Analysis Log from the Tools menu 34 PyroMark Q24 Analysis Software User Guide 01 2009 Use modified assay in other runs Changes made at the Analysis Setup tab will not be saved in the original assay file To use the modified assay in other runs 1 In the plate navigator select a well that is using the modified assay and click Save Assay The Save Assay As dialog appears 2 Save the changes to the original file or save the modified assay as a new file Select destination folder from the Save in drop down list Enter filename in the File name text box and click Save View analysis results By selecting a well in the plate navigator its Pyrogram is
26. can be pasted into applications that support Enhanced Metatile EMF images Variable positions tab The Variable Positions tab contains information about the variable positions Note If the sequence to analyze is changed and a new dispensation order is generated the variable position parameters are reset to their detault values Position The location of the variable position in the sequence to analyze counting from left to right Name The name of the variable position To change the name either select the text box the current contents will be selected or double click the text box Type CpG assays only The type of variable position SNP or CpG site Note The CoG mode does not support analysis of SNPs PyroMark Q24 Analysis Software User Guide 01 2009 19 Analyze It this option is checked the variable position will be analyzed Note This option is not available for variable positions that cannot be analyzed using the selected analysis mode Methylation ranges The expected CpG methylation Setting this parameter for all the CpG sites allows easy identification of sites in the analysis results that are outside the expected methylation range Note The expected methylation cannot be set for CpG sites with the Analyze option cleared The light green area is below the expected range The green area is within the expected range This area can be moved to the left or to the right by holding down the left mous
27. collapse the Note field click or SSS SOE PyroMark Q24 Analysis Software User Guide 01 2009 33 Analysis setup tab At the Analysis Setup tab in the analysis view it is possible to modity how the analysis is performed Change analysis settings Moditied analysis settings can be applied to selected wells or all wells that share the same assay and dispensation order as the displayed well 1 In the plate navigator or overview select the well or wells see Select Wells page 36 for which you wish to change the analysis settings Only select wells with the same assay and dispensation order Note If you wish to change the analysis settings for all wells with the same assay and dispensation order you only have to select one of the wells 2 Change analysis settings at the Analysis Setup tab To enable or disable variable positions turn the Analyze option on or off and or change expected methylation ranges only CpG assays see Variable Positions Tab page 19 To enable or disable reference peaks and or bisulfite treatment controls only CpG assays or adjust the heights of the histogram bars see Histogram pagel 7 To change other analysis settings see Analysis Parameters Tab page 21 Note It is not possible to change the assay name dispensation order or assay note 3 When finished click Apply The Apply Analysis Setup dialog appears 4 To apply the changes to all wells that sh
28. cted well select Analysis Log from the Tools menu Export Pyrogram as image Pyrogram can be copied as an image to the clipboard by right clicking the Pyrogram area and selecting Copy as Image trom the context menu The image can be pasted into applications that support Enhanced Metatile EMF images Well information area The Well Information area shows the following information about an analyzed well that is selected in the plate overview or navigator Well name Assay name Sample ID if entered Well note if entered E Any analysis warnings If several wells are selected in the plate overview or navigator the information for the first selected well the well with the orange selection frame is shown n Note Wells with a high substrate peak will be marked with an intormation icon in the plate overview This will not affect the quality assessments Analysis warnings An analysis warning affects the quality assessment for either all variable positions or a single position If several warnings were triggered only the mos serious ones are displayed in the Well Intormation area PyroMark Q24 Analysis Software User Guide 01 2009 t 39 For some of the warnings the criteria for occurrence and the effect on the quality assessment can be moditied by the user see Analysis Parameters Tab page 21 Note If a dispensation warning is triggered we recommend replacing the reagent car
29. d above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Software and or its components For updated license terms see www giagen com 2009 QIAGEN all rights reserved www giagen com Australia Orders 03 9840 9800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 m Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 0086 21 3865 3865 Fax 0086 21 3865 3965 Technical 800 988 0325 800 988 0327 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 m Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 02 33430 420 Fax 02 33430 426 Technical 800 787980 Japan
30. de and change C TG to A C TG in the Sequence to Analyze text box and analyze the variable position In the same way C TG A can be analyzed by typing C TG in the Sequence to Analyze text box and TCGA in the Dispensation Order text box After analysis of the CpG sites switch to the AQ mode and change C TG to C TG SA in the Sequence to Analyze text box and analyze the variable position Dispensation order A dispensation order for the entered sequence to analyze is generated by the software by clicking the Generate Dispensation Order button The generated dispensation order includes blank dispensations to ensure that the correct sequence has been obtained When creating CpG assays the dispensation order should also include bisulfite treatment controls These controls have to be added manually by the user after the dispensation order has been generated see Histogram page 17 E PyroMark Q24 Analysis Software User Guide 01 2009 15 It desired the dispensation order can be entered manually Note When clicking Generate Dispensation Order any existing dispensation order will be overwritten Note When a base position is selected in the sequence to analyze the corresponding dispensation is highlighted with a gray background color and vice versa Sequence to Analyze fr GYGGTGYGTATYGTTTGRGAT Generate Dispensation Order Dispensation Order GTCTGTCG TAGTCGCTGATCOTAGTEGS Dispensation wa
31. displayed in the Pyrogram area and well information including any analysis warnings is listed in the Well Information area If several wells are selected in the plate navigator information for the first selected well the well with the orange selection frame is shown For more information see Well Information Area 39 and Pyrogram Details page 37 Color legend Quality assessments E Quality bars in the plate overview at the Overview tab If the quality bars are not shown click one of the three at the top right corner and select from the drop down list E The background color of the analysis results the allele frequencies or the methylation percentages in Pyrogram for example 25 If a quality assessment has been edited by the user this is displayed in Pyrogram by a border around the analysis result for example F and in the plate overview or navigator by a black square in the well for example For instructions on how to edit a quality assessment see Edit Quality Assessments page 39 Off white means not analyzed either analysis is not supported by the software e g SNP when in the CoG mode or the variable position has been deselected by the user blue means passed yellow means check red means failed PyroMark Q24 Analysis Software User Guide 01 2009 35 Methylation levels When in the CoG mode a methylation bar at the Overview tab shows the methylation level tor each CpG site in the well To view th
32. e a copy of the current file Select location and name of the file in the dialog box Select Exit to shut down the software Select Instrument Methods to view the settings for the instrument methods and if necessary import or set up new methods according to settings supplied by Pyrosequencing see Manage Instrument Methods page 47 Select Pre Run Information to view the required volumes of the enzyme mix substrate mix and nucleotides and the plate setup for the current run file To print the report click amp Note To print the report in color turn on the Print background colors and images option in the Internet Explorer Tools Internet Options Advanced Printing Select Run Information to view and print the run parameters and a run log for the current run file To print the report click amp Select Export Peak Heights to save the peak heights of all used wells as a text file Select Export Environment Data to save the mixer block temperature and pressure readings as a text tile The temperatures of the environment the process chamber lid and the cooler are also listed Select Export Raw Data to save the intensities and dispensation substances and moments as a text file Select Analysis Log to save the log with all saved analyses performed on the selected well as a text file or an HTML file Each analysis is logged with the used analysis settings analysis
33. e button while moving the area with the mouse The dark green area is above the expected range The arrows can be used to increase or decrease the expected range Note You can also increase or decrease the expected range by E Positioning the mouse pointer over the left or the right end of the green area so that the pointer changes trom a white arrow to E Moving the mouse to the left or the right while holding down the left mouse button To edit all methylation ranges simultaneously hold down the Shift key while changing one of the ranges eee 20 PyroMark Q24 Analysis Software User Guide 01 2009 Example of methylation ranges il I 100 100 2 F ails _ q gt dab dep 4 Ex 1 Expected methylation 100 Ex 2 Expected methylation 0 100 Ex 3 Expected methylation 30 70 default Ex 4 Expected methylation 0 To reset the parameters at the Variable Positions tab and the Analysis Parameters tab to their default values click Revert to Default Analysis parameters tab It applicable after the assay validation the following analysis parameters can be edited at the Analysis Parameters tab Ensure changes are validated see Appendix B of the PyroMark Q24 User Manual page B 1 Unsuccesstul bisultite These parameters state the highest acceptable treatment CpG assays percentages of unconverted sequence to achieve 1 only Passed quality assessment
34. e has never been saved select location and name of the file in the dialog that appears eee 12 PyroMark Q24 Analysis Software User Guide 01 2009 Validation of assays Validate your assays using reference samples see Appendix B of the PyroMark Q24 User Manual page B 1 Assay Setup Workflow Hew AQ Assay Mew CpG assay 1 Click in the toolbar and select New AQ Assay or New CpG Assay A new assay file is created 2 Enter the sequence to analyze see Sequence to Analyze page 14 3 Click the Generate Dispensation Order button see Dispensation Order page 15 4 Optional If creating a CpG assay enter the Sequence Before Bisulfite Treatment This information is useful when adding bisulfite treatment controls 5 Recommended If creating a CpG assay add bisulfite treatment controls preferable in the beginning of the sequence see Histogram page 17 6 Optional Enter information about the assay in the Note text box 7 Optional Set up the variable positions see Variable Positions Tab page 19 8 Before running your samples validate your assay using reference samples see Appendix B of the PyroMark Q24 User Manual page B 1 Note In the shortcut browser you can create a new assay file by right clicking the folder you wish to place it in and selecting New Assay and then AQ Assay or CpG Assay from the context menu Enter the filename and press Enter
35. e methylation bars in the plate overview click one of the three w at the top right corner and select from the drop down list E Light green Below the defined range E Green Within the defined range E Dark green Above the defined range Select wells To select a single well simply click on it CX OOO 00000000 BO OOOOOOO To select a rectangular group of wells for example A2 A3 B2 B3 and C2 C3 E Press and hold down the left mouse button while dragging the mouse pointer from well A2 to C3 or E Select well A2 and press and hold down the Shift key while selecting well C3 or E Select well A2 and press and hold down the Shift key while pressing the Right Arrow key once and the Down Arrow key twice 100000000 To add wells to the selection above for example wells BZ and C7 press and hold down the Ctrl key while selecting the wells PEPEPEPE 00000000 BO OOK FO To deselect a well press and hold down the Ctrl key while selecting the well Note It several wells are selected in the plate overview or navigator information for the first selected well the well with the orange selection frame is shown in the Well Information area Pyrogram area etc 36 PyroMark Q24 Analysis Software User Guide 01 2009 Pyrogram details A5 YGGATAGYGATTTTTAAYGYGTAAGYGTATA zoom Cur lal Copy as Image Show Histogram Show Reference Peaks E 5 amp 5 TCECGACTATET C G
36. ed for the run see the instructions supplied with the products Note We recommend that only methods supplied by QIAGEN are used 24 PyroMark Q24 Analysis Software User Guide 01 2009 Plate ID Optional Enter ID of the PyroMark Q24 Plate Note It you position the mouse pointer over a run file in the shortcut browser a tooltip displays the entered plate ID Barcode Optional Enter a barcode number for the plate or if you have a barcode reader connected to your computer place the mouse cursor in the Barcode text box by clicking the box and scan the barcode Reagent ID Optional Enter the lot number tor the PyroMark Q24 Gold Reagents to be used The lot number can be found on the product label Note We recommend this is entered so that any unexpected problems with the reagents can be traced Estimated run time The estimated run time Run name The name of the run is given when the file is saved Renaming the tile also changes the name of the run Run note Optional Enter a note about the contents or purpose of the run Plate setup The Well Information area shows the following information about a well that is selected in the Plate Setup E Well name Type of assay AQ or CpG Assay name Sample ID if entered Sequence to analyze Dispensation order E Well note if entered If several wells are selected in the Plate Setup the information for the first selected well is shown E
37. en in the CoG mode Note Wells with a high substrate peak will be marked with an intormation icon in the plate overview This will not affect the quality assessments Print or export plate overview as image The plate overview can be printed or copied as an image to the clipboard by right clicking the plate overview and selecting Print or Copy as Image from the context menu The image can be pasted into applications that support Enhanced Metafile EMF images Compare Pyrogram of different wells To compare Pyrogram of a specitic well displayed in the Pyrogram area with Pyrogram of one or several wells displayed in the histogram area 1 Select the well or wells see Select Wells page 36 you wish to open in the histogram area the lower area 2 Right click the selection and select Open from the context menu 3 Select the well you wish to open in the Pyrogram area the middle area Al LE oO Analyze Selected Ls pnn Lal Copy as Image If Pyrogram of several wells are displayed in the lower area use the scroll bar to change Pyrogram within the selection For more intormation and instructions on how to zoom in and out of Pyrogram see Pyrogram Details page 37 To close Pyrogram in the lower area and view the histogram for the selected well click x in the upper right corner of the Pyrogram area Enter analysis note In the lower right corner you can enter an analysis note To expand or
38. esult depends on the magnitude of the deviation Sum deviation in The deviation between the measured sum of all the variable positions peaks in the variable position and the theoretical sum based on the single peak height It the deviation is higher than the stringency level allows the warning Uncertain Failed due to high sum deviation in variable position is triggered during the analysis Whether the warning will yield a Check or Failed quality assessment for the analysis result depends on the magnitude of the deviation Parameters A peak The tactor by which the A peak intensities are reduction factor multiplied by to account for the fact that A peaks are higher than other peaks The default value is 0 86 For more information Appendix B of the PyroMark Q24 User Manual page B 1 To reset the parameters at the Variable Positions tab and the Analysis Parameters tab to their default values click Revert to Default PyroMark Q24 Analysis Software User Guide 01 2009 23 Run setup Workflow 1 Click in the toolbar A new run file is created Note It you wish to base your run on a previous run right click the processed run file in the shortcut browser and select Copy and Rerun from the context menu The process and analysis data will not be copied 2 Enter the run parameters see Run Parameters page 24 3 Set up the plate i e add an assay and if desired enter a sample ID and n
39. form the following steps see Section 5 5 of the PyroMark Q24 User Manual 1 Prepare your samples 2 Fill the PyroMark Q24 Cartridge with the required volumes of reagents 3 Load the reagent cartridge and the PyroMark Q24 Plate into the instrument 4 Insert the USB stick containing the run file into the USB port at the front of the instrument 5 Select the run file and start the run 6 When the run has been completed and data transferred to the USB stick remove the USB stick 7 Unload the plate and the reagent cartridge Analysis Workflow 1 Insert the USB stick containing the processed run file into computer s USB port Note The computer must have PyroMark Q24 Software installed 2 Move the run file from the USB stick to the desired location on the computer using Windows Explorer 3 Open the run file in PyroMark Q24 Software either by selecting Open in the File menu or double clicking the file in the shortcut browser To update the contents of a folder in the shortcut browser right click it and select Refresh from the context menu 30 PyroMark Q24 Analysis Software User Guide 01 2009 4 To analyze the run and get an overview of the results compare the Pyrogram of different wells or enter an analysis note see Overview Tab page 31 5 To modify how the analysis is performed see Analysis Setup Tab page 34 6 To generate analysis reports see Reports page 40 Note The more
40. he AQ mode If you still want to analyze in the CoG mode enter extra Gs in the Sequence to Analyze text box and set the expected heights of the extra Gs to zero 0 see Adjust Heights of Histogram Bars page 18 Description Description Adenine Tor G Cytosine TorA Guanine CorG Thymine C T or G not A Uracil A T or G not C Purine A or G A T or C not G Pyrimidine C or T A C or G not T CorA Any base A C G or T Note S B V and N are not valid after bisulfite treatment Valid patterns in a CpG assay Patterns that cannot exist after bisulfite treatment are not valid in a CpG assay for example GC TGAC G as C TG is a forward CpG site and C G cannot exist after bisulfite treatment 14 PyroMark Q24 Analysis Software User Guide 01 2009 The following CpG site and SNPs can be included in a torward sequence E CpG site C TG E SNPs A T A G G T and A T G i e C cannot be included The following CpG site and SNPs can be included in a reverse sequence E CpG site CG A E SNPs A T A C C T and A T C i e G cannot be included Note The software does not support analysis of CpG sites that include an additional variable position for example A C TG Note A CG before bisulfite treatment can be analyzed by typing C TG in the Sequence to Analyze text box and ATCG in the Dispensation Order text box Proceed with the run as usual After analysis of the CpG sites switch to the AQ mo
41. n experiments with replicates are performed PyroMark Q24 Analysis Software User Guide 01 2009 41 Passed check The analysis results to be included The calculations can be performed on results with passed and or check quality assessment Note It all passed and check results are included in the report check the Passed and Check boxes you can exclude results within this group by turning off the Analyze option for these positions at the Analysis Setup tab see Variable Positions Tab page 19 If you wish to view the report before you save or print it click Preview Analysis results report Reports Window Help AQ Analysis Statistics AQ Analysis Statistics AQ Analysis Results AQ Pyrogram Report AQ Pyrogram Report AQ Full Report AQ Full Report SNP Analysis Results CpG Analysis Statistics SNF Pyrogram Report SNF Pyrogram Report CpG Analysis Results SNF Full Report SMP Full Report CpG Pyrogram Report SNP Overview Report SNP Overview Report CpG Full Report The Analysis Results report includes the following information for all or selected wells see Select Wells page 36 E Well information assay name Sample ID and if desired well note E The allele frequencies AQ report genotypes SNP report or methylation percentages CpG report only E The mean methylation percentage and the standard deviation of all CoG sites except for tailed in a well CpG report only E The highest
42. or Show Reference Peaks from the context menu PyroMark Q24 Analysis Software User Guide 01 2009 37 Zoom Pyrogram It is possible to zoom in on Pyrogram by selecting a stretch of Pyrogram with the left mouse button Zoom out either by right clicking the Pyrogram area and selecting Zoom Out from the context menu the zoom is set to the previous level or by double clicking the Pyrogram area the zoom is set to 100 If comparing Pyrogram at the Overview tab see Compare Pyrogram of Ditterent Wells page 33 Pyrogram with the same sequence to analyze can be zoomed simultaneously i e linked zooming by clicking in the top right corner of the Pyrogram area View peak heights To view the height of a peak position the mouse pointer over the top of the peak A tooltip displays the height Enable or disable reference peaks and controls Reference peaks and controls can be enabled or disabled for CoG assays only either in Pyrogram or in the histogram at the Analysis Setup tab For instructions see Enable or Disable Reference Peaks and Controls in the Assay Setup page 18 Note If you wish to edit a selection of wells that share the same assay and dispensation order select the wells see Select Wells page 36 in the plate overview or navigator Apply changes 1 To apply changes made in Pyrogram click the green button This button is enabled when a change has been made 2 To apply changes m
43. ote for each used well 4 When the run is set up and ready to run on the PyroMark Q24 Print a list of required volumes of enzyme mix substrate mix and nucleotides and the plate setup Select Pre Run Information trom the Tools menu and when the report appears click amp Close the run file and copy it to one of the USB sticks supplied with the system using Windows Explorer To open Windows Explorer right click the folder containing the run file in the shortcut browser and select Explore from the context menu For more information press the F1 key to open Windows online help To run the plate on the PyroMark Q24 see Run page 30 Note In the shortcut browser you can create a new run file by right clicking the desired tolder and selecting New Run trom the context menu Enter the filename and press Enter To add a shortcut to a folder or drive click Add Folder Shortcut Note The more frequently the file is saved the more information is recovered if there is a power failure or similar problem while the file is open To save the file click ll in the toolbar If the file has never been saved select location and name of the file in the dialog that appears Run parameters Run name The name of the run is given when the file is saved Renaming the file also changes the name of the run Instrument method Select instrument method according to the reagents and cartridge that will be us
44. rnings It the dispensation order contains a warning this is displayed by a red exclamation mark at the end of the text box It is possible to run an assay with a dispensation warning but the warning must be considered when evaluating the analysis result If you position the mouse pointer over the exclamation mark a tooltip will display an explanation of the warning Warning Suggested action Sequence uncertain due The problem may be resolved by either entering to lack of terminal more sequence information or reducing the sequence information number of dispensations Sequence not in phase The problem may be resolved by adjusting the at the end of the dispensation order manually or by clicking dispensations Generate Dispensation Order or entering more sequence information Note If the problem is not resolved the out of phase stretch will not be analyzed The generated It possible enter more sequence information and dispensation order increase the number of dispensations For the best contains less reference possible quality assessment of the results we peaks than required recommend five or more reterence peaks 16 PyroMark Q24 Analysis Software User Guide 01 2009 Histogram Histogram l Copy 45 Image Show Reference Peaks i ot wf eS l o T T T 6 T A 6 T C o CT GA T gt G T A G T C OA 5 10 15 20 25 The following icons and colors are used in the histogram E Variable positions are highlighted
45. s The Full Report includes run parameters run log and well information and analysis results including Pyrogram for all or selected wells The SNP Overview Report includes genotypes and quality assessments for all SNPs The information is presented in plate overviews with one plate per position number The report options are only available for processed runs For more information on the reports see the Reports section Note In order to view the Full Report Pyrogram Report and SNP Overview Report you must have Adobe Acrobat Reader installed on your computer Adobe Acrobat Reader is available on the PyroMark Q24 Software CD but can also be downloaded at www adobe com Toggle between open files in the software using the Window menu Select PyroMark Q24 Software Help or press the F1 key to open the online help PyroMark Q24 Analysis Software User Guide 01 2009 11 Analysis toolbar Click and select Analyze All Wells or Analyze Selected Wells see Select Wells page 36 for the current run file Analyze All Wells eee Click 8 to view and print the run parameters run name instrument method plate ID barcode reagent At ID and run note and a run log for the current run AQ l i ae file To print the report click amp p Select AQ or CpG in the toolbar to toggle between the analysis applications General Hints and Tips All saved analy
46. s AQ report genotypes SNP report or methylation percentages CpG report is displayed above each variable position for example 26 The background color shows the quality assessment of the analysis result see the Color Legend section Note Deselected by the user 4 in white The software does not support analysis for example SNP when in the CoG mode W in red Not possible to analyze due to lack of data E Bisulfite treatment controls are highlighted with a light yellow background color E f desired the variable positions are highlighted with a blue gray background color Report options In the Pyrogram Report dialog you have the following options All wells Selected wells The wells to be included in the report Number of The number of columns and rows of Pyrogram on rows columns each sheet Sort by row column The sorting order of the wells Portrait landscape The paper orientation Highlight variable It this option is checked the variable regions are regions highlighted with a blue gray background color Paper size The paper size If you wish to view the report before you save or print it click Preview 44 PyroMark Q24 Analysis Software User Guide 01 2009 Note In order to view the report you must have Adobe Acrobat Reader installed on your computer Adobe Acrobat Reader is available on the PyroMark Q24 Software CD but can also be downloaded at www adobe com Full report AQ
47. s Software in the following sections M Introduction PyroMark Q24 Analysis Software Analysis modes Shortcut browser Main menu and toolbars General hints and tips Assay setup Run setup Run Analysis Reports Manage instrument methods 6 PyroMark Q24 Analysis Software User Guide 01 2009 PyroMark Q24 Analysis Software The PyroMark Q24 system is a complete solution comprising instrument chemistry and software The integrated software is designed with two analysis modes AQ for a variety of quantification studies and SNP analysis and CoG for methylation analysis AQ and CpG assays can be performed on the same plate The main advantages of PyroMark Q24 are E High resolution quantification of individual sites Built in quality control for bisulfite treatment in methylation assays Analysis of methylation in the presence of SNPs Quantification of di tri or tetra allelic mutations All assays use sequence context as built in quality control Analysis Modes PyroMark Q24 Software has two analysis modes New 40 assay AQ A variety of quantification studies and SNP analysis Hew Cpt Assay i CpG Methylation analysis of multiple consecutive CpG a sites at CoG AQ assays and CpG assays can be performed on the same PyroMark Q24 Plate To toggle between the analysis modes in the analysis view select AQ or CpG in the toolbar PyroMark Q24 Analysis Software User Guide 01 2009 7 Shortcut Bro
48. ses performed are logged with analysis settings used analysis mode AQ or CpG and analysis version results including any warnings date and time of the analysis and who performed the procedure For information on who performed an analysis and who created an assay or run file to be correct all users must log on to Windows using their own user accounts For more information about user accounts and logging on and off see Windows online help or contact your system administrator Protection of files If you want to protect a file from being edited by another user save the file in a folder that can only be accessed by you contact your system administrator for more information If you want to protect a file from being accidentally overwritten by you or another user set the read only attribute for the file using Windows Explorer 1 Close the file in PyroMark Q24 Software 2 Open Windows Explorer and locate the file This can be done by right clicking the folder containing the file in the shortcut browser 3 In Windows Explorer right click the file and select Properties from the context menu 4 When the Properties dialog box appears turn on i the Read only attribute and click OK A backup should be performed frequently The more frequently a file is saved the more information is recovered if there is a power failure or similar problem while the file is open To save a file click ll in the toolbar If the fil
49. ssay to create a New Run Cre new AQ or CpG assay see Assay Setup page 13 ya Open Chrl 0 Select New Run or click the green icon in the toolbar to create a new run file see Run Setup page 24 Import el Save Cerl 5 Save As Ctrl Shift 5 Exit Select Open or click amp in the toolbar to open a saved assay or run Tile Select Create New Run trom Sample Layout File from the Import submenu to create a new run using a plate layout for Sample IDs and notes optional defined in a tab or comma delimited text file tsv or csv Select Create New AQ CpG Assay from Assay Design File trom the Import submenu to create a new AQ or CpG assay based on an assay file xml created with Pyrosequencing Assay Design Software The software will import the sequence to analyze and the names of the variable positions Hew AQ assay Hew CpG assay PyroMark Q24 Analysis Software User Guide 01 2009 9 Tools menu for unprocessed run files Window Instrument Methods Pre Run Information Tools menu for processed run files Reports Window Help Instrument Methods Run Information Export Peak Heights Export Environment Data Export Raw Data Analysis Log 10 Select Save or click H in the toolbar to save the changes in the current tile If the tile has never been saved select the location and name of the file in the dialog box Select Save As to sav
50. stics AQ Analysis Results AO Pyrogram Report 42 Full Report Renart O SNP Pyrogram Report CpG Analysis Results SNF Full Report CpG Pyrogram Report SNP Overview Report CpG Full Report The Analysis Statistics report includes the following statistics for variable positions with the same assay and position number and if desired with the same sample ID in all or selected wells see Select Wells page 36 E The mean allele frequencies AQ report or mean methylation percentage CpG report E The highest and the lowest allele frequencies AQ report or methylation percentage CpG report E The standard deviation The number of values used in each calculation The report can be saved as a text file tsv or csv or an HTML file html The report can be imported into Microsoft Excel or other applications that can handle text files tsv or csv with data that is separated by semicolons or tabs This is useful when doing further calculations on the data Report options In the Analysis Statistics Report dialog there are the tollowing options All wells selected wells The wells to be included in the report Assay assay and The analysis results allele frequencies or methylation sample percentages can be grouped according to ID E Assay that is results with the same assay will be compared Assay and sample ID that is results with the same assay and sample ID will be compared Can be useful whe
51. strument method PyroMark Q24 Analysis Software User Guide 01 2009 47 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www giagen com FAQ FAQLIist aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www qiagen com Comments and suggestions Analysis software related errors a Red cross over wells in Contact QIAGEN Technical Services the Overview tab during analysis b Exception dialog Save the error report and send to QIAGEN appears Technical Services for information Click Continue to proceed with analysis If the dialog remains click Quit and reopen the run c Could not create assay Ensure a valid assay tile type is being imported from specified Pyrosequencing Assay Design Software file References QIAGEN maintains a large up to date online database of scientific publications utilizing QIAGEN products Comprehensive search options allow you to find the articles you need either by a simple keyword search or by specifying the application research area title etc For a complete list of references visit the QIAGEN Reference Database online at www
52. tormation trom the Tools menu and when the report appears click 3 Note It you want to print the report in color turn on the Print background colors and images option in the Internet Explorer Tools Internet Options Advanced Printing Define sample ID and note externally By using the Import Insert Sample Layout File or Paste Sample Layout feature you can easily use the same layout in several runs and reuse intormation available in existing documentation Using the import insert sample layout file feature You can for example generate layout files from your Laboratory Information Management Systems LIMS Sample and note layout files can also be created in Microsoft Excel Notepad and similar applications The layout tile must have two or three columns Well Sample ID and Note optional Each column must be separated by a tab comma or semicolon and each line must be delimited by a line break Save the file as a tab or comma delimited text file tsv or CSV 28 PyroMark Q24 Analysis Software User Guide 01 2009 The sample and note layout tile can be imported into E An existing run tile by right clicking a well in the Plate Setup and selecting Insert Sample Layout File trom the context menu E A new run tile by selecting Import and then Create New Run trom Sample Layout File from the File menu ey File Edit View Insert Format Tools Data Window Help D
53. tridge Reports PyroMark Q24 Software offers the following reports for processed runs Reports menu for CpG MM Analysis Statistics Report This includes analysis runs statistics for all or selected wells Window E Analysis Results Report This includes well intormation and analysis results tor all or selected wells Reports Help CpG Analysis Statistics Coo Analysis Results Cos Pyragram Report E Pyrogram Report This includes well information CpG Full Report and Pyrogram for all or selected wells Reports for AQ runs E Full Report This includes run parameters run log and well information and analysis results including Pyrogram for all or selected wells Reports Window Help AQ Analysis Statistics 40 Analysis Results E SNP Overview Report This includes genotypes and quality assessments for all SNPs The intormation is presented in plate overviews with one plate per position number AQ Pvrogram Report AQ Full Report SMP Analysis Results SMFP Pyroagram Report SMP Full Report Note In order to view the Full Report Pyrogram Report and SNP Overview Report you must have Adobe Acrobat Reader installed on your computer Adobe Acrobat Reader is available on the PyroMark Q24 Software CD but can also be downloaded at www adobe com SMP Overview Report 40 PyroMark Q24 Analysis Software User Guide 01 2009 Analysis statistics report Reports Window Help AQ Analysis Stati
54. ulfite controls are Cs converted to Ts read as Gs and As in a reverse assay or not PyroMark Q24 Analysis Software User Guide 01 2009 17 View reference peaks Non variable peaks i e peaks that are not a part of a variable position including blank dispensations are referred to as reference peaks Reference peaks are used in the analysis both as reterences when calculating the single peak height and as internal controls when assessing the quality To toggle between viewing and hiding reference peaks in the histogram right click the histogram and select Show Reference Peaks from the context menu Enable or disable reference peaks and controls By lett clicking a reference peak diamond the peak is either enabled or disabled as a reference peak depending on the previous state The diamond displays the status E Filled blue diamond Enabled as a reference peak E Hollow blue diamond Disabled as a reference peak By lett clicking a bisulfite treatment control diamond CpG assays only the control is either enabled or disabled as a control and or a reference peak depending on the previous state The diamond displays the status E Filled orange diamond Enabled both as a bisulfite treatment control and a reference peak E Filled blue diamond Enabled as a reference peak but disabled as a bisulfite treatment control E Hollow orange diamond Disabled both as a bisulfite treatment control and a reference peak Position
55. ur technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products If you have any questions or experience any ditticulties regarding the PyroMark Q24 or QIAGEN products in general please do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helptul to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please see our Technical Support Center at www giagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www qiagen com E PyroMark Q24 Analysis Software User Guide 01 2009 5 Introduction About this user guide This user guide provides information about the functions and features of the PyroMark Q24 Analysis Software Please refer to the PyroMark Q24 User Manual tor complete information about the proper care maintenance and use of the instrument This user guide describes the features of the software and associated tools and enables the user to manage and modify files and analyses This user guide provides intormation about the PyroMark Q24 Analysi
56. wser The shortcut browser provides a quick and easy way to access folder contents and commonly used files PyroMark Q24 Fie Tools E GJ Shortcuts H Example Files B Lagerholm OFTO12 1 ril 2 gt 071023 d AG Assay 1 J AQ Assay 2 CpG Assay 1 pG Assay 2 Explore Refresh lt Mew assay New Run Remove Shortcut Properties Add a shortcut to a folder or drive by clicking Add Folder Shortcut or right click the Shortcuts folder and select Add Folder Shortcut trom the context menu Add a shortcut to a file by clicking Add File Shortcut or right click the Shortcuts folder and select Add File Shortcut from the context menu Remove a shortcut by right clicking the shortcut and selecting Remove Shortcut from the context menu A folder shortcut s tiles and subfolders cannot be removed separately Update the contents of a folder by right clicking it and selecting Retresh from the context menu View file or folder properties e g run parameters by right clicking the file or folder and selecting Properties from the context menu Creating opening and copying tiles Create a new assay tile by right clicking the desired folder Select New Assay and then AQ Assay or CpG Assay trom the context menu Enter the tilename and press Enter To set up the assay see Assay Setup page 13 Create a new run file by right clicking the desired tolder

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