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PyroMark® Q48 Autoprep User Manual

1. 1 A sequencing primer is hybridized to a single stranded PCR amplified DNA template 2 The template is incubated with enzymes and substrates 3 One nucleotide is added to the reaction If the nucleotide is complementary to one or more bases in the template strand it will be incorporated into the DNA strand by the DNA polymerase 4 Each incorporation event is accompanied by release of pyrophosphate PPi in an equimolar quantity to the amount of nucleotides incorporated Polymerase DNA dNTP DNA PPi 5 ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5 phosphosulfate PyroMark Q48 Autoprep User Manual 10 2015 Zl This drives the conversion of luciferin to oxyluciferin by luciferase generating visible light in amounts proportional to the amount of ATP and thus incorporated nucleotides Light is detected using a photomultiplier tube PMT and seen as a peak in the Pyrogram Each light signal is proportional to the number of nucleotides incorporated Sulfurylase 62 light APS PPi ATP luciferin oxyluciferin s Luciferase fy ATP Light time nucleotide incorporation generates light seen as a peak in Pyrogram Apyrase a nucleotide degrading enzyme continuously degrades unincorporated nucleotides and ATP When degradation is complete another nucleotide is added dNTP EPS dNDP dNMP phosphate ATP PES ADP AMP phosphate Nucleotides
2. PyroMark Q48 Autoprep Software offers the following analysis reports for processed runs Analysis Statistics Report This includes analysis statistics for all or selected wells Analysis Results Report This includes well information and analysis results for all or selected wells PyroMark Q48 Autoprep User Manual 10 2015 119 Pyrogram Report This includes well information and Pyrogram for all or selected wells Full Report This includes run parameters run log well information and analysis results including Pyrogram for all or selected wells Note To view reports generated in PDF format a PDF reader must be installed on the computer Adobe Reader can be downloaded at www adobe com 7 16 1 Analysis statistics report only AQ and CpG modes Reports Window Help Reports Window Help CpG Analysis Statistics AQ Analysis Statistics CpG Analysis Results AQ Analysis Results CpG Pyrogram Report AQ Pyrogram Report CpG Full Report AQ Full Report The Analysis Statistics report includes the following information for variable positions in all or selected wells see section 7 7 The mean allele frequencies AQ report or mean methylation percentage CpG report The highest and the lowest allele frequencies AQ report or methylation percentage CpG report e The standard deviation The number of values and the wells used in each calculation f analysis parameters or quality assessments have been ed
3. 7 Before running your samples validate your assay using a reference DNA sample see Appendix A Assay design and validation 8 Optional If applicable during the assay validation edit the analysis parameters 9 Optional Lock the assay for editing by clicking the Lock Assay button at the bottom of the assay setup window A locked assay A that has been run on the PyroMark Q48 Autoprep Instrument cannot be unlocked i e it will not be possible to edit the analysis parameters or results after the assay has been processed 10 Click lei in the toolbar to save the file If the file has never been saved select location and enter the filename in the dialog box that opens 7 11 2 Enter the sequence to analyze Type or paste the sequence to analyze into the Sequence to Analyze text box If creating a CpG assay we recommend entering the sequence before bisulfite treatment if it is known see Enter the sequence before bisulfite treatment Note It is possible to add assays to a well in the disc layout without a sequence to analyze PyroMark Q48 Autoprep User Manual 10 2015 88 The following rules apply when entering the DNA Sequence to Analyze in the software The allowed characters for sequence input are A C G and T as well as IUPAC codes Variable positions can be entered using either IUPAC codes or a forward slash as a separator between the two potential bases e g C T InDels should be entered usin
4. PyroMark Q48 Autoprep por Gj x Sequence Cleaning Tools The instrument is now ready for use 4 4 Installation of the analysis software 4 4 1 Installing PyroMark Q48 Autoprep Software To install or upgrade PyroMark Q48 Autoprep Software 1 Ensure that the computer meets the minimum requirements see section 3 5 Close any programs running on the computer Insert the PyroMark Q48 Autoprep Software CD into the CD drive In the CD menu click Install PyroMark Q48 Autoprep Software a e p e If the CD menu does not appear automatically perform the following steps Select My Computer in the Windows Start menu Right click the CD drive with the software CD and select Explore Double click the file autorun exe 6 When the software has been successfully installed click Exit Setup in the CD menu 7 Please use Windows Update www update microsoft com to check for any critical updates to the NET Framework 4 0 Note If the computer is connected to a network network policy settings may prevent you from completing this procedure For more information contact your system administrator To view reports generated by PyroMark Q48 Autoprep Software in PDF format a PDF reader must be installed on the computer Adobe Reader can be downloaded at www adobe com PyroMark Q48 Autoprep User Manual 10 2015 33 4 4 2 Uninstalling PyroMark Q48 Autoprep Software 1 Select Control Panel in the Windows Start menu In the li
5. Quality colors in section 7 15 3 e f a compensated Pyrogram is included the peaks are colored according to their quality assessments Compensated Pyrograms are only shown for analyzed wells Report options Al AQ Pyrogram Report ee Selection Al wells gt Selected wells Layout Number of rows Number of columns Sort by Orientation Options V Highlight variable regions Portrait P Landscape Paper size A4 bf Paper size A4 Selection Al wells Selected wells Layout Number of rows 3 Number of columns 1 Sort by Row Column Orientation Portrait C Landscape Options Show peak levels Raw pyrogram C Compensated pyrogram Preview Save Cancel Preview In the Pyrogram Report dialog box there are the following options Option All wells selected wells Number of rows columns Sort by row column Portrait Landscape PyroMark Q48 Autoprep User Manual 10 2015 Description The wells to be included in the report The number of columns and rows of a Pyrogram on each sheet The sorting order of the wells The paper orientation 125 Option Description Highlight variable regions If this option is checked the variable regions are highlighted with a blue gray background color This option is not available for the SEQ report If this option is checked the calculated peak levels are Show peak levels shown in the
6. noted as missing peaks in Pyrogram Reagents incorrectly dissolved or stored PyroMark Q48 Autoprep User Manual 10 2015 Comments and suggestions Check the PCR samples using an agarose gel to confirm there is one strong specific band If not rerun PCR with high quality DNA The PyroMark PCR Kit is recommended for highly specific amplification of bisulfite converted DNA and gDNA from various sources Check the PCR samples using an agarose gel to confirm there is one strong specific band If not reoptimize PCR Check assay design see Appendix A Assay design and validation Use a recommended primer supplier Ensure the biotinylated primer is HPLC purified or similar Follow the recommendations for amount of template see Appendix A Assay design and validation Use less PCR product Be sure to add sufficient reagents see information shown on the display prior to the run Perform the injector test see section 8 5 Be sure to follow the instructions in the handbook supplied with PyroMark Q48 Autoprep Reagents and PyroMark Q48 Autoprep CpG Reagents Include an empty well Annealing Buffer only in the run setup to check whether background peaks are coming from nucleotides 140 Comments and suggestions Incorrect sequence to analyze Correct the sequence to analyze and if necessary rerun the samples Background peaks in Pyrogram Follow the recommendations in Appendix A Assay design and validati
7. 20 pl typical reaction volume 10 pl 60 RPM maximum 2500 RPM during wash cycle Rotor vibration frequency of 50 Hz 3 injector cartridges each with 4 x 3 8 ml injectors 100 10 nl dispensation dATPaS dCTP dGTP and dTTP 500 10 nl dispensation Primer 1 Primer 2 Primer 3 Primer 4 Binding Buffer 500 10 nl dispensation Denaturation Solution Enzyme Substrate Annealing Buffer 28 0 5 C Absorbent waste capture strip with a maximum allowable volume capacity of 8 ml Operating conditions AC Input Power consumption 100 240V AC 50 60 Hz Mains supply voltage fluctuations are not to exceed 10 of the nominal supply voltages Max 100 W PyroMark Q48 Autoprep User Manual 10 2015 162 Fuse type Overvoltage category Air temperature Relative humidity Altitude Place of operation Pollution level Environmental class F3 15A 250V 5 mm x 20 mm Fast Acting Fuse adhering to local regulations 18 30 C 64 86 F 20 80 noncondensing Up to 2000 m 6500 ft For indoor use only in a draftfree location No exposure to direct sunlight 2 3K2 IEC 60721 3 3 3M2 IEC 60721 3 3 Unless otherwise specified herein Transport conditions Air temperature Relative humidity Environmental class 25 C to 60 C 13 F to 140 F in manufacturer s packaging Max 75 noncondensing 2K2 IEC 60721 3 2 2M2 IEC 60721 3 2 Unless otherwise specified herein Storage conditio
8. During this operation do not expose the PMT to strong light i e do not use a flashlight to check if the window is clean 8 3 Cartridge care To ensure that the injectors are dispensing volumes within specification it is important that routine cleaning is conducted following a workday However an annual service of the cartridges at a QIAGEN service center is highly recommended to test that the cartridges are to specification and recalibrated if necessary Servicing of the cartridges requires them to be removed from the instrument PyroMark Q48 Autoprep User Manual 10 2015 131 8 3 1 Removing cartridges from instrument 1 Ensure that the reservoirs are empty and the cartridge lid is locked prior to removing the cartridge 2 Turn the instrument off 3 Take out the thumb screws holding the cartridge in place Place the screws in a safe place to avoid losing them Note Putting back the thumb screws without cartridges installed can damage the instrument 4 Gently pull the cartridge out from its slot 5 Always transport the cartridges in the dedicated cartridge boxes PyroMark Q48 Autoprep User Manual 10 2015 132 8 3 2 Installing injector cartridges into instrument 1 To install an injector cartridge slot the cartridge into place ensuring that the air pin and electrical connectors align Make sure that cartridges are connected properly by carefully exerting pressure on the rear part of the cartridge blac
9. Zeh OO een 128 ZAGA PO ECO Ole een es 129 7 18 5 Protection of analysis results 129 8 Ma neigen ee bang sarge censemdpaneceeasoet 130 8 1 Cleaning the PyroMark Q48 Autoprep cceeeeeeeeeeeeeeeeeeeeeeeeeeasaneeeeeeeeees 130 8 2 Light detector maintenance eer eee ae nee aetna eae Per ee 131 GO CE ee ee 131 8 3 1 Removing cartridges from instrument 132 8 3 2 Installing injector cartridges into instrument 133 8 33 Cleaning oie lg des nee ne eee eo nee ones ee 133 8 34 Exchanging CAES a Ra 133 8 4 CO Nieten ee 133 PyroMark Q48 Autoprep User Manual 10 2015 Ga LE SCC ee ea ee use 134 86 Pyrophosph fe clean nenn 135 9 Toubes hoo 1116 Be SS EEE EEE NEE E suena ae 138 2 Checkingihe ron Deere nee 138 9 2 Copying SUPPORTE en ee ee ee ee 139 93 Analysisteled Gols epee een r eier 140 GA Analysis software related erotik 141 9 A 1 Messages of the PyroMark Q48 Autoprep Software 142 9 5 Instrumentrelated SOS ee 150 OO veg arate sacw baci eesane S 152 11 Appendix Assay design and validation 0 cccccccceeesseeeeeeeeeeeeeeeeeeeeeeeeeeaaees 155 PSS CO recaps sheen ces E A ces ee ee 155 Re ENEE 155 PER PIE a EE ee 155 ADO ee bg 155 SEQUENCING primer aneeessseenssennssennssennsnennenennnnnennneenneennneennenennennneenneen nenn 156 RSS a Ro ms 156 SO Er Ig1le e SPPRPEEUEERENERERTERPENERERFSUREHEUEEENEPFFERFEHEE
10. national state and local health and safety regulations and laws 2 4 Chemical safety WARNING Hazardous chemicals W7 A The Denaturation Solution contains sodium hydroxide which is irritating to eyes and skin Always wear safety glasses gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the operators are not exposed to hazardous levels of toxic substances chemical or biological as defined in the applicable Safety Data Sheets SDSs or OSHA ACGIH or COSHH documents For more information visit www qiagen com safety Venting for fumes and disposal of wastes must be in accordance with all national state and local health and safety regulations and laws OSHA Occupational Safety and Health Administration United States of America t ACGIH American Conference of Government Industrial Hygienists United States of America t COSHH Control of Substances Hazardous to Health United Kingdom PyroMark Q48 Autoprep User Manual 10 2015 2 5 Mechanical hazards WARNING Moving parts w8 A To avoid contact with moving parts during operation of the PyroMark Q48 Autoprep the instrument must be operated with the injector cover closed Do not remove the cover panels since there are no user serviceable parts inside If there is a problem with the PyroMark Q48 Autoprep contact QIAGEN Technical Services immediate
11. 7 18 1 Validation of assays Validate your assays using reference DNA samples see Appendix A Assay design and validation 7 18 2 Run log A log is maintained for each run detailing events and warnings that occur during a run This log is available in the Run Information window which can be accessed from the Tools menu 7 18 3 Analysis log All analyses performed are logged with analysis settings used analysis mode AQ SNP CpG or SEQ analysis version results including analysis warnings date and time of the analysis and who performed the analysis 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 To view the analysis log for a selected well select Analysis Log from the Tools menu PyroMark Q48 Autoprep User Manual 10 2015 128 7 18 4 Protection of files 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 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 the PyroMark Q48 Autoprep Software 2 Open Windows Explorer and locate the file This can be d
12. 7 Uberhitzungsgefahr WARNUNG A WARNUNG A HeiBe Oberflache W10 Die Disc Heizung im Inneren des PyroMark Q48 Autoprep Ger ts kann Temperaturen von bis zu 95 C erreichen Ber hren Sie sie nicht wenn sie aufgeheizt ist Uberhitzungsgefahr C6 Vergewissern Sie sich dass ein Mindestabstand von 5 cm und hinter dem Ger t ein Freiraum von 20 cm bleibt damit eine ausreichende Bel ftung des Ger ts gew hrleistet ist Die L ftungsschlitze und ffnungen die f r die Be und Entl ftung des PyroMark Q48 Autoprep bestimmt sind d rfen nicht verdeckt werden PyroMark Q48 Autoprep User Manual 10 2015 182 16 8 Verbrauchsartikel ACHTUNG Nicht unterst tzte Verbrauchsartikel A Verwenden Sie keine anderen Verbrauchsartikel oder Zubeh rteile und schlie en Sie keine anderen externen Ger te an als die die in diesem Handbuch angegeben sind 16 9 Abfallentsorgung ACHTUNG Entfernen des Absorberstreifens C8 A W hrend des Laufs nimmt der Absorberstreifen Denaturierungsl sung auf die Natriumhydroxid enth lt Dieses wirkt reizend auf Augen und Haut Tragen Sie beim Entfernen des Absorberstreifens immer Schutzbrille Laborhandschuhe und Laborkittel Bei der Entsorgung des Absorberstreifens m ssen alle Bestimmungen und Gesetze zu Gesundheitsschutz und Sicherheit am Arbeitsplatz auf Ubernationaler nationaler und regionaler Ebene eingehalten warden ACHTUNG Entsorgung von Kunststoffverbrauchsmaterial C9 A G
13. HSOs is referred to as bisulfite or hydrogen sulfite In the bisulfite reaction DNA is treated with sodium bisulfite to convert cytosine residues to uracil under conditions whereby methylated cytosines remain non reactive Pyrosequencing assays can contain an internal control to assess successful bisulfite treatment C bases that are not followed by G in the sequence are normally 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 Analysis mode used for analysis of CoG methylation Analysis capability within CpG mode for analysis of methylation at CpN sites A repetitive dispensation order for nucleotide dispensation Normally used in Pyrosequencing technology for sequencing unknown DNA sequences For example CTGA or TCGA can be used and repeated for the desired number of times Non cyclic order of dispensation that follows the known sequence It can be used in Pyrosequencing technology when you know the sequence to be analyzed For example the sequence TCCAGAA can be analyzed with the dispensation order TCAGA Defines the nucleotides and the order in which they should be dispensed in Pyrosequencing runs A continual decrease in peak height normally seen in the Pyrogram A pro
14. I CpG assay file IX SEQ assay file EM A run file that has not been processed EM A run file that has been processed X Broken shortcut This may be due to a network server that is temporarily inaccessible or that the file or the folder has been moved renamed or deleted outside the software The following are instructions to add and remove shortcuts to update the contents of a folder and to view file and folder properties e Add a shortcut to a folder or drive by clicking Add Folder Shortcut or right click the Shortcuts folder and select Add Folder Shortcut from the context menu e 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 rightclicking the shortcut and selecting Remove Shortcut from the context menu The files and subfolders in a shortcut folder cannot be removed separately Update the contents of a folder by right clicking it and selecting Refresh from the context menu or by pressing the F5 key eo View file or folder properties e g run parameters by right clicking the file or folder and selecting Properties from the context menu Note If the mouse pointer is positioned over a file or a folder in the shortcut browser a tooltip displays the file or folder pathway the file name the assay note for assay files and the disc ID and run note for run files if entered PyroMark Q48 Autoprep User M
15. ID Sample 1 is copied and incremented by column 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 file 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 txt or csv PyroMark Q48 Autoprep User Manual 10 2015 107 The sample and note layout file can be imported into An existing run file by right clicking a well in the Disc Setup and selecting Insert Sample Layout File from the context menu Anew run file by selecting Import followed by Create New Run from Sample Layout File from the File menu 17 a ne be D en re TC a h E K a Eee JS Samp av out Example le i z r cr Hoi Ins Pac For Dat Rev Vie BE gt oe 23 iTi i _ bette T F AF B Yo p ER F m F a T Paste Font Alignment Number Styles Cells F F 7 7 7 wir Clipboard Editing a f 1 EF H A B C 1 Well Sample ID Note 2 A1 DNA 342 Notes for well Ai 3 DNA 360 Notes for well A3 4 DNA 360 Notes for well AS The result when importing the sample and
16. Lysis Buffer Proteinase K Bisulfite Solution DNA Protect Buffer MinElute DNA Spin Columns Carrier RNA and Buffers For 50 preps Lysis Buffer Proteinase K Bisulfite Solution DNA Protect Buffer MinElute DNA Spin Columns Carrier RNA and Buffers For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor PyroMark Q48 Autoprep User Manual 10 2015 Cat no 974203 979203 978703 978801 59824 59844 59864 168 14 Appendix D Legal Information Warranty statement Thank you for your purchase of QIAGEN instrumentation Your instrument has been carefully tested to ensure optimum operating efficiency and reproducibility of results QIAGEN warrants that all new instrumentation manufactured by QIAGEN will correspond to the product specifications and be free from defects in workmanship and materials for a period of twelve 12 months from the original date of shipment Repair or replacement of defective parts will be provided to the purchaser during this time period provided the QIAGEN instrumentation is operated under conditions of normal and proper use but not for damage caused by the customer If any part or subassembly proves to be defective it will be repaired or replaced at QIAGEN s sole option subseque
17. Manual 10 2015 1 Introduction Thank you for choosing the PyroMark Q48 Autoprep We are confident it will become an integral part of your laboratory Before using the PyroMark Q48 Autoprep it is essential that you read this user manual carefully and pay particular attention to the safety information The instructions and safety information in the user manual must be followed to ensure safe operation of the instrument and to maintain the instrument in a safe condition 1 1 About this user manual This user manual provides information about the PyroMark Q48 Autoprep system in the following sections Introduction Safety Information General Description Installation Procedures Instrument Administration Performing a Run PyroMark Q48 Autoprep Software Maintenance Procedures N WR WON Troubleshooting 10 Glossary The appendices include the following Assay design and validation Technical data and specifications Ordering information Legal informationSafety information in French FR Safety information in German DE PyroMark Q48 Autoprep User Manual 10 2015 1 2 General information 1 2 1 Technical assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Services Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products If y
18. Photomultipliers PMT vorsichtig zu reinigen Verwenden Sie keine Papiert cher Weitere Anweisungen finden Sie in Abschnitt 8 2 Besch digung des Ger ts C13 Setzen Sie den Photomultiplier PMT w hrend der Wartung nicht starkem Licht aus Reinigung der Injektoren C14 Die Injektoren m ssen innerhalb von 12 Stunden nach dem letzten Lauf gereinigt werden um die ordnungsgem Be Leistung des Injektors ber l ngere Zeit sicherzustellen Wenn die rechtzeitige Reinigung unterbleibt kann es zur Verstopfung der Injektoren kommen PyroMark Q48 Autoprep User Manual 10 2015 184 Index Aborting a run 59 About this user manual 10 Absorber strip 45 64 Accessories 167 Add assay files to the disc 104 Analysis 64 AQ analysis results 67 CpG analysis results 67 Reports 69 SEQ analysis results 67 SNP analysis results 69 View the analysis results 65 Analysis modes 70 Analysis reports Analysis results reports 122 Analysis statistics reports 120 Full reports 126 Pyrogram reports 124 Analysis Reports 119 Analysis Setup tab 80 Analysis software 28 Analysis software related errors 141 Analysis Toolbar 77 Analysis view 78 Analysis related errors 140 Analyze a run 110 Analysis warnings 114 Edit base called sequences 119 Homopolymers and uncertain bases 115 Methylation levels 115 Modified assay in other runs 118 Quality assessments 114 View and compare Pyrogram 116 View the analysis results 113 Zo
19. Pipet 3 pl PyroMark Q48 Magnetic Beads into the required wells of the PyroMark Q48 Disc immediately after resuspension Note Position the pipet tip in the middle of the bead slurry to ensure consistency of each aliquot Note Avoid excess liquid on the outside of the pipet tip Excess liquid can be removed by gently touching the pipet tip against the top of the bead tube when removing the tip Use the image below as a visual reference example for required bead amount image was taken after templates were also loaded into the wells Well 1 contains too few beads well 2 contains the correct amount of beads well 3 contains too many beads mn Ff fF Note It is not necessary to equally distribute the beads within the well At the beginning of the sequencing run the disc will be shaken resulting in an equal distribution of beads Repeat the resuspension step between each pipetting step 5 After dispensing beads into all required wells discard the remaining beads in the pipet tip using the purge function Store the beads immediately after use according to the manufacturer s guidelines to ensure a longer shelf life Pyrosequencing template 1 Pipet 10 pl biotinylated PCR product into the correct wells of the PyroMark Q48 Disc The software indicates the well position for each sample Alternatively use the Pre run print out from the PyroMark Q48 Autoprep Software as a sample position guide Note If using less than 1
20. Pyrogram This option is only available for the SEQ report Raw Pyrogram The type of Pyrogram to be included in the report Compensated Pyrogram This option is only available for the SEQ report Paper size The paper size A4 A3 letter or tabloid To view the report before saving or printing it click Preview Note In order to view the report a PDF reader must be installed on the computer Adobe Reader can be downloaded at www adobe com 7 16 4 Full report Reports Window Help AQ Analysis Statistics Reports Window Help AQ Analysis Results SNP Analysis Results AQ Pyrogram Report SNP Pyrogram Report AQ Full Report RE u SNP Full Report Reports Window Help CpG Analysis Statistics Reports Window Help CpG Analysis Results SEQ Analysis Results CpG Pyrogram Report SEQ Pyrogram Report cos FulRepor Serum The full report includes the following information for all or selected wells see section 7 7 Run parameters run name run date and time protocol instrument name serial number operator disc ID barcode reagent ID and run note and a run log Well information well name assay name sample ID and well note analysis version AQ SNP or CpG assay sequence to analyze Pyrogram For information on icons and colors used in the Pyrogram area see section 7 16 3 PyroMark Q48 Autoprep User Manual 10 2015 126 Allele frequencies AQ report genotypes SNP report methylation p
21. SDS oder den Vorschriften der OSHA ACGIH oder COSHH festgelegt sind nicht berschritten werden Weitere Informationen finden Sie unter www qiagen com safety Beim Betrieb eines Abzugs und bei der Entsorgung von Abfallstoffen m ssen alle Bestimmungen und Gesetze zu Gesundheitsschutz und Sicherheit am Arbeitsplatz auf bernationaler nationaler und regionaler Ebene eingehalten werden 16 5 Chemikalien ARNUNG Gef hrliche Chemikalien W7 Die Denaturierungsl sung enth lt Natrium hydroxid das auf Augen und Haut reizend wirkt Tragen Sie immer eine Schutzbrille Laborhandschuhe und einen Laborkittel Die verantwortliche Person z B der Laborleiter muss alle erforderlichen Vorsichtsma nahmen treffen um sicherzustellen dass die unmittelbare Umgebung des Arbeitsplatzes sicher ist Auch d rfen die Grenzwerte in Bezug auf infekti se Erreger die in den entsprechenden Sicherheitsdatenbl ttern SDS oder den Vorschriften der OSHA ACGIHt oder COSHH festgelegt sind nicht berschritten werden Weitere Informationen finden Sie unter www qiagen com safety Beim Betrieb eines Abzugs und bei der Entsorgung von Abfallstoffen m ssen alle Bestimmungen und Gesetze zu Gesundheitsschutz und Sicherheit am Arbeitsplatz auf bernationaler nationaler und regionaler Ebene eingehalten werden OSHA Occupational Safety and Health Administration Vereinigte Staaten von Amerika t ACGIH American Conference of Government Ind
22. This will show if the template can loop back on itself and give a background signal in Pyrosequencing reactions Sequencing primer without any PCR product This will show if the sequencing primer can form duplexes or hairpins and give background signal in Pyrosequencing reactions Biotinylated primer without any PCR product This will show if the biotinylated primer can form duplexes or hairpins and give background signal in Pyrosequencing reactions Sequencing primer and biotinylated primer together without PCR product This will show if the sequencing primer and the biotinylated primer can form duplexes and give background signal in Pyrosequencing reactions Pyrograms from these controls should not show any significant peak after any nucleotide addition Quality assessment The user will be warned if something in the assay may reduce the quality of the result given by the analysis software The ultimate goal for a well optimized assay is that all variable positions in an AQ SNP or CpG assay or the sequence in the quality control window of a SEQ assay have the quality assessment Passed when using default or more stringent analysis parameters Such results will be shown as blue in the quality bar in the well after analysis Results of lower quality are indicated as Check yellow or Failed red together with error messages PyroMark Q48 Autoprep User Manual 10 2015 160 Analysis results For samples and
23. Use of other reagents and substances may lead to fire or explosion Insertion of absorber strip C3 Ensure that the absorber strip is inserted into the PyroMark Q48 Autoprep as described in section 6 2 2 to prevent liquid from entering the chamber In case of emergency switch off the PyroMark Q48 Autoprep at the power switch and unplug the power supply from the power outlet PyroMark Q48 Autoprep User Manual 10 2015 2 2 Electrical safety Note Disconnect the line power outlet before servicing WARNING Electrical hazard W5 A Any interruption of the protective conductor earth ground lead inside or outside the instrument or disconnection of the protective conductor terminal is likely to make the instrument dangerous Intentional interruption is prohibited Lethal voltages inside the instrument When the instrument is connected to line power terminals may be live Opening covers or removing parts is likely to expose live parts Avoid spilling liquid onto or into the instrument In case of spilling liquid over the instrument immediately disconnect the instrument from the mains power To ensure satisfactory and safe operation of the PyroMark Q48 Autoprep follow the guidelines below The line power cord must be connected to a line power outlet that has a protective conductor earth ground Keep mains plug easily accessible in case the equipment needs to be disconnected quickly from mains power Use onl
24. adobe com 7 3 4 Window menu Window Help 1 SEQ Run Analysis C Example Files Example run Read Only 2 Run Setup C Desktop test Toggle between open files in the software using the Window menu PyroMark Q48 Autoprep User Manual 10 2015 7 3 5 Help Menu 7 Pyromark Q48 Autoprep Software Help Fl About Pyromark Q48 Autoprep Select PyroMark Q48 Autoprep Software Help or press the FI key to open the online help 7 3 6 Analysis Toolbar A File Tools Reports Window Help a oF 19 gt 40 3 2 Shortcuts gt AQ 2 Example Files gt SNP AQ 1 Z CpG 1 gt CpG ghd Example run gt SEQ ves SEQ 1 Ze SNP 1 ij My PyroMark Files Click BA to analyze selected wells see section 7 7 for the current run file Click LB to view the run parameters and a run log for the current run file To print the report click 8 Select AQ SNP CpG or SEQ in the toolbar to toggle between the analysis modes 7 4 Workflow views PyroMark Q48 Autoprep Software is organized into views that reflect the Pyrosequencing workflow Assay Setup Run Setup and Analysis The active view is indicated in the status bar at the top of the window 7 4 1 Assay Setup view n D This view becomes active when creating a new assay The color of the workflow arrow Assay Setup in the status bar reflects the type of assay selected In this view the user specifies the assay name the sequence to analyze and can option
25. be moved manually when a run is not being performed PyroMark Q48 Autoprep User Manual 10 2015 PyroMark Q48 Autoprep rear view Chamber lid Software controlled motor operated access to reaction chamber Injector cartridges Three cartridges of four injectors each A Nucleotide cartridge B Primer cartridge C Reagent cartridge Injector cover Opens manually allowing access to the cartridges Ethernet connection For network connection Power switch Instrument on off switch 24 3 4 1 Injector cartridges The instrument has a total of 12 injectors divided into three cartridges of four injectors per cartridge Each cartridge consists of a lid lid lock and four injectors containing a reservoir filter injector nozzle and drop sensor 1 Cartridge lid Closes and seals the reagent reservoir 2 Reservoir label For identification of reagents in each injector 3 Cartridge lid lock Secures the cartridge lid to seal the reagent reservoir for pressurization 4 Hole for thumb screws Thumb screws hold cartridges in place Remove only when cartridges need to be serviced 5 Cartridge serial number 6 Injector Dispenses reagent into the disc 7 Injector drop sensor Monitors the quality of each dispensation and delivers feedback during injector priming and injector cleaning operations 8 Connector pins Connect the cartridge electronics to the instrument 9 Reservoir Storage of nucleotides reagen
26. be sequenced ahead of the rest of template sequences Minus shift A small proportion of the template sequences that fails to incorporate a nucleotide will be sequenced subsequent to the rest of template The ratio of the signal height and the noise height An indication of the clarity of the data The higher the ratio the better the data SNPs involve the change of one DNA base to another SNPs and point mutations are structurally identical differing only in their frequency Variations that occur in 1 or less of a population are considered point mutations while those occurring in more than 1 are SNPs A protein that can bind very strongly to biotin A molecule acted upon by an enzyme Pyrosequencing technology uses a mixture of the substrates adenosine 5 phosphosulfate APS and luciferin in the sequencing reaction A region in the sequence that varies at one or more variable bases In the PyroMark Q48 Autoprep Software the variable positions are highlighted with a blue gray background color in the histogram and Pyrogram PyroMark Q48 Autoprep User Manual 10 2015 154 11 Appendix A Assay design and validation Assay design For design of Pyrosequencing assays use the latest version of PyroMark Assay Design Software ADSW The program automatically generates primer sets that include both PCR and sequencing primers Each primer set is given a quality score based on several parameters that are specific for Pyrosequencing
27. 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 A in the Sequence to Analyze text box and analyze the variable position 7 11 3 Generate the 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 that are used as negative controls When creating CpG assays the dispensation order should also include bisulfite treatment controls If the user enters the sequence before bisulfite treatment the sequence to analyze will be automatically generated and one bisulfite control will be automatically selected if possible If the user directly enters the sequence to analyze the bisulfite control will need to be added manually by the user after the dispensation order has been generated see section 7 11 4 PyroMark Q48 Autoprep User Manual 10 2015 91 If desired the dispensation order can be entered or adjusted 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
28. control for bisulfite treatment in methylation assays Base calling with quality assessment 7 1 Analysis modes PyroMark Q48 Autoprep Software has four analysis modes New AQ Assay Ctri A New SNP Assay Ctri N New CoG Assay Ctrl G New SEQ Assay Ctrl Q e AQ A variety of quantification studies of mutations such as SNPs and InDels SNP Genotype analysis of SNPs and InDels This mode does not provide quantification of alleles as in AQ mode CpG Methylation analysis of single or multiple CpG sites For methylation analysis at CoN sites a CpG assay is used with CoN mode enabled during the assay setup SEQ Base calling of unknown sequences PyroMark Q48 Autoprep User Manual 10 2015 70 The four different types of analysis can be performed on the same PyroMark Q48 Disc To toggle between the analysis modes in the Analysis view select AQ SNP CpG or SEQ in the toolbar 7 2 Shortcut browser The shortcut browser provides a quick and easy way to access folder contents and commonly used assay and run files A File Tools Reports Window Help a oo 2 Shortcuts 3 Example Files AQ 1 Z CpG 1 gid Example run vei SEQ 1 Waa SNP 1 Ca E My PyroMark Files Explore Refresh F5 New Assay gt BE New Run Remove Shortcut Properties PyroMark Q48 Autoprep User Manual 10 2015 71 The following icons are used to display information about the files AQ assay file I SNP assay file
29. d alimentation situ l arri re de l appareil et d brancher le cordon d alimentation de la prise de courant PyroMark Q48 Autoprep User Manual 10 2015 172 15 3 S curit lectrique Avant l entretien d brancher les cordons d alimentation des prises de courant AVERTISSEMENT Danger lectrique W5 A Toute interruption du conducteur de protection conducteur de terre de masse l int rieur ou l ext rieur de l appareil ou toute d connexion de la borne du conducteur de protection est susceptible de rendre l appareil dangereux Toute interruption intentionnelle est interdite Tensions mortelles l int rieur de l quipement Lorsque l quipement est reli l alimentation les bornes peuvent tre sous tension et l ouverture de capots ou le retrait de pi ces risque d exposer des l ments sous tension Ne pas r pandre de liquide dessus ou l int rieur de l instrument En cas d coulement accidentel de liquide sur l instrument d brancher imm diatement l instrument de sa source principale d alimentation lectrique Afin que le PyroMark Q24 Instrument fonctionne de mani re satisfaisante et en toute s curit suivre les conseils suivants Les cordons d alimentation de l appareil doivent tre branch s sur des prises d alimentation munies d un conducteur de protection terre masse Les fiches d alimentation reli es au secteur doivent tre facilement accessibles s il est n cessai
30. hand side of the absorber strip cavity 9 o clock position Absorber strip removal CAUTION Removal of absorber strip C15 A Upon completion of the run the absorber strip will contain Denaturation Solution which contains sodium hydroxide This can be irritating to the eyes and skin Always wear safety glasses gloves and a lab coat when removing the absorber strip Disposal of the absorber strip should be in accordance with all national state and local health and safety regulations and laws PyroMark Q48 Autoprep User Manual 10 2015 46 1 Remove the absorber strip by pulling it out Ensure that excessive amounts of liquid do not drip into the chamber The chamber cavity can be cleaned with ethanol to remove any excess waste left behind 6 2 3 Cartridge loading and injector priming Injector priming ensures the correct operation of the injectors by loading up the injector system with liquid and removing all the air During injector priming some of the liquid in the reservoir will be used Therefore it is important that the minimum loading volume be adhered to to ensure that adequate levels of reagent remain to achieve the sequencing run PyroMark Q48 Autoprep User Manual 10 2015 A7 1 Open the injector cover and then open each individual cartridge lid Set Up Injectors Fun Hame dBA amp 1 Select injectors to prime all used injectors willbe tested 2 Select if this is the last run today 3 L
31. highlighted with a gray background color and vice versa Sequence to Analyze TGTTN SAMATGATNCAG A Generate Dispensation Order Dispensation Order 1 CS TAGCACATGCATAGCA The arrow in the sequence to analyze the dispensation order and the histogram show the position of the cursor Note If the last variable position in the sequence to analyze is a long InDel dispensation will only be performed until three variable peaks are found providing the requirement of five reference peaks is fulfilled To dispense the whole InDel add a variable position after the InDel or adjust the dispensation order manually Note If it is not possible for the sequence to come in phase before 32 alleles are dispensed the dispensation order will not be completed For example the sequence ACTCDDDDG will have the dispensation order ACTC since the four D polymorphisms will generate an out of phase stretch over too many alleles Dispensation warnings If the dispensation order contains a warning this is displayed by an information symbol at the end of the Dispensation Order 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 PyroMark Q48 Autoprep User Manual 10 2015 92 the mouse pointer over the exclamation mark a tooltip will display an explanation of the warning Warning Suggested action Sequence uncertain due to lack of te
32. kits indicated for use with the PyroMark Q48 Autoprep for the applications described in the kit handbooks PyroMark Q48 Autoprep User Manual 10 2015 If the PyroMark Q48 Autoprep is used with other than QIAGEN kits it is the user s responsibility to validate the performance of such product combination for any particular application The PyroMark Q48 Autoprep System is intended for use by professional users trained in molecular biological techniques and the operation of the PyroMark Q48 Autoprep 1 4 Requirements for PyroMark Q48 Autoprep users This table covers the general level of competence and training necessary for transportation installation use maintenance and servicing of the PyroMark Q48 Autoprep Task Delivery Installation Routine use running protocols Assay design and validation Cartridge maintenance Preventive maintenance Servicing PyroMark Q48 Autoprep User Manual Personnel No special requirements Laboratory technicians or equivalent Laboratory technicians or equivalent Scientist or equivalent Must be performed at a QIAGEN Service Center Laboratory technicians or equivalent QIAGEN service personnel or service technicians of an authorized agent 10 2015 Training and experience No special requirements Appropriately trained and experienced personnel familiar with use of computers and automation in general Appropriately trained and experienced personnel familiar with use o
33. of the run the absorber strip will contain Denaturation Solution which contains sodium hydroxide This can be irritating to the eyes and skin Always wear safety glasses gloves and a lab coat when removing the absorber strip Disposal of the absorber strip should be in accordance with all national state and local health and safety regulations and laws Disposal of plasticware Used plasticware e g PyroMark Q48 Disc may contain hazardous chemicals or contagious biohazardous materials Such wastes must be collected and disposed of properly according to local safety regulations 2 9 Maintenance safety Perform the maintenance as described in section 8 QIAGEN charges for repairs that are required due to incorrect maintenance WARNING CAUTION A WARNING CAUTION A Risk of personal injury and material damage W11 Only perform maintenance that is specifically described in this user manual Risk of electric shock W12 Do not open any panels on the PyroMark Q48 Autoprep Only perform maintenance that is specifically described in this user manual PyroMark Q48 Autoprep User Manual 10 2015 18 CAUTION A CAUTION A CAUTION A A CAUTION A Damage to the instrument C10 Do not use solvents or reagents containing acids alkalis or abrasives to clean the PyroMark Q48 Autoprep Damage to the touchscreen and computer C11 Do not pour or spray liquids e g cleaning agents on to the PyroM
34. reagents e High purity water Milli Q 18 2 MQ x cm or equivalent Sterile pipet tips with filters for the supplied electronic multi dispense pipet please refer to the user manual of the electronic multi dispense pipet 6 2 1 DNA amplification biotinylated PCR product serves as the template for the Pyrosequencing reaction Amplify the DNA to be analyzed by PCR with one of the primers biotinylated To receive valid analysis data see Appendix Assay design and validation 6 2 2 Absorber strip The absorber strip is an absorbent strip inside the instrument which collects liquid waste spun out from the disc wells during a wash cycle and liquid waste from the priming and cleaning steps PyroMark Q48 Autoprep User Manual 10 2015 45 Important The absorber strip has a maximum capacity of 8 ml If a run exceeds this limit in terms of calculated waste disposed the software will inform the user to replace the strip prior to conducting another run prime or clean Not replacing the absorber strip as recommended could result in some or all of the following issues e Oversaturation of the absorber strip and liquid overflow into the instrument chamber Condensation within the instrument due to high moisture content in the absorber strip e Microbial growth Absorber strip insertion Insert the absorber strip into the absorber strip cavity by sliding it into place ensuring that it sits level Ensure that the ends meet on the left
35. results with other dispensation orders Where possible SEQ assays that involve resolution of different sequences should be designed such that resolution does not depend on accurate sequencing of homopolymers In addition it may be useful to have a few known bases at the beginning of the sequence preferably single peaks These can be used as reference peaks to aid the setting of the peak levels in difficult assays Ensure that the initial DNA sample is pure or that the assay is capable of specifically amplifying and or sequencing only one target sequence in the sample The assay may otherwise generate mixed sequence that cannot be analyzed Pay attention to tips and warnings indicated by the red icon and make suitable modifications when the icon appears PyroMark Q48 Autoprep User Manual 10 2015 159 Validation of a new assay Controls All new assays have to be validated by the user Use a reference DNA sample when testing a new assay and ensure that appropriate analysis parameters in the PyroMark Q48 Autoprep Software are used Interactions between primers or loops formed on single stranded DNA can serve as priming sites for base incorporation by DNA polymerase The following controls should be included when an assay is analyzed for the first time PCR without template DNA This will show if the primers interact to give a background signal in Pyrosequencing reactions PCR with template DNA but with no sequencing primer
36. see section 7 12 2 3 Optional Enter information about the assay in the Assay Note text box Note An assay note can be displayed in a tooltip in the shortcut browser by positioning the mouse pointer over the assay file 4 Before running your samples validate your assay using a reference DNA sample see Appendix A Assay design and validation 5 Optional If applicable during the assay validation edit the analysis parameters 6 Optional Lock the assay for editing by clicking the Lock Assay button at the bottom of the assay setup window A locked assay A that has been run on the PyroMark Q48 Autoprep Instrument cannot be unlocked i e it will not be possible to edit the analysis parameters or results after the assay has been processed 7 Click ball in the toolbar to save the file If the file has never been saved select location and enter the filename in the dialog box that opens 7 12 2 Enter the dispensation order Type the dispensation order into the Dispensation Order text box The following rules apply when entering the dispensation order in the software The allowed characters for input are A C G and T To repeat a group of bases use numbers in combination with parentheses e g 3 CTGA corresponds to CTGACTGACTGA PyroMark Q48 Autoprep User Manual 10 2015 99 If the dispensation order contains an error this is displayed by a red exclamation mark at the end of the text box Position the mouse
37. see section 7 13 5 5 To enter a sample ID or note select the cell and enter the text A selected cell is highlighted with a blue background color Assay Name Assay Mare PyroMark Q48 Autoprep User Manual 10 2015 A3 6 In the protocol drop down list select the desired protocol for the run Note There are 2 available protocols for running the PyroMark Q48 Autoprep which are selected in the PyroMark Q48 Autoprep Software during run setup Standard protocol The Standard protocol is used as the default P4 Extra protocol The P4 Extra protocol enables the use of the fourth sequence primer injector P4 When using this protocol the binding buffer must be loaded manually by the user because the BB P4 injector will be used to dispense sequence primer instead of binding buffer 7 Sequencing primers can be added manually or automatically Either manual or automatic primer loading must be selected in the Run Setup In the latter case one injector can be chosen for each assay Primer Loading Manual Automatic 8 Click hell in the toolbar 9 Print the disc setup by selecting Pre Run Information from the Tools menu When the report opens click 9 10 Close the run file and copy it to the USB stick supplied The run file can now be processed by inserting the USB stick into the USB port at the front of the PyroMark Q48 Autoprep see section 3 4 Alternatively the run file can be saved on the networ
38. seting UP an oE GLASIO Seesen lee 99 7 12 1 Workflow to set up an SEQ Assay nn 99 7 12 2 Enter the dispensation Ode ee 99 7 12 3 Edit analysis parameters cers ee een ee ee 100 7 18 DOI UP RO nace ea ae ce ae en E 102 7 13 1 Workflow to set up a Un 102 7 13 2 Enter run parameters 103 7 13 3 Add assay Tiles to the dise ee 104 PyroMark Q48 Autoprep User Manual 10 2015 7 13 4 Print or export disc setup as image un ein nen 105 7 13 5 Define sample ID and note externally ccc cccecceeseeeeeceeeeeeeeeeeeeeeeaaaes 105 743 6 Review IG disc SEND a be ee 109 7 14 FOSSES ee ee ee ee 110 7 5 PUM ZU Cree 110 TVS Workliow loanalyze ati 110 7 92 Pralyzeseeee wel 111 7 15 3 View the analysis test bannen a anaes 113 7 15 4 Edit analysis parameter g scents ad came en a ee 117 7 15 5 Edit base called sequences ccmnnnessssssseennnnnnnneseeeennnnnnnnnenennenn 119 7 16 Viewing printing and saving Analysis Reports ssseseeeeeeeeeeeeeeeeeeees 119 7 16 1 Analysis statistics report only AQ and CpG modes 120 7 16 2 Analysis results TON constanessahncsudienstansisdumnesnddandeandecdtanst dasnanannconns 122 7 10 3 Pyrogram report an sense 124 PO O PPPBUEERRIUERERTERFENEREREFEREHEEEEEEERUEUREEHEEUDENEEUEUEEEREEREINERERERPEHERTUUERERER 126 ENF SU DOMES een een 128 7 18 General Hints and ibn 128 7 18 1 VOOR esi 128 O2 RC ee een eae steer 128
39. shortcut browser rightclick it and select Refresh from the context menu or press the F5 key Note It is also possible to open the run file by double clicking it in Windows Explorer 3 All wells on the disc with an Assay Setup corresponding to the selected analysis mode will be automatically analyzed 4 View the analysis results see section 7 15 3 5 Optional If applicable modify how the analysis is performed see section 7 15 4 6 Optional Enter an analysis note in the Note text box in the Overview tab Note To expand or collapse the Note field click or 5 7 To save the analysis results click lal in the toolbar Note A dialog box will request confirmation of any modifications made in the Overview or Analysis Setup tabs prior to switching from one tab to another or upon selecting orange outline another well Note It is not possible to edit the analysis parameters or enter an analysis note for a locked assay Ah 7 15 2 Analyze selected wells Analysis of selected wells can be initiated by clicking the Analyze Selected Wells button Ml in the Overview tab It is also possible to rightclick the selection and select Analyze Selected from the context menu see section 7 7 During the analysis a progress dialog box is shown This dialog box 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 The progress dialog box closes when the ana
40. 0 pl of PCR product add high purity water to the sample to give a final volume of 10 yl Samples with no names will be displayed as No sample ID PyroMark Q48 Autoprep User Manual 10 2015 54 Load Template Load Syl of beads and 10yl of the indicated template Well Assay Sample A1 AQI lt No Sample ID E A2 AQI lt No Sample D gt AJ AQ 1 No Sample D gt Ad AQ 1 lt No Sample ID A5 AQI No Sample D gt AG AQ 1 lt No Sample ID AT AQ 1 No Sample ID AS AQI lt No Sample D gt Note When running the P4 extra protocol the user will be prompted by the instrument to additionally pipet 5 pl PyroMark Binding Buffer into each well 2 Ensure that the template thoroughly covers the beads inside the well 3 Ensure that the disc is correctly inserted into the instrument and locked in place see section 6 2 4 6 3 Starting a run 1 After the template has been loaded begin the run by selecting the Start button 2 Aconfirmation message will display Close injector cover Start run To start the run close the injector cover and select Yes To go back and make any adjustments or to confirm any setting select No If the injector cover is not closed at the beginning of the run the following warning will appear Injector cover check Higher than expected signal detected Check the injector cover is closed and select retry 6 4 Monitoring a run When a run is started the instrumen
41. 167 Orderingilniormalion 28 die 167 14 Appendix D Legal Inom ation ten 169 Warranty statement 169 0 10 1 Inge 0 1 1 FR RER RE ER 169 15 Appendix E Safety Information French FR 171 15 1 Consignes de s curit ee eier 171 15 2 Utilisation appropri e ide 171 I5 CCU HS Eee ee 173 154 CRC DO eer Bene 174 15 5 Produns CHIMIQUES a ee ee een 174 15 6 Dangers TES AMIS a de eg 175 IS Danger esa lachdlei anne Pe eee ee 175 138 CSS OR ee 176 15 9 Traitement des d chets ee ee ee ne 176 15 10 S curit relative la maintenance cceeccccccccceeeeeeeeeeeeseaeeeeeeeeeeeeeeeeees 176 16 Appendix F Safety Information German DE 178 16 1 SIENEINE SEE ene ee 178 16 2 Sachgem fe Handhabung c cc cecccseeeeececccccceeeceeeessssseeeeeeseeeeeeeeeeeees 178 PyroMark Q48 Autoprep User Manual 10 2015 103 Schulz VON Ss OSG MN ee ee 180 164 Biologische Sicherheit en 181 105 CME RING e EA EE ee ee 181 16 6 Gefahren durch mechanische Teile cccccccccccceceeeeessssseeeeeeeeeeeeeeees 182 16 7 berhitzungsgefohr sed ace cgeeccnta ee ee 182 10 9 Mmmm T Re er ne ere anemone 183 19 7 ADR SCOR ere eee erie near oe ee eee 183 16 10 Sicherheitshinweise Wartungsarbeiten 183 A NIE NENNE EERURNCHBUERIUERR E E PERF DEREN NE OREET DEERWERE 185 PyroMark Q48 Autoprep User
42. 3 nee 74 7 3 2 Tools menu for processed run files 75 723 3 RECO S TO WU scoparia nn nts vies ores pre ee 76 7A Window SA er ese 76 7233 ee ee 77 730 Anasi Toll ee 77 PyroMark Q48 Autoprep User Manual 10 2015 7 4 Workflow views cccccccecceccecceceecuccuceececcuceecceuceesescaseuceesaseseesecseuceneusenseneas 77 RSS ON I ENEE ENESE EENE TEN EES cen 77 ZA 2 BUNDES Wear D ATAA 78 2A PCN SIG MEN ees sete oon ese een ale 78 7 3 NEN OR eee eee ere ee eee nee ee 78 70 PANS Sep D ee 80 Er lt 6 1016 Ba eee ee nee a a eae eee eee eee ee 81 7 9 1511020 ae ar ee ee ee 82 PCM COMIN SHOW ces es sect ee en ee 83 7 8 2 Export the histogram as an image ccccccceceeeeeseeeceeeeeeeessaeeeeeeeeeeaaaeeees 83 Fee MRO sce ge pees teats ones prt cae eo ee once se epee daar aoe eve ee steps eae ec bane 83 79 1 AQ SINP and Sessel 84 1 0 SE OS ONE ee a de 85 7 93 LOOM Pylogram Sn een 86 7 9 4 Export Pyrogram as an image 86 7 10 Starting the software 86 7 11 Setting up an AQ SNP or CpG Assay 87 7 11 1 Workflow to set up an AQ SNP or CpG Assay nennen 87 7 11 2 Enter the sequence to analyze 88 7 11 3 Generate the dispensation order cccccccccceeeseseeeeeeeeeeeesaeeeeeeeeeesaaaee ees 91 7 11 4 Add or remove bisulfite treatment controls CoG assays 93 7 11 5 Setup variable positions de en nn 94 7 11 6 Edit analysis parameters ndlr 96 7 12
43. AL T CAL TOA 5 ID LS 20 75 ct 35 an 45 Eu 55 Ao 65 70 75 a0 ES 0 as 100 105 Lia 115 13 Fun Mile bc hea bo enter Hun Motes gbh rii iseis cee eee ee eee eee eee ee eee ee eee eee eee ee ee eee eee eee eee eee reed eee eee eee eee eee CGR TAC TRACTS TAC PSC TC TRE SG PG TOUS TC Te TOC TC TOC TAC PSS CGA Ta TGS TOM TOC Te TOC TOC To 5 La 15 20 5 a 35 a a5 Eu 55 65 70 75 B AS Su GS 100 105 110 125 1 i 6 8 1 Quality assessments The disc overview in the Overview tab gives a quick overview of the quality assessments The color bar mm shows the quality assessment of all variable positions in the well or of all the bases in the base called sequence Quality colors L_ Blue Passed Yellow Check I Red Failed _ White Not analyzed Either analysis is not supported by the software e g analysis of SNP when in the CpG mode or the variable position has been deselected by the user AQ SNP and CpG assays only PyroMark Q48 Autoprep User Manual 10 2015 66 6 8 2 AQ analysis results A G 4 The allele frequencies are displayed in Pyrogram for example and af 4s InDel The quality assessment is displayed by the background color of the result 6 8 3 CpG analysis results The methylation percentage 26 or average methylation percentage Arszege 25 are displayed in the Pyrogram The quality assessment is displayed by the background color of the result A methylation bar in the dis
44. CAUTION Damage to the instrument C10 Do not use solvents or reagents containing acids alkalis or abrasives to clean the PyroMark Q48 Autoprep CAUTION Damage to the touchscreen and computer C11 Do not pour or spray liquids e g cleaning agents on to the PyroMark Q48 Autoprep Use a tissue moistened with water only for cleaning Important We recommend wiping the PyroMark Q48 Autoprep with a damp cloth only PyroMark Q48 Autoprep User Manual 10 2015 130 The following disinfectants and detergents are recommended for cleaning the PyroMark Q48 Autoprep Note If cleaning agents different from those recommended are used ensure that their compositions are similar to those described below If in doubt please contact QIAGEN Technical Service prior to use General cleaning of the PyroMark Q48 Autoprep e Mild detergents e 70 ethanol or isopropyl alcohol 8 2 Light detector maintenance To avoid decreased signal levels we recommend checking the light detector every 2 4 months and cleaning the photomultiplier tube PMT window when dirty To clean the PMT follow these steps Manually move the chamber lid to the fully open position all the way to the back Turn the instrument onto its side Clean the PMT window with a lint free tissue suitable for cleaning optical lenses CAUTION Damage to the instrument C13 A Do not expose the photomultiplier PMT to strong light during maintenance Important
45. D and permits the user to select wells see section 7 7 The Well Information pane summarizes all information associated with the well selected in the disc overview The Pyrogram of the selected well is displayed at the bottom of the tab Details about the assay of a selected well are visible in the large main pane including assay name sequence to analyze nucleotide dispensation order histogram and notes entered during assay setup In addition three tabs display the analysis setup variable positions analyzed and the analysis parameters used 7 7 Selecting wells The following methods for selecting wells can be used in the disc overview of both the Overview and the Analysis Setup tabs The color schematic for wells applies to both tabs To select a single well simply click on it OCTO 888 To select a rectangular group of wells for example A2 A3 B2 B3 and C2 C3 Press and hold down the left mouse button while dragging the mouse pointer from well A2 to C3 or Select well A2 and press and hold down the Shift key while selecting well C3 or Select well A2 and press and hold down the Shift key while pressing the Right Arrow key twice and the Down Arrow key twice Ja ae 5 8 PyroMark Q48 Autoprep User Manual 10 2015 81 To add wells to the selection above for example wells B7 and C7 press and hold down the Ctrl key while selecting the wells A A SOC poa de OOO rE lt ADHOOO0
46. ERESULEREUREEUELERIENKERERBELEFEREEEERER 156 Quality of starting template cccccccccccccccceccceeeeeeeeseeeeeeeeeeeeeeeeeeeeeeeeeaaaaaaas 156 Quality of starting template when performing CpG assays 156 Quantity of starting template ccccccccccccccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeaaaaaaas 157 PER ORMMUIZONO 1 PER ee ee ee 157 Equal amplification of both alleles in AQ and CpG assays ueeeeeeeeeeeeeeeeeeeeeeeen 157 Templale pre Bar le ee ee ee 158 Pyroseguencing analysis ee ee een 158 POS Reese ee ee ee ee 158 TAG SPORE a ET 158 Bisulfite treatment controls in CpG assays ccccccccccccceeceeeeeeeeessseseeeeeeeeeeeeeeees 159 Sl eo E ne EN errand eee ern tere ere eee ne ens ern ee 159 Validation of a news nee 160 CSU IS ee see ss E semen eee 160 PyroMark Q48 Autoprep User Manual 10 2015 UM a ses 2 A PR En 160 Analysis TOURS E ance ees 161 l2 Appendix Technical Pia een een ee ee 162 Mechanical data and hardware features 162 OBeralng Con ons rennen een 162 Tampon CORAIS eera een re ee 163 orade COCA en ne eee rem ee ere eee ee 163 SRE cei eee a crenata ewe E 164 CR ee VENEN ee ee E 164 Waste Electrical and Electronic Equipment WEEE ccccccceeeeeeesseeeeeeeeeesaeee ees 164 pO Dee la ol ee ee ee ee ee 165 legal NARUTO te tt et a ee 166 13 Appendix C PyroMark Q48 Autoprep Accessories cccceeeececeeeeeeesseeeeeeeeeeeaas
47. Name User Name Password O Obtain an IP address automatically O specify an IP address IP Address 0 0 0 0 Default Gateway 0 0 0 0 i Subnet Mask 0 0 0 0 3 DNS Server 1 0 0 0 Not Connected Test Save Changes Touching any of the text boxes will result in a keyboard being displayed in the bottom half of the screen that can be used for setting entry The visible portion of the settings window can be scrolled by touching and dragging the window The keyboard can be dismissed by touching the blank background of the visible portion of the setting window Network Path serveripath I Domain Name DOMAIN User Name user m Ed a wlelr t v ul ilo pik LX 1 MW A ES ak j Miia i l o mem ajsidjfjqgj hijfik l Once the settings have been entered touch the save changes button to save the settings It may take a minute for the instrument to successfully reconnect to the network if you changed the IP Address Settings see section 5 7 4 If the instrument has successfully connected to the network it will be possible to test the settings entered by touching the test button If the instrument was able to read and write to the specified PyroMark Q48 Autoprep User Manual 10 2015 38 network share a success message will be presented otherwise an error message will be displayed Contact the network administrator for assistance if
48. OC on on To deselect a well press and hold down the Ctrl key while clicking the well Note If several wells are selected in the disc information for the well with the orange selection frame in the Analysis view is shown in the Well Information area etc Colors of wells in disc overview diagrams Selected well a dark blue outline in the Overview tab or a black outline in the Assay Setup tab Most recently selected well an orange outline e Active analyzed wells a light blue outline and pale blue background Active unanalyzed wells a light blue outline and gray background Inactive wells a gray outline and background Inactive wells cannot be selected Well with an error a red cross 7 8 Histogram Histogram showing a theoretical CpG assay result When setting up an AQ SNP or CpG assay the theoretical representation of the expected Pyrosequencing peak pattern is presented in the Histogram area The following icons and colors are used in the histogram PyroMark Q48 Autoprep User Manual 10 2015 82 Variable regions which contain one or more variable positions are highlighted with a blue gray background color When showing reference peaks blue diamonds are displayed above the reference peaks A filled blue diamond indicates the reference peak is enabled and a hollow blue diamond indicates the reference peak is disabled Reference peaks may be disabled or disabled by leftcl
49. October 2015 PyroMark Q48 Autoprep User Manual Sample to Insight Contents POT e EEEE E AE EE E T E ENTE A EE ETT 10 1 1 About this user many ser een 10 LAC Se OI SSI tenec 11 AE Technical assistante a no an nen a ee eer er ee ee 11 E22 PONS NS 1 1 ee ee 11 1 253 Version management eee pra 11 1 3 Intended use of the PyroMark Q48 Autoprep 11 1 4 Requirements for PyroMark Q48 Autoprep users ccccecc cece eee ssssteeeeeeseeeeeeeees 12 sarei Alla SOA saa sece dpa E EEE E E EE 13 Dall ROOST Se NE E E E E E E 13 22 CO oe re 15 293 Bologa Sa ea ne oe ee ee 16 ZA Chen a ee 16 2 5 NC ee ee 17 20 ea NG ZS cee ee ee ne 17 PC OS E nee ee eee REN 18 NE ee ee 18 29 NOIR SCC Sale ee ee lee te el ct ee 18 2 10 Symbols on the PyroMark Q48 Autoprep products 20 General Descr piOn EE ee 21 3 1 PyroMark Q48 Autoprep definitions cccccccccccccccececeeeeeeeeaeseeeeeeseeeeeeeeeeeees 21 3 2 Pyrose vencing PrINCIpI s a pee 21 3 3 PyroMark Q48 Autoprep principle ccccccecceccccceeeeesseeeeeeeeessaaeeeeeeeeeeeaaas 23 3 4 PyroMark Q48 Autoprep instrument ccccceeeeeccceeeeeeeseeeeeeeeeeesaaeeeeeeeeeeeaaas 24 OAD Ineco Catan nee 25 SAD OGE a ee en ee 27 3 9 MGI SIS SO AI apps ess pastes aces nee meinen 28 Stall On TPS AUR A gsc eeepc eect ee ee ee 29 Al SySlEn EVER e e E A E A ee 29 PyroMark Q48 Autoprep User Manual 10 2015 22
50. PyroMark Q48 Autoprep Software CD Multi dispense pipet Reagents and other accessories can be ordered separately visit www giagen com 4 2 Requirements 4 2 1 Installation site The PyroMark Q48 Autoprep must be located out of direct sunlight away from heat sources and away from sources of vibration and electrical interference Refer to Appendix B Technical Data for the operating conditions temperature and humidity The site of installation should be free of excessive drafts excessive moisture excessive dust and not subject to large temperature fluctuations Refer to Appendix B Technical Data for the weight and dimensions of the PyroMark Q48 Autoprep Ensure that the workbench is level dry clean vibration proof and has additional space for accessories Approximately 40 cm 15 8 in clearance above the workbench is required to accommodate the PyroMark Q48 Autoprep with the chamber lid open Allow at least 20 cm 8 in of free space behind the instrument to allow for opening of the chamber lid Operation of the PyroMark Q48 Autoprep lid is driven by a motor controlled by the software Insufficient space to operate the chamber lid may result in damage to the lid on opening PyroMark Q48 Autoprep User Manual 10 2015 The PyroMark Q48 Autoprep must be placed within approximately 1 5 m 59 in of a properly grounded earthed AC power outlet The power lines to the PyroMark Q48 Autoprep should be voltage regulated and sur
51. Pyrograms of different wells in section 7 15 3 Above the Well Information pane are dropdown menus to specify the type of information to be displayed for each well and an easy access button to initiate analysis of selected wells PyroMark Q48 Autoprep User Manual 10 2015 79 Right clicking on the disc overview in this tab opens a context menu with the following options Analyze Selected Open in Lower Area Copy as Image Print Run Information e Analyze Selected initiates analysis of all selected wells Open in Lower Area replaces the histogram with one or more Pyrograms of selected wells Copy as Image an image of the full disc layout is copied to the clipboard Print an image of the full disc layout is formatted for printing e Run Information the Run Information window is displayed see section 7 15 3 7 6 Analysis Setup tab The Analysis Setup tab displays information specific to the analysis performed on a specific well or selection of wells SNP Overview Analysis Setup Setup Variable Postions Analysis Parameters Assay Name SNP 1 Sequence to Analyze 2 we LINN FCCGATTICAT AN nn Well Information A5 Dispensation Order Assay SNP 1 TCGATCA Sample ID Replicate 1 ve Note Histogram 5 T CCGATTTCAT PyroMark Q48 Autoprep User Manual 10 2015 80 In the upper left pane is a schematic of the disc overview that displays the disc I
52. RE EN Se ENIA 29 ROIS OS een een 29 4 2 2 Power T QUIT M NIS nennen 30 423 Grounding feo dike Me Misch ae en n a ae 30 43 Unpacking nes oO testes ee 30 4 4 Installation of the analysis software 33 4 4 1 Installing PyroMark Q48 Autoprep Software 33 4 4 2 Uninstalling PyroMark Q48 Autoprep Software 34 2 Instrument Administration ccceccececceeeeeceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeesesssaaaeneeeeeeeeeeeees 35 5 1 SSM daie and Re ee 35 52 Copying ON a ee ee nee eee 35 5 3 Viewing software and hardware version ccccccccceee esse eeseeeeeeeeeeeeeeeeeeeeeeaaaaaas 36 5 4 Upgrading the instrument software ueeeeeccaesenssseseeeeeennnnnnnnnnnnnnnnneneneneeenn 36 5 5 Reset Haraw re nenne entered her 37 9 0 WEIS SOL lock ee ee ee Bee 37 9 7 Connecting to a network a nsicdnoncsinccnstanecdudsavedaentawsisdeerennenduduavasecstarsiniuoncsneototeests 38 5 7 1 Network share SEMNMS ice saisine 39 572 NN OS Palhier a aE er 39 5 7 3 User Name Domain Name and Password c cscceccecceceecescuscescescasens 39 57A IFAddess EINS eee ee eee en ee een 40 6 POS RO a ona pa ee mn oi 41 Ol Preparing HIN een a a at N Doit Al 6 1 1 Starting PyroMark Q48 Autoprep Software nennnnennnnnnnnnn A1 OR Selina Up GIN SSA Re pte re eese ee ee 4 Oko SL ole MV co EE A ETE 42 6 2 Preparing templates and reagents uen eneee ae 44 e 2 DNA OP AS E EO 45 022 ABDe P ee nee ee me ner 45 6 2 3 Cartridge loading and injector p
53. SEMENT Le terme AVERTISSEMENT signale des situations risquant d entra ner des N accidents corporels dont l utilisateur ou d autres personnes pourraient tre victime Les d tails concernant ces circonstances sont donn s dans un encadr identique celui ci ATTENTION le terme ATTENTION signale des situations risquant d entra ner des N d t riorations de l appareil ou de tout autre mat riel Les d tails concernant ces circonstances sont donn s dans un encadr identique celui ci Les conseils donn s dans ce manuel ont pour but de venir compl ter les exigences de s curit habituelles en vigueur dans le pays de l utilisateur et non de s y substituer 15 2 Utilisation appropri e AVERTISSEMENT Risque d accident corporel et de d t rioration du mat riel w1 ATTENTION L utilisation inappropri e du PyroMark Q48 Autoprep peut entra ner des A accidents corporels ou une d t rioration de l appareil L utilisation du PyroMark Q48 Autoprep est r serv e exclusivement au personnel qualifi ayant t convenablement form L entretien du PyroMark Q48 Autoprep doit tre effectu uniquement par des sp cialistes de l entretien sur site QIAGEN ou par des techniciens d un agent agr PyroMark Q48 Autoprep User Manual 10 2015 171 ATTENTION A ATTENTION A AVERTISSEMENT ATTENTION A AVERTISSEMENT ATTENTION A ATTENTION A ATTENTION A D t rioration de l instrument C1 L
54. Treatment This enables the software to automatically generate the sequence to analyze and select the most appropriate bisulfite treatment control 3 Click the Generate Dispensation Order button If creating an SEQ assay enter the Dispensation Order PyroMark Q48 Autoprep User Manual 10 2015 A1 4 Click lll in the toolbar to save the assay Note Before running your samples validate your assay using a reference DNA sample see Appendix A Note When using QIAGEN kits use the settings stated in the kit handbooks Optional If desired enter a note about the assay in the Assay Note box Set up the variable positions in the Variable Positions tab AQ SNP and CpG assays only Lock the assay for editing by clicking the Lock Assay button at the bottom of the assay setup window If creating a CpG assay check that the software has selected a bisulfite treatment control If no bisulfite treatment control has been automatically selected add one manually if possible preferably at the beginning of the sequence A bisulfite treatment control can be added manually by adding a C before or after a T that was a C before bisulfite treatment in a forward assay or by adding a G before or after an A that was a G before bisulfite treatment in a reverse assay in the dispensation order a ogra Let i i il k i E u i i 1 LE Lil A T G A T E A G T C G T A T C 5 7 A T G A T ite k i0 je di 6 1 3 Setting up a run 1 C
55. a homopolymer Wrong input format PyroMark Q48 Autoprep User Manual 10 2015 Comments and suggestions The dispensation order is very long more than 200 Shorten the dispensation order to less than 200 The dispensations used to analyze the InDel will give a high signal Quantification may be uncertain Check results carefully One of the characters used is not recognized by the software Use characters from the IUPAC code A B C D G H K M N R S T V W Y and L 142 Message text Sequence not in phase at the end of dispensations The sequence contains less reference peaks than required The generated dispensation order contains less reference peaks than required General analysis messages Uncertain due to low peak height or Failed due to low peak height Uncertain due to wide peaks or Failed due to very wide peaks Uncertain due to high peak height deviation in variable position or Failed due to high peak height deviation in variable position Uncertain due to high peak height deviation at dispensation or Failed due to high peak height deviation at dispensation Uncertain due to baseline drift or Failed due to baseline drift PyroMark Q48 Autoprep User Manual 10 2015 Comments and suggestions One allele is sequenced ahead of the other s and the two or more alleles are not in phase with the last dispensation Add more sequence information i e lengthen th
56. ally enter an assay note A nucleotide dispensation order is generated by the software The Variable Positions tab displayed upon PyroMark Q48 Autoprep User Manual 10 2015 77 generating the dispensation order lists the variable positions in the sequence entered and allows the user to name the positions and indicate which should be analyzed By default all variable positions supported in the used analysis mode are selected for analysis The Analysis Parameters tab is used to specify parameters for the data analysis refer to Edit analysis parameters in the Analysis Parameters tab in section 7 11 6 The Revert to Default button resets default assay parameters and the Lock Assay button locks entered assay parameters so they cannot be altered during analysis see section 7 15 All changes made to an assay file are recorded in a Change Log which can be accessed by clicking the Show Change Log button 7 4 2 Run Setup view A new run file is created in the Run Setup view This view serves to enter run specific information such as disc ID Reagent ID and a note about the run Using the Disc Setup assays are added to individual or a group of disc wells 7 4 3 Analysis view Su EEE gt When a run is completed on the PyroMark Q48 Autoprep the Analysis view opens Alternatively this view becomes active when any processed run file is opened The color of the workflow arrow Analysis in the status bar reflects the selected analysis mod
57. an SEQ Assay 99 Shortcut browser 71 Sicherheitshinweise 178 Software Add assay files to the disc 104 Analysis log 128 Analysis modes 70 Analysis reports 119 Analysis Setup tab 80 Analysis Toolbar 77 Analysis view 78 Analyze a run 110 Analyze selected wells 111 Assay Setup view 77 Edit base called sequences 119 File menu and toolbars 74 General hints and tips 128 Help menu 77 Histogram 82 Homopolymers and uncertain bases 115 Install analysis software 33 Main menu and toolbars 74 Overview tab 78 Process a run 110 186 Protection of analysis results 129 Protection of files 129 Pyrogram 83 Quality assessments 114 Reports menu 76 Review the disc setup 109 Run log 128 Run Setup view 78 Select wells 81 Set up a Run 102 Set Up an AQ SNP or CpG Assay 87 Set up an SEQ assay 99 Shortcut browser 71 Start 86 Supported files 128 Tools menu 75 Uninstall analysis software 34 Upgrade instrument software 36 View and compare Pyrogram 116 View software version 36 View the analysis results 113 Window menu 76 Workflow views 77 Zoom Pyrogram 117 PyroMark Q48 Autoprep User Manual 10 2015 Standard protocol 44 Support packages 139 Supported files 128 Technical assistance 11 Technical data Mechanical data and hardware features 162 Operating conditions 162 Storage conditions 163 Transport conditions 163 User software 164 Template loading 53 Tools menu 75 Transpor
58. analysis Ensure you use the correct assay type in PyroMark ADSW PCR For PCR amplification we recommend using PyroMark PCR Kit QIAGEN which is specifically optimized for Pyrosequencing analysis and enables highly specific and unbiased amplification of template DNA for various Pyrosequencing applications such as mutation detection SNP analysis methylation analysis and base calling The convenient master mix format enables specific amplification of various starting materials such as genomic DNA from a variety of species as well as bisulfite converted DNA using only one protocol PCR primers One of the primers must be biotin labeled to enable immobilization to magnetic streptavidin coated beads during the preparation of a single stranded DNA template The orientation of the assay can either be forward or reverse The primer that needs to be biotinylated is indicated by the PyroMark ADSW The biotinylated primer should be purified by HPLC or an equivalent procedure since free biotin will compete with the biotinylated PCR product for binding sites on PyroMark Q48 Magnetic Beads Amplicon length The optimal amplicon length for Pyrosequencing assays is between 80 and 200 bp although products up to 500 bp may work well Amplicons for CpG assays should ideally be shorter than 200 bp PyroMark Q48 Autoprep User Manual 10 2015 155 Sequencing primer Design sequencing primers using PyroMark ADSW When designing an InDel assay it is hi
59. anual 10 2015 12 The following are instructions to create open and copy files and to view the run log for a processed run Create a new assay file by right clicking the desired folder and selecting New Assay and the desired assay type from the context menu Enter the filename and press Enter To set up the assay see section 7 11 for an AQ SNP or CpG assay or section 7 12 for a SEQ Assay Create a new run file by right clicking the desired folder and selecting New Run from the context menu Enter the filename and press Enter To set up the run see section 7 13 Copy a processed run file and rerun it by right clicking the run file and selecting Copy and Rerun from the context menu Note Only the run setup not the run and analysis data will be copied Copy a file by right clicking the folder containing the file and selecting Explore from the context menu Windows Explorer opens For more information press the F1 key to open the online help for Windows Explorer Note To avoid losing data do not copy a file that is open in PyroMark Q48 Autoprep Software e Open a file by double clicking it or right clicking the file and selecting Open from the context menu To open a processed run file select Open followed by the analysis mode AQ SNP CpG or SEQ View the run parameters and a run log for a processed run file by right clicking the file and selecting Run Information from the context menu PyroMark Q48 Autop
60. are added one at a time Note Deoxyadenosine alpha thio triphosphate dATPaS is used instead of natural deoxyadenosine triphosphate dATP since it is used efficiently by the DNA polymerase but not recognized by the luciferase As the process continues the complementary sequence is built up and the nucleotide sequence is determined from the pattern of peaks in the Pyrogram nucleotide sequence G C A GG cc T nucleotide added PyroMark Q48 Autoprep User Manual 10 2015 3 3 PyroMark Q48 Autoprep principle 1 The PyroMark Q48 Autoprep performs DNA sequencing using Pyrosequencing technology 2 The USB stick containing the run file created using PyroMark Q48 Autoprep Software is inserted into the USB port at the front of the instrument Alternatively the run file can be opened via network drive 3 The PyroMark Q48 Disc is placed into the instrument and the PyroMark Q48 Cartridges are filled with PyroMark Q48 Autoprep Reagents 4 The run is then started by the user 5 Reagents are dispensed into the absorber strip to ensure that the dispensation capillaries are flushed and filled with solution 6 Preparation of single stranded DNA is performed in an automatic process within the instrument This process includes a heating step chamber lid will open for this step to reduce the volume in the well a template binding step and a denaturation step to separate the DNA strands During this process several wash and centrifugat
61. ark Q48 Autoprep Use a tissue moistened with water only for cleaning Light detector maintenance C12 Use lint free tissues to carefully clean the window of the photomultiplier PMT Do not use paper tissues See section 8 2 for further instructions Damage to the instrument C13 Do not expose the photomultiplier PMT to strong light during maintenance Injector cleaning C14 Injectors must be cleaned within a 12 hour period following the last run to ensure long term injector performance Failure to comply can result in injectors blocking PyroMark Q48 Autoprep User Manual 10 2015 2 10 Symbols on the PyroMark Q48 Autoprep products Location Type plate on the back of the instrument Type plate on the back of the instrument Type plate on the back of the instrument Type plate on the back of the instrument Type plate on the back of the instrument Type plate on the back of the instrument Reagents solutions disc Reagents solutions disc Reagents solutions disc Reagents cartridge Inside the instrument cartridge Inside the instrument Cartridge wal R Fe amp 4 BH A A x PyroMark Q48 Autoprep User Manual 10 2015 Description CE mark for European Conformity Legal manufacturer Waste Electrical and Electronic Equipment WEEE mark for Europe FCC mark of the United States Federal Communications Commission RoHS mark for China the restriction of the use of certain hazar
62. ature is 60 C and 56 C for genomic DNA and bisulfite treated DNA respectively when using PyroMark ADSW 2 0 Addition of Q Solution provided with the PyroMark PCR Kit can improve PCR yield and specificity for difficult templates that for example have a high degree of secondary structure or templates that are GC rich For all PCR optimization tests analyze 5 pl of a 25pl PCR on an agarose gel and aim for one strong specific band with minimal excess of primers Please refer to the PyroMark PCR Handbook for further troubleshooting Equal amplification of both alleles in AQ and CpG assays Reliable results in quantification assays depend on equal amplification of both alleles and this must be carefully tested To ensure equal amplification in a CpG assay unmethylated DNA can be mixed with increasing proportions of completely methylated DNA We recommend using EpiTect Control DNAs which provide bisulfite treated completely methylated and unmethylated DNA in ready to use solutions Regression analysis of the frequency of one allele measured in the PyroMark Q48 Autoprep as a function of the input expected allele should give an R2 value greater than 0 9 PyroMark Q48 Autoprep User Manual 10 2015 157 For an AQ assay the allelic variants including the variable position can be mixed at different ratios similar to the procedure for a CpG assay If the variable position in an AQ assay is a SNP the easiest way to test for equal am
63. be used to increase or decrease the expected range You can also increase or decrease the expected range by 1 Positioning the mouse pointer over the left or the right end of the green area so that the pointer changes from a white arrow to 2 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 Examples of methylation ranges Il de gt 45 4 42 deb 45 42 42 1 Expected methylation 100 2 Expected methylation 0 100 3 Expected methylation 30 70 default 4 Expected methylation 0 To reset the parameters in the Variable Positions tab and the Analysis Parameters tab to their default values click Revert to Default PyroMark Q48 Autoprep User Manual 10 2015 7 11 6 Edit analysis parameters The default analysis settings have been set to give optimal analysis results for most assays If applicable during the assay validation the results may be improved by editing the analysis parameters e Edit analysis parameters in the Analysis Parameters tab Enable or disable reference peaks Enable or disable bisulfite treatment controls Adjust heights of histogram bars e Enable or disable variable positions and or change expected methylation ranges only CpG assays see section 7 11 5 Ensure changes are validated see Appendix A Assay design and validati
64. been idle for over a week To reduce the potential build up of material we highly recommend that intermittently used injectors be cleaned regularly Injectors with a green tick do not require a clean but can be selected if desired PyroMark Q48 Autoprep User Manual 10 2015 61 Clean Injectors P Step 1 7 Select injectors to clean X X X X 6 x x X 4 v A Cc G T DS E AB vw vw vw vw P1 P2 PJWB P4BB er 2 Remove the disc from the chamber 3 Check that an absorber strip is inserted refer to section 6 2 2 Important The absorber strip must be present to avoid liquid being emptied into the chamber during the clean 4 Pipet 200 pl of high purity water into the reservoirs of the injectors selected to be cleaned 5 Close and lock the cartridge lids 6 Select the Start button to begin the first clean Confirm Empty Injectors by selecting Yes During the first clean the chamber lid will move each injector into position over the front half of the absorber strip and begin dispensing the liquid out of the injector until the drop sensor detects that there is no more liquid in the injector Injectors that fail to fully empty will be indicated by a warning message describing the issue Note Depending on the run performed the residual volume in the injector may be too high to be dispensed within one cycle In this case a warning message will appear on the touchscreen Select Retry to start a second rou
65. ble site Not analyzable due to lack of data Assay setup messages in SNP mode The InDel position contains a homopolymer Genotyping may be uncertain the variable position contains a homopolymer Sequence uncertain due to lack of PyroMark Q48 Autoprep User Manual 10 2015 in a different application mode Two or more variable sites share dispensations and can therefore not be distinguished If possible use recommended dispensation order According to the sequence before bisulfite treatment this is not a possible bisulfite treatment control position Uncertain or failed bisulfite treatment controls are hints for incomplete bisulfite conversion Review the bisulfite conversion process Check nucleotide degradation Normal Pyrosequencing specific background may trigger this warning although bisulfite treatment was complete Peak heights deviate from expected levels Even at a variable position the software expects a certain pattern e g a certain sum of the peak heights Check sequence to analyze Check for unknown SNPs The software has detected that all four nucleotides have been dispensed but no signal was detected Clean the cartridge see section 8 3 3 in case there was a dispensation error If problem persists perform injector test see section 8 5 No peaks detected Check PCR yield Check sequencing primer Clean cartridges Check reagents Check correct usage of buffers The dispensations used to a
66. by the user AQ SNP and CpG assays only Homopolymers and uncertain bases For sequences containing homopolymers quantification of the base at the end of the homopolymer may be uncertain and should be checked by the user Uncertain bases are indicated in these ways By yellow in the quality bar of the disc overview By yellow in the quality bar above the base called sequence By surrounding the uncertain base in the base called sequence By a yellow bar above the blue bar in the histogram See section 7 15 5 to add or remove the uncertain base If the uncertain base is added to the base called sequence the disappear and the added base is shown in lowercase If the uncertain base is deleted the and the base are both deleted If an analysis containing uncertain bases is exported to a FASTA formatted report uncertain bases with an intensity of 1 5 or higher will be included while those with an intensity of less than 1 5 will not be included Methylation levels In the CpG mode a methylation bar in the Overview tab shows the methylation level for each CpG site or CpN site if CoN mode was used in the well see Get an overview of the results this section Methylation colors Light green Below the expected range Green Within the expected range Dark green Above the expected range PyroMark Q48 Autoprep User Manual 10 2015 115 View and compare Pyrogram By selecting an analyzed well in the O
67. c overview shows the methylation level for each CpG site in the well Light green Below the expected range ID Green Within the expected range D Dark green Above the expected range 3 GAYGGGYGGGTTYGTTTTTTTGAGAGYGAYGYGAGTTAATGGGAAGGTTTTGGGGTGATATTATGGGTTATTTTTAGGG 32 35 100 358 354 78 5 10 15 20 25 30 35 Example Pyrogram for a CpG assay Variable positions in AQ and CpG assays are highlighted with a blue gray background color and bisulfite treatment controls in CpG assays with a light orange background color 6 8 4 SEQ analysis results The base called sequence is displayed in the Overview tab The bases in the base called sequence and the peaks in the compensated Pyrogram are colored according to their quality assessments PyroMark Q48 Autoprep User Manual 10 2015 67 ee ITETGTGETTTCCAAGACCCAATGTGCAAATGTGATTTTCTAATAATATGTATTCACTGTGAAGGAAGTAAGTTGGTT le m j t E E E ET ml EAEE ee E PN A ELT ETTE ET TT EEE TTE E Qu A 5 10 15 26 25 30 35 40 45 50 55 60 65 70 75 60 BS 90 S5 100 105 110 115 120 125 130 135 140 145 150 155 160 IA F Li Example of a base called sequence and Pyrogram for a SEQ assay PyroMark Q48 Autoprep User Manual 10 2015 68 6 8 5 SNP analysis results The SNP is displayed in Pyrogram for example E E for heterozygote SNP and A for a homozygote SNP The quality assessment is displayed by the background
68. color of the result BE ATOGATIGAGTIAGT 1 Capac a ita EE ess 22 RS N me EEE IEEE EAEE EESE aa E PEA AE AE AE le ER ee re a a faeces Rene eae wane rt at beet s Example Pyrogram for a SNP assay 6 8 6 Analysis reports To generate a report select the desired report from the Reports menu For more information about the reports see section 7 16 Reports Window Help AQ Analysis Statistics Reports Window Help AQ Pyrogram Report SNP Pyrogram Report AQ Full Report SNP Full Report Window Help CpG Analysis Statistics Reports Window Help SEQ Pyrogram Report SEQ Full Report CpG Pyrogram Report CpG Full Report To view reports generated in PDF format a PDF reader must be installed on the computer Adobe Reader can be downloaded at www adobe com PyroMark Q48 Autoprep User Manual 10 2015 7 PyroMark Q48 Autoprep Software The PyroMark Q48 Autoprep Software enables rapid and straightforward assay and run setup for the PyroMark Q48 Autoprep instrument as well as accurate analysis of run results Instrument software and other components of the PyroMark Q48 Autoprep System afford High resolution quantification of di tri or tetra allelic mutations Genotyping and quantification of InDels Built in quality control for AQ SNP and CpG assays using sequence context Analysis of methylation at CpN sites Analysis of methylation in the presence of SNPs Built in quality
69. creen 5 5 Reset Hardware In very rare circumstances the instrument may freeze up with the chamber lid closed and locked and or with the heater ring in the up position Upon restarting the instrument the Reset Hardware button can be used to unlock the lid and return the heater ring to the home position enabling the instrument for another run 5 6 Transport lock Within the Systems folder the user can prepare the instrument for transport by selecting the Lock button Before transporting the device ensure that the cartridges are empty and removed from the instrument that there is no disc or lock nut for the disc inside the device and that the absorber strip has been removed Once the chamber lid is locked into place attach the transport lock screw and select OK to confirm Current Device Time 26 11 2014 11 02 36 a oma When starting the instrument after it has been transported select the Unlock button to open the lid The user must then remove the transport lock screw and confirm by selecting Yes in order for the chamber lid to open PyroMark Q48 Autoprep User Manual 10 2015 37 5 7 Connecting to a network The PyroMark Q48 Autoprep can be connected to a TCP IP network through the Ethernet port at the rear of the instrument The network page in the tools controls the configuration of this feature The network administrator should be consulted before configuring the network settings Network Path Domain
70. ction 7 7 you wish to open in the lower area 2 Rightclick the selection and select Open in Lower Area from the context menu Two or more Pyrograms are now displayed in the Pyrogram pane In the upper area of the pane is the Pyrogram of the well in the disc overview displaying an orange outline This Pyrogram is fixed In the lower area are the one or more Pyrograms of the additional wells PyroMark Q48 Autoprep User Manual 10 2015 116 selected for the comparison The Pyrograms are linked meaning that if the Y axis value for one of the Pyrograms is set manually the entered value is used for both Pyrograms The Pyrograms in the lower area are displayed one at a time 3 Select the well in the disc overview that should appear as the fixed Pyrogram in the upper area of the Pyrogram pane 4 Use the scroll bar in the lower area of the Pyrogram pane to change the Pyrogram displayed Analyze Selected Open in Lower Area Copy as Image Print Run Information To close the Pyrogram list in the lower area click X in the upper right corner of the lower area Zoom Pyrogram and view description of icons and colors For information on icons and colors used in the Pyrogram area and how to zoom see section 7 9 7 15 4 Edit analysis parameters The default analysis settings have been set to give optimal analysis results for most assays If changing these settings ensure the changes are validated see Appendix A Assay design and vali
71. d or the current quality assessments for the variable positions 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 specified well PyroMark Q48 Autoprep User Manual 10 2015 122 Report options A AQ Analysis Results Report Selection Sort By Al wells Row Selected wells Column Preview Save Cancel Options Note column Y Wamings column E Analysis version column E Position name column 4 Quality column E Original quality column A SEQ Analysis Results Report Sr Selection Wells All wells Row Selected wells Column Included Sequence Al Passed Passed Check Only Quality Window Preview Save Options E Note column W Waming column E Analysis version column W Quality columns In the Analysis Results Report dialog box there are the following options Option All wells selected wells Sort by row column All Passed Passed Check Only Quality Wind
72. dation Note When using QIAGEN kits use the settings stated in the kit handbooks Note It is not possible to edit the analysis parameters for a locked assay 9 1 Select the well or wells see section 7 7 for which you wish to edit the analysis parameters Note The changes will only be applied to wells that share the same assay and dispensation order as the displayed well To edit the analysis parameters for all wells with the same assay and dispensation order you only have to select one of the wells PyroMark Q48 Autoprep User Manual 10 2015 117 Edit analysis parameters in the Analysis Setup tab To enable or disable variable positions and or change expected methylation ranges only CpG assays see section 7 11 5 To edit other analysis parameters for an AQ SNP or CpG assay see section 7 11 6 To edit the analysis parameters for an SEQ assay see section 7 12 3 Note It is not possible to change the assay name dispensation order or assay note When finished click Apply In the resulting message box select if changes should be applied to all wells with the same assay name and dispensation order To all or to only selected wells with the same assay name and dispensation order To selected Note It is also possible to enable or disable reference peaks and bisulfite treatment controls CpG assays only in the Pyrogram in the Overview tab see Enable or disable reference peaks and bisulfite treatment controls
73. ddress Address Default Gateway Subnet Mask DNS Server Current IP 192 168 1 31 PyroMark Q48 Autoprep User Manual 10 2015 6 Performing a Run This section describes the workflow for how to prepare run and analyze a run on the PyroMark Q48 instrument 6 1 Preparing a run Before preparing a run we recommend reading and familiarizing yourself with the safety information in section 2 Detailed instructions on setting up a run can be found in section 7 13 or by using the F1 help function in the PyroMark Q48 Autoprep Software 6 1 1 Starting PyroMark Q48 Autoprep Software In the Windows Start menu select All Programs PyroMark PyroMark Q48 Autoprep 6 1 2 Setting up an assay 1 Click EX in the toolbar and select the required assay A new assay file is created el BF New AQ Assay Ctri A New SNP Assay Ctrl N New CpG Assay Ctri G New SEQ Assay Ctrl Q LEL IE For analyzing methylation at CpN sites select New CpG Assay and then enable the CpN mode by checking the box next to CpN mode enabled Alternatively you can create a new assay file in the shortcut browser by right clicking the folder you wish to place it in and selecting New Assay followed by the desired assay type from the context menu Note To add a shortcut to a folder in the shortcut browser click Add Folder Shortcut 2 Enter the sequence to analyze Note If creating a CpG assay we recommend entering the Sequence Before Bisulfite
74. des lunettes de protection des gants et une blouse de laboratoire pour retirer la bandelette absorbante L limination de la bandelette absorbante doit se faire conform ment toutes les r glementations et lois nationales r gionales et locales relatives la sant et la s curit limination des mat riaux en plastique C9 Les produits en plastique usag s tels que le PyroMark Q48 Disc peuvent contenir des produits chimiques ou des agents infectieux dangereux Ces d chets doivent tre convenablement collect s et mis au rebut conform ment aux r gles de s curit locales 15 10 S curit relative la maintenance Proc der la maintenance comme d crit la section 8 QIAGEN facture les r parations rendues n cessaires suite une maintenance inappropri e AVERTISSEMENT ATTENTION A AVERTISSEMENT ATTENTION A Risque d accident corporel et de d t rioration du mat riel W11 Effectuer uniquement la maintenance qui est d crite sp cifiquement dans le pr sent manuel d utilisation Risque de d charge lectrique W12 Ne pas ouvrir les panneaux du PyroMark Q48 Autoprep Effectuer uniquement la maintenance qui est d crite sp cifiquement dans le pr sent manuel d utilisation PyroMark Q48 Autoprep User Manual 10 2015 176 ATTENTION D t rioration de l instrument C10 A Ne pas utiliser de solvants ni de r actifs contenant des acides des agents alcalins ou des produits abra
75. diamond and a tooltip will describe the consequence of a click Note To toggle between viewing and hiding reference peaks in the histogram right click the histogram and select Show Reference Peaks from the context menu Adjust heights of histogram bars This feature can be used when previous experiences have shown a reproducible deviation in the measured pattern from the theoretical pattern Use this feature with care 1 Press and hold down the Ctrl 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 opens or increase or decrease the height by using the arrows next to the text box 3 To apply the new height press Enter Dork orange morenced height light orange decreased heighi PyroMark Q48 Autoprep User Manual 10 2015 98 7 12 Setting up an SEQ Assay 7 12 1 Workflow to set up an SEQ Assay New AQ Assay Ctrl A New SNP Assay Ctrl M New CpG Assay Ctrl 6 New SEQ Assay Ctrl Q LEA ox pod 1 Click in the toolbar and select New SEQ Assay A new assay file is created Alternatively you can create a new assay file in the shortcut browser by right clicking the folder you wish to place it in and selecting New Assay and New SEQ Assay from the context menu Enter the filename and press Enter To add a shortcut to a folder or drive click Add Folder Shortcut 2 Enter the dispensation order
76. dous substances in electrical and electronic equipment RCM mark for Australia New Zealand supplier identification N17965 Identification of production batch lot Use by date Temperature limitation Read the manual Warning consult user manual Warning hot surface Irritant substance Denaturation solution NaOH 20 3 General Description The PyroMark Q48 Autoprep uses proven real time sequence based Pyrosequencing technology for sequence based detection and quantification in genetic analysis and epigenetic methylation studies The system can analyze up to 48 samples simultaneously An easy to use automated protocol is used to prepare single stranded DNA samples after PCR This protocol uses magnetic streptavidin coated Sepharose beads PyroMark Q48 Magnetic Beads which bind to the biotinylated PCR strand Annealing of sequencing primers can be automated for up to four different sequencing primers If more sequencing primers are used the primers can be manually added to the single stranded DNA samples 3 1 PyroMark Q48 Autoprep definitions PyroMark Q48 Autoprep Instrument Instrument only PyroMark Q48 Autoprep Software Software only PyroMark Q48 Autoprep Instrument software and installation PyroMark Q48 Autoprep System All of the above plus any PyroMark Q48 Kits 3 2 Pyrosequencing principle Pyrosequencing uses sequencing by synthesis for accurate and quantitative analysis of DNA sequences
77. e Quality colors in section 7 15 3 Select to show the mean methylation percentage of all CpG sites or CpN sites if CoN 4 mode was used in the well Select to show the methylation bar The methylation bar shows the methylation level for each CpG site or CpN site if CoN mode was used in the well See Methylation colors in section 7 15 3 Note Wells with a high substrate peak will be marked with an information icon in the disc overview This will not affect the quality assessments Note If analysis parameters quality assessments or base called sequence for SEQ results have been edited by the user the well is marked with a warning icon Ay Note If an assay is locked the well is marked with the icon PyroMark Q48 Autoprep User Manual 10 2015 113 Print or export disc overview as an image The disc overview can be printed or copied as an image to the clipboard by right clicking the disc 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 Analysis warnings By selecting an analyzed light blue well the analysis warnings if any are listed in the Well Information area An analysis warning affects the quality assessment in the following way AQ SNP and CpG assays Affects the quality assessment for either all variable positions or a single position If several warnings of the same kind were tr
78. e This view is used to manage the analysis of individual or groups of disc wells including making changes to analysis parameters for unlocked assays Information for analysis is displayed in two tabs the Overview tab see section 7 5 and the Analysis Setup tab see section 7 6 Note A dialog box will request confirmation of any modifications made in the Overview or Analysis Setup tabs prior to switching from one tab to another or upon selecting orange outline another well 7 5 Overview tab The Overview tab in the Analysis view displays the disc overview with well specific information Directly below in the Pyrogram pane is the Pyrogram of the well selected in the disc overview The corresponding histogram is displayed beneath the Pyrogram To the right is the Well Information pane and the Run note which display information specific to the well selected in the disc overview PyroMark Q48 Autoprep User Manual 10 2015 78 M O Assay ce or Erz poron oep na g EB Replicate 1 i i Well Information Sample ID Replicate 1 Note Click here to enter Run Notes The Pyrogram of the selected well always appears in the upper area of the Pyrogram pane and the histogram for AQ SNP and CpG assays or compensated Pyrogram for SEQ assays appears in the lower area It is also possible to replace the histogram in the lower area with one or more Pyrograms of selected wells see Simultaneously view
79. e As to save a copy of the current file Select location and enter the filename in the dialog box that opens Select Exit to shut down the software PyroMark Q48 Autoprep User Manual 10 2015 7 3 2 Tools menu for processed run files Tools Reports Window Help JE Kun Information x Export Peak Heights Export Environment Data Export Raw Data Export As FASTA eee m eee je Analysis Log F Select Run Information to view the run parameters and a run log for the current run file To print the report click 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 speed block temperature and pressure readings as a text file 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 data as a text file Select Export As FASTA to save base called sequences in FASTA format SEQ assays only In the dialog that opens select the wells to be included all or selected the sorting order of the wells row or column and the bases in the sequences to be included all passed passed check or only quality control window Select Analysis Log to view or save the log with all analyses performed on the selected well as an HTML file Each analysis is logged with the used analysis settings analysis mode AQ SNP CpG or SEQ analysis version result
80. e Clean to begin 2 Ensure that the injectors are empty before beginning If the injectors are not empty run the Clean protocol first refer to section 6 5 2 PyroMark Q48 Autoprep User Manual 10 2015 135 3 Select the nucleotide injectors to clean Only nucleotide injectors can be selected All the nucleotide injectors are selected by default 2s tte Pyrophosphate Clean lt ea Step 1 Select nucleotide injectors to clean 4 6 va 6 A c G T 4 Manually pipette 100 pl of pyrophosphatase cleaning solution into selected injectors and then select the Next button 5 Check that the absorber strip is inserted 6 Start clean The instrument will automatically prime the injectors to ensure that the cleaning solution is in contact with the entire injector flow channel The software will begin a 30 minute countdown timer to allow the solution to incubate Once started the pyrophosphate clean procedure cannot be terminated At the completion of the pyrophosphatase incubation the software will notify the user to continue to the next step with an audible beep u Pyrophosphate Clean x Step 4 Incubate pyrophosphatase e Incubation time remaining 29min 59sec AB P1 P2 P3 NB P4 BB PyroMark Q48 Autoprep User Manual 10 2015 136 7 Empty 1 of 4 Manually pipette 200 pl of high purity water into the selected nucleotide injectors and then select the Start button The injectors will be emp
81. e available parameters are listed below Note If the sequence to analyze is changed and a new dispensation order is generated the variable position parameters are reset to their default values Parameter Description 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 The type of variable position SNP InDel CoG or CpN site Analyze If this option is checked the variable position will be analyzed Note This option is not available for variable positions that cannot be analyzed for the current assay type Methylation ranges The expected CpG or CpN methylation Setting this parameter for all the CpG assays only CpG or CpN sites allows easy identification of sites in the analysis results that are outside the user expected methylation range The light green area is below the expected range The green area is within the expected range The dark green area is above the expected range PyroMark Q48 Autoprep User Manual 10 2015 94 Parameter Description Note The expected methylation cannot be set for CpG sites with the Analyze option unchecked The expected range area can be moved to the left or to the right by holding down the left mouse button while moving the area with the mouse The arrows can
82. e normal safety requirements prevailing in the user s country 2 1 Proper use WARNING CAUTION A Risk of personal injury and material damage W1 Improper use of the PyroMark Q48 Autoprep may cause personal injuries or damage to the instrument The PyroMark Q48 Autoprep must only be operated by appropriately trained and experienced personnel Servicing of the PyroMark Q48 Autoprep must only be performed by QIAGEN service personnel or service technicians of an authorized agent PyroMark Q48 Autoprep User Manual 10 2015 13 CAUTION A CAUTION A WARNING CAUTION A WARNING CAUTION A CAUTION A Damage to the instrument C1 Direct sunlight may bleach parts of the instrument and the cartridges and cause damage to plastic parts The PyroMark Q48 Autoprep and the cartridges must be located out of direct sunlight away from heat sources and away from sources of vibration and electrical interference Damage to the instrument C2 Avoid spilling water or chemicals onto the PyroMark Q48 Autoprep Damage caused by water or chemical spillage will void your warranty Risk of personal injury and material damage w2 Do not attempt to move the PyroMark Q48 Autoprep during operation Explosive atmosphere The PyroMark Q48 Autoprep is not designed for use in an explosive atmosphere Risk of explosion W4 The PyroMark Q48 Autoprep is intended for use with reagents and substances supplied with QIAGEN kits
83. e of buffers Comments and suggestions Ensure that the lab is within the operating temperature range 18 30 C Send support package to QIAGEN Technical Services see section 9 2 Issue with disc heater Send support package to QIAGEN Technical Services see section 9 2 Ensure that the injector cover is closed Check the injector cover is closed and select retry Run lid movement test see section 8 4 Select Re try button and avoid moving the lid during injector priming or cleaning Ensure that no object is impeding the lid to open or close Ensure that the cartridges are inserted 150 Loaded wrong nucleotide into injector position Poor drop sensor data on dispensation Reported in PyroMark Q48 Autoprep Software Pump will not reach pressure Spurious peaks on negative control nucleotide dispensations Injector Priming failure Injector Clean failure Other Liquid or beads outside of the wells lt no sample ID gt No disc detected No run setup file Comments and suggestions Run Injector Clean for that injector see section 6 5 2 Ensure priming was completed successfully Ensure sufficient liquid was loaded into the cartridge reservoir Send support package to QIAGEN Technical Services see section 9 2 Check the cartridge lids are closed Check cartridges are tirmly seated If problem persists contact QIAGEN Technical Services Run Pyrophosphate Clean see section 8 6 Check
84. e section 7 11 2 Note If creating a CpG assay we recommend entering the Sequence Before Bisulfite Treatment see Enter the sequence before bisulfite treatment in section 7 11 2 This enables the software to automatically generate the sequence to analyze and select the most appropriate bisulfite treatment control Note When CpN mode is enabled each CpN position in the sequence before bisulfite treatment is indicated by a small check box above the position The user should select which position s to analyze by individually clicking the appropriate check boxes or by clicking Select all or Deselect all Selected boxes are indicated by blue color and deselected boxes are indicated by white color All of the selected CpN positions will be considered during the analysis as long as they are not deselected in the Variable Positions tab 3 Click the Generate Dispensation Order button see section 7 11 3 4 If creating a CpG assay check that the software has selected a bisulfite treatment control If no bisulfite treatment control has been automatically selected add one manually preferably at the beginning of the sequence see section 7 11 4 5 Optional Enter information about the assay in the Assay Note text box Note An assay note can be displayed in a tooltip in the shortcut browser by positioning the mouse pointer over the assay file 6 Optional Set up the variable positions in the Variable Positions tab see section 7 11 5
85. e sequence to analyze For reliable results the software needs at least 5 dispensations with known results reference peaks Do not remove reference peaks For reliable results the software needs at least 5 dispensations with known results reference peaks Add more sequence information i e lengthen the sequence to analyze Signal intensity too low Check PCR yield Ensure that the correct amount of beads is pipetted into wells of the PyroMark Q48 disc and correct concentration of sequence primer is used Verify that the enzyme and substrate mixtures are not expired are stored correctly and are completely dissolved Wide peaks indicate bad chemistry Check reagent storage conditions Check shelf life of reagents Verify that PyroMark Q48 Autoprep Reagents were used Redesign the assay sometimes peak width is assay dependent Peak heights deviate from expected levels Even at a variable position the software expects a certain pattern e g a certain sum of the peak heights Check sequence to analyze Check for unknown SNPs Peak height deviates from expected level at indicated dispensation Check sequence to analyze Check for unknown SNPs Too high increase or decrease in baseline Verify that the environmental temperature did not change e g air conditioner cycling on or off and remains under 32 C during the run Ensure that reagents have been adapted to room temperature prior to the run 143 Message text U
86. ebrauchtes Kunststoffverbrauchsmaterial z B die PyroMark Q48 Disc kann gef hrliche Chemikalien oder infekti ses Material biologische Gefahrstoffe enthalten Solche Abf lle m ssen unter Einhaltung der rtlichen Sicherheitsvorschriften gesammelt und entsorgt werden 16 10 Sicherheitshinweise Wartungsarbeiten F hren Sie alle Wartungsarbeiten gem den Anweisungen in Abschnitt durch QIAGEN stellt alle Reparaturen in Rechnung die nachweislich auf eine inkorrekte Wartung zur ckzuf hren sind WARNUNG Gefahr von Verletzungen und Sachsch den ACHTUNG Es d rfen nur Wartungsarbeiten ausgef hrt werden die in diesem Benutzerhandbuch konkret beschrieben sind WARNUNG Gefahr durch Stromschlag ACHTUNG ffnen Sie keine Abdeckplatten des PyroMark Q48 Autoprep Es d rfen nur Wartungsarbeiten ausgef hrt werden die in diesem Benutzerhandbuch konkret beschrieben sind PyroMark Q48 Autoprep User Manual 10 2015 183 ACHTUNG A A ACHTUNG A Besch digung des Ger ts Verwenden Sie zur Reinigung des PyroMark Q48 Autoprep keine L sungsmittel und keine s ure laugen oder scheuermittelhaltigen Reagenzien Besch digung des Touchscreens und Computers C11 Sch tten oder spr hen Sie keine Fl ssigkeiten z B Reinigungsmittel auf den PyroMark Q48 Autoprep Verwenden Sie zur Reinigung ein nur mit Wasser angefeuchtetes Tuch Wartung des Lichtdetektors C12 Verwenden Sie fusselfreie T cher um das Fenster des
87. ector Ensure correct volume of nucleotide was loaded without any air bubbles If problem persists run injector diagnostics see section 8 5 Ensure injectors are cleaned daily No drops have been dispensed from the indicated injector Ensure correct volume of nucleotide was loaded without any air bubbles If problem persists run injector diagnostics see section 8 5 Ensure injectors are cleaned daily The indicated injector dispensed drops with unusual shape Ensure correct volume of nucleotide was loaded without any air bubbles If problem persists run injector diagnostics see section 8 5 Ensure injectors are cleaned daily Variable position was deselected for analysis by the user The last dispensation is the terminal base of the sequence to analyze Add at least one more base at the end of the sequence to analyze so that one or more additional base dispensations are generated The added base must be different from the base currently at the end of the sequence to analyze The last dispensation is included in terminal variable position Add at least one more base at the end of the sequence to analyze so that one or more additional base dispensations are generated The added base must be different from the base currently at the end of the sequence to analyze One allele is sequenced ahead of the other s and the two or more alleles are not in phase with the last dispensation Add more sequence information i e
88. egin injector priming and testing 7 A confirmation message Start prime and test will appear To confirm select Yes All injectors selected for priming will undergo the prime protocol The chamber will rise to the prime position and the injectors will be pressurized If the instrument reports a problem with the pump reaching pressure please check that the cartridge lids are not open PyroMark Q48 Autoprep User Manual 10 2015 A9 The chamber lid will move the injectors over the front half of the absorber strip and dispense the solution to prime the injectors All injectors that have undergone a prime will be displayed with a question mark Accidentally moving the chamber lid during injector priming will pause injector priming and deliver a warning message indicating that the lid has moved Select Retry to continue with the prime An Abort icon in the top right corner can be used to stop injector priming A confirmation message Abort prime will appear Select Yes to stop injector priming or No to continue Following the abort the injector status will be updated to denote the current state of each injector s v v v S x Xe vy A c G T Ds E s AB v BB Following the prime all required injectors will undergo a number of test shots The test shots ensure that the injectors are operating correctly by observing the drop sensor data Injectors that pass the test will be displayed with a green tick Once inject
89. elf life of reagents Verify that PyroMark Q48 Autoprep Reagents were used Redesign the assay sometimes peak width is assay dependent A certain peak width is needed for correct determination of peak heights Peaks become wider over time which is normal Check results carefully The software has detected that all four nucleotides have been dispensed but no signal was detected Clean the cartridge see section 8 3 3 in case there was a dispensation error If problem persists perform injector test see section 8 5 149 Message text High noise level Very high noise level Not analyzable due to lack of data 9 5 Temperature Chamber not at temperature reported in PyroMark Q48 Autoprep Software Fail to reach annealing temperature Optics High baseline signal on start acquisition Higher than expected signal detected Chamber Lid Lid not moving Lid movement during priming or clean Lid movement impeded Injectors No cartridge detected PyroMark Q48 Autoprep User Manual 10 2015 Instrument related errors Comments and suggestions The noise of the baseline generated by the instrument is too high Check instrument lid Contact QIAGEN Technical Services The noise of the baseline generated by the instrument is too high Check instrument lid Contact QIAGEN Technical Services No peaks detected Check PCR yield Check sequencing primer Clean cartridges Check reagents Check correct usag
90. encing reaction performed using Pyrosequencing technology Incorporated nucleotides are shown as peaks in the Pyrogram Gives an overview of the quality at the end of a defined number of bases in the base called sequence A setting in the SEQ analysis mode Nonvariable peaks i e peaks that are not a part of a variable position are referred to as reference peaks Reference peaks are used in the analysis both as references when calculating the single peak height level and as internal controls when assessing the quality Relative Light Unit entity used in Pyrosequencing to define peak heights in Pyrogram Analysis mode used for base calling of unknown sequences A short part of a DNA sequence in your sample starting directly after the sequencing primer which contains one or several variable positions to be analyzed using Pyrosequencing instrument platforms The sequencing primer is annealed to the template during the template preparation The 3 end of the sequencing primer serves as the starting point for the extension by the DNA polymerase PyroMark Q48 Autoprep User Manual 10 2015 153 Term Shift Signal to noise ratio Single nucleotide polymorphism SNP Streptavidin Substrate Variable position Description Plus shift A small proportion of the template sequences that incorporates more than one type of nucleotide at a time if for example there are residues left from the dispensation before and will
91. equence to analyze can be added in the Analysis Setup tab 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 a CpG assay can be analyzed in the AQ mode using the sequence to analyze used in the CpG setup To exclude the CpG sites in the SNP reports select the Analysis Setup tab and uncheck the Analyze option for these positions in the Variable Positions tab To analyze SEQ assays in any other analysis mode a sequence to analyze must be entered in the Analysis Setup tab PyroMark Q48 Autoprep User Manual 10 2015 112 7 15 3 View the analysis results By selecting an analyzed light blue well in the Overview tab the corresponding Pyrogram is displayed in the Pyrogram area and the well information including analysis warnings is listed in the Well Information area If several wells are selected in the disc overview information for the well with the orange selection frame is shown Get an overview of the results Sample ID Note Quality AQ SNP and SEQ assays CpG assays The following well information can be viewed in the disc overview in the Overview tab Select to show the assay name Select to show the sample ID hal Select to show the well note iii Select to show the quality bar The quality bar shows the quality assessment of all variable positions in the well or of all the bases in the base called sequence Se
92. er ID of the PyroMark Q48 Disc Note If you position the mouse pointer over a run file in the shortcut browser a tooltip displays the entered disc ID Optional Enter a bar code number for the disc or if you have a bar code reader connected to your computer place the mouse cursor in the Bar code text box and scan the bar code Optional Enter the lot number for the PyroMark Q48 Advanced Reagents or PyroMark Q48 Advanced CpG Reagents to be used The lot number can be found on the product label Note We recommend entering the reagent ID so that any unexpected problems with the reagents can be traced Optional Enter a note about the contents or purpose of the run PyroMark Q48 Autoprep User Manual 10 2015 103 7 13 3 Add assay files to the disc To add assays to wells you can either Select the assay in the shortcut browser and press and hold down the left mouse button while you drag the assay to the well Right click the well and select Load Assay from the context menu this option is only available when one well is selected e Add an assay to several wells by selecting the wells and dragging the assay to the selection To select several wells either click and drag the cursor across the wells to be selected or hold down the Ctrl key as you click wells Note It is not possible to add an assay with no dispensation order or add two or more assays with the same assay name but have different dispensation orders N
93. ercentages CpG report or base called sequences SEQ report and the quality assessments Analysis warnings f analysis parameters or quality assessments have been edited by the user the affected wells are listed Report options A SEQ Full Report 28 Selection All wells A AQ Full Report _ A D Selected wells Selection Options All wells Raw pyragram Selected wells l O Compensated pyrogram Preview Save Cancel Preview Save Cancel In the Full Report dialog box there are the following options Option Description All wells selected wells The wells to be included in the report Raw Pyrogram The type of Pyrogram to be included in the report Compensated Pyrogram This option is only available for the SEQ report To view the report before saving or printing it click Preview Note In order to view the report a PDF reader must be installed on the computer Adobe Reader can be downloaded at www adobe com PyroMark Q48 Autoprep User Manual 10 2015 127 7 17 Supported files PyroMark Q48 Autoprep Software supports the following file types AQ assay qaq SNP assay qsnp CpG assay acpg SEQ assay qseq Disc setup qset Processed run qrun Files can be opened by either selecting Open in the Files menu or clicking Note Files created in PyroMark Q24 2 0 Software or in PyroMark Q96 2 5 Software are not supported 7 18 General Hints and Tips
94. es and buttons become enabled again once the errors in the Sequence Before Bisulfite Treatment are corrected IUPAC codes Description Description Adenine TorA Cytosine CorG Guanine C T or G not A Thymine 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 A G T R Y M K TorG Note S B V and N are not valid after bisulfite treatment PyroMark Q48 Autoprep User Manual 10 2015 90 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 is not valid since C TG is a forward CpG site and C G cannot exist after bisulfite treatment The following CpG sites CpN sites and SNPs can be included in a forward assay e CpG site C TG CpN site C TA C TC C TG and C TT e SNPs A T A G G T and A T G i e C cannot be included The following CpG sites CpN sites and SNPs can be included in a reverse assay e CpG site CG A CpN site AG A CG A GG A and TG 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 These kinds of SNPs 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 mode and
95. es for further help and instructions esse Injector Test Diagnostic or Step 5 Follow these instructions to complete the test 1 Inspect the disc 2 Visually check if drops are present and record in table below 3 Copy and send results to your support representative Well A1 A2 A4 A5 AG AT AB A9 A10 A11 A12 T P1 P2 P3WBP4BB DS E S AB ARARA E AE AEAEE IEICE VIVIVMIN X X X X X A3 Injector A c G Drop Sensor WY YW A Visual Check WH A Copy 8 6 Pyrophosphate clean Pyrophosphate contamination may occur and is typically observed as small peaks in control dispensations where no peaks are expected Such contamination will have a negative influence on the final analysis result Pyrophosphate contamination only affects the nucleotide injectors Removal of the contamination is achieved using the enzyme pyrophosphatase and a thorough clean with high purity water Use the following table to prepare the pyrophosphatase cleaning solution based on the number of injectors selected to be cleaned Each injector will require 100 pl Component Final concentration Volume per injector PyroMark Advanced Annealing buffer 95 pl Pyrophosphatase 5 U ml 5 pl 100 U mi For example Pyrophosphatase Inorganic from New England Biolabs cat no MO361L t In case of higher concentrated stock solutions of Pyrophosphatase dilute Pyrophosphatase solution to 100 U ml with PyroMark Advanced Annealing Buffer 1 Select Pyrophosphat
96. et WARNUNG Der Begriff WARNUNG WARNING weist Sie auf Situationen hin in A denen eine Verletzungsgefahr f r Sie selbst oder andere Personen besteht N here Einzelheiten ber diese Situationen werden in einem Textfeld wie diesem beschrieben ACHTUNG Der Begriff ACHTUNG CAUTION weist Sie auf Situationen hin in denen das Ger t oder andere Ger te besch digt werden k nnten A N here Einzelheiten ber diese Situationen werden in einem Textfeld wie diesem beschrieben Die in diesem Handbuch enthaltenen Hinweise stellen eine Erg nzung und keinen Ersatz der blichen Sicherheits anforderungen dar die im jeweiligen Land gelten 16 2 Sachgem e Handhabung WARNUNG Verletzungsgefahr und Besch digung des Ger ts w1 ACHTUNG Die unsachgem e Bedienung des PyroMark Q48 Autoprep kann zu einer A Verletzung des Benutzers oder zur Besch digung des Ger ts f hren Die Bedienung des PyroMark Q48 Autoprep darf nur durch qualifiziertes entsprechend geschultes Personal erfolgen Die Instandhaltung des PyroMark Q48 Autoprep darf nur durch einen Servicespezialisten des QIAGEN Au endiensts oder Servicetechniker eines autorisierten Vertreters durchgef hrt werden PyroMark Q48 Autoprep User Manual 10 2015 178 ACHTUNG Besch digung des Ger ts C1 A Direktes Sonnenlicht kann zum Ausbleichen von Teilen des Ger ts und der Kartuschen f hren und Sch den an Kunststoffteilen verursachen Das PyroMa
97. ety of sources For more information about QlAprep QlAamp DNeasy and RNeasy products contact one of our Technical Service Departments see back cover or visit www qiagen com Quality of starting template when performing CpG assays Critical parameters for a successful PCR using bisulfitetreated DNA templates include complete bisulfite conversion and DNA fragments that are long enough for PCR EpiTect Bisulfite Kit provides a fast and reliable procedure for efficient bisulfite conversion and a unique DNA Protect PyroMark Q48 Autoprep User Manual 10 2015 156 Buffer prevents DNA fragmentation during the bisulfite conversion reaction For more information about EpiTect products contact one of our Technical Service Departments see back cover or visit www giagen com Quantity of starting template The annealing efficiency of a primer to the template is an important factor in PCR Owing to the thermodynamic nature of the reaction the primer template ratio strongly influences the specificity and efficiency of PCR and should be optimized empirically If too little template is used primers may not be able to find their complementary sequences Too much template may lead to an increase in mispriming events PCR optimization The PyroMark PCR Kit will produce satisfactory results in most cases However if a higher Mg concentration is required use of the 25 mM MgCl provided in the kit is recommended The recommended annealing temper
98. exposition la lumi re solaire directe peut provoquer le blanchiment de certains l ments de l appareil et des cartouches et d t riorer les pi ces en plastique L appareil PyroMark Q48 Autoprep et les cartouches ne doivent pas tre expos s la lumi re solaire directe et doivent tre loign s des sources de chaleur des sources de vibration et des interf rences lectriques D t rioration de l instrument C2 viter de renverser de l eau ou des produits chimiques sur le PyroMark Q48 Autoprep La d t rioration due la projection d eau ou de produits chimiques annulera la garantie Risque d accident corporel et de d t rioration du mat riel W2 Ne pas essayer de d placer le PyroMark Q48 Autoprep pendant qu il est en marche Atmosph re explosive W3 Le PyroMark Q48 Autoprep n est pas con u pour tre utilis dans une atmosph re explosive Risque d explosion WA Le PyroMark Q48 Autoprep a t con u pour l utilisation des r actifs et substances fournis par les kits QIAGEN L utilisation de r actifs et de substances autres que celles indiqu es peut entrainer un risque d incendie ou d explosion Insertion de la bandelette absorbante S assurer que la bandelette absorbante est ins r e dans l instrument PyroMark Q48 Autoprep comme d crit dans la section 6 2 2 pour emp cher le liquide de p n trer dans la chambre En cas d urgence teindre le PyroMark Q48 Autoprep l aide de l interrupteur
99. f computers and automation in general Appropriately trained and experienced personnel familiar with molecular biological techniques Regularly trained certified and authorized by QIAGEN Appropriately trained and experienced personnel familiar with use of computers and automation in general Trained and authorized by QIAGEN 2 Safety Information Before using the PyroMark Q48 Autoprep it is essential that you read this user manual carefully and pay particular attention to the safety information The instructions and safety information in the user manual must be followed to ensure safe operation of the PyroMark Q48 Autoprep and to maintain the PyroMark Q48 Autoprep in a safe condition Note Translations of the Safety Information in French and German are available in Appendix E Safety Information French FR and Appendix F Safety Information German DE The following types of safety information appear throughout this user manual WARNING A CAUTION A The term WARNING is used to inform you about situations that could result in personal injury to you or other persons Details about these circumstances are given in a box like this one The term CAUTION is used to inform you about situations that could result in damage to the instruments or other equipment Details about these circumstances are given in a box like this one The advice given in this manual is intended to supplement not supersede th
100. g QIAGEN Technical Services Check the run log in the Run Information report to assess if the system was working properly during the run Consult the Troubleshooting sections below Recommended Verify proper installation and operation of your system using PyroMark Control Oligo see Appendix A Assay design and validation 9 1 Checking the run log It is advisable to check the run log to assess if the system was working properly during the run Open the run file using the PyroMark Q48 Autoprep software Select Run Information from the Tools menu or right click the file in the shortcut browser and select Run Information from the context menu The Run Information report is opened Check the run log at the end of the report for any problems during the run PyroMark Q48 Autoprep User Manual 10 2015 138 e f deviations from the preset block temperature pressure and or mixer speed values are noticed several times during a run and for longer time periods or in repeated runs please contact QIAGEN Technical Services If requested to send an Environment Data file Select Export Environment Data from the Tools menu e Select the destination folder for the data file from the Save in drop down list Enter the filename in the File name text box and click Save 9 2 Copying support packages Support packages contain information useful to QIAGEN Technical Services A support package is created for every run clean py
101. g square bracket notation e g AT The sequence should not include more than 400 characters or 100 variable positions Variable positions involving a combination of SNPs and InDels should be entered using a combination of or IUPAC codes and For example T A or W represents a tri allelic polymorphism where the possible alleles are a T an A or neither deletion It is not possible to have a combination of a single nucleotide polymorphism and constant bases within an InDel e g A TC Nested InDels are not supported e g ATT C G 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 marked in red in the sequence to analyze Sequence to Analyze aie Wrong nput format Use characters from the IUPAC code 4 B D 6 H EM N RS T MW Yand f fs mot allowed at the end L must have content applies only to AQ and SNP As T T is not a valid variable position it causes an error In SNP assays a warning message will appear if the sequence to analyze contains multiple variable sites within the same variable region that will generate the same sequence pattern This will make it difficult to discriminate genotypes Enter the sequence before bisulfite treatment CpG a
102. g the run file in the shortcut browser If several assay types are included select analysis mode in the dialog box that opens The analysis modes displayed in the dialog box are limited to the assay types on the disc Note To update the contents of a folder in the shortcut browser right click it and select Refresh from the context menu or press the F5 key Note It is also possible to open the run file by double clicking it in Windows Explorer PyroMark Q48 Autoprep User Manual 10 2015 In the Overview tab analyze selected wells with a valid analysis setup for the current analysis mode Analysis modes PyroMark Q48 Autoprep Software has four analysis modes AQ SNP CpG and SEQ To toggle between the modes select AQ SNP CpG or SEQ in the toolbar Note How the analysis is performed can be modified using the Analysis Setup tab 6 8 Viewing the analysis results By selecting an analyzed well in the Overview tab the corresponding Pyrogram is displayed in the Pyrogram area and the well information including analysis warnings is listed in the Well Information area PyroMark Q48 Autoprep User Manual 10 2015 65 co Assay 0 J Sarme Iik Semple Hobe TCTGTGCTTTCCAAGACCCAATGTGCAAATGTGATTTTCTAATAATATGTATTCACTGTGAAGGAAGT 1E pe mn m wa 75 j TOLL TOAL TOA TGA TOAL TOC TGA TOAL TAL MAL GAC TOAL TGA TOAL TOAL TOUT CAL GEL TG MAL TOAL PGS TOAL TICAL CAL GAC TOAL C
103. ge protected Note It is recommended to plug the instrument directly into its own power outlet and not to share the power outlet with other lab equipment Do not place PyroMark Q48 Autoprep on a vibrating surface or near vibrating objects To ensure proper ventilation maintain a minimum clearance of 5 cm 2 in at the sides and 20 cm 8 in at the rear of the PyroMark Q48 Autoprep Slits and openings that ensure the ventilation of the PyroMark Q48 Autoprep must not be covered Note It is recommended to use the instrument only in post PCR environment 4 2 2 Power requirements The PyroMark Q48 Autoprep operates at input 100 240V AC 50 60Hz 2 5A For details please refer to Appendix B Technical Data Make sure that the voltage rating of the PyroMark Q48 Autoprep is compatible with the AC voltage available at the installation site Mains supply voltage fluctuations are not to exceed 10 of nominal supply voltages 4 2 3 Grounding requirements To protect operating personnel the PyroMark Q48 Autoprep must be correctly grounded earthed The PyroMark Q48 Autoprep is equipped with a 3 conductor AC power cord IEC 60320 type C14 To preserve this protection feature do not operate the PyroMark Q48 Autoprep from AC power outlets that have no ground earth connection 4 3 Unpacking and installation Note We recommend installing the cartridges before switching on the instrument If no cartridges have been installed or the ca
104. ghly recommended that the sequencing primer is located a few bases before the variable position PCR setup PCR reactions of 25 pl are set up using the PyroMark PCR Kit Ensure that you follow the instructions provided in the PyroMark PCR Handbook Run the PCR at the optimal annealing temperature for 45 cycles Using fewer cycles may give insufficient yield and cause background problems in Pyrosequencing reactions due to excess unused biotinylated primer The PCR product should give one strong band with minimal excess of primers when analyzed on an agarose gel Starting template The yield and quality of PCR product is affected by both the quality and quantity of the nucleic acid starting template This is particularly true for amplification of long regions from DNA that has been fragmented by bisulfite treatment or extracted from paraffin embedded material Quality of starting template Since PCR consists of multiple rounds of enzymatic reactions it is more sensitive to impurities such as proteins phenol chloroform salts ethanol EDTA and other chemical solvents than single step enzyme catalyzed processes QIAGEN offers a complete range of nucleic acid preparation systems ensuring the highest quality templates for PCR These include the QlAprep system for rapid plasmid purification the QlAamp and DNeasy systems for rapid purification of genomic DNA and viral nucleic acids and the RNeasy system for RNA preparation from a vari
105. gionales et locales relatives la sant et la s curit OSHA Occupational Safety and Health Administration Etats Unis d Am rique Administration pour la sant et la s curit du travail t ACGIH American Conference of Government Industrial Hygienists tats Unis d Am rique Conf rence am ricaine des hygi nistes industriels gouvernementaux t COSHH Control of Substances Hazardous to Health Royaume Uni Contr le des substances dangereuses pour la sant PyroMark Q48 Autoprep User Manual 10 2015 174 15 6 Dangers m caniques AVERTISSEMENT A AVERTISSEMENT A ATTENTION A ATTENTION A Pieces mobiles w8 Pour viter tout contact avec des pi ces en mouvement pendant le fonctionnement du PyroMark Q48 Autoprep l appareil doit tre utilis avec le capot d injecteur ferm Ne pas d monter les panneaux du capot Ils ne renferment aucune pi ce r parable par l utilisateur En cas de probl me avec l appareil PyroMark Q48 Autoprep contacter imm diatement les Services techniques de QIAGEN Pi ces mobiles w9 Le capot de la chambre de l appareil PyroMark Q48 s ouvre et se ferme automatiquement pendant le fonctionnement afin d emp cher l accumulation d humidit dans l instrument Insertion du disque C4 Le PyroMark Q48 Disc doit tre verrouill en place afin d viter de renverser le contenu du disque dans la chambre de l instrument S assurer que le disque est verrouill e
106. icking the peak The bisulfite treatment control is highlighted with an orange background color and the base is displayed in orange When showing reference peaks orange diamonds are displayed above the bisulfite treatment controls CpG assays only 7 8 1 Zoom histogram It is possible to zoom in on the histogram by selecting a stretch 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 It is possible to adjust the heights of histogram bars see Adjust heights of histogram bars in section 7 11 6 7 8 2 Export the histogram as an 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 can be pasted into applications that support Enhanced Metafile EMF images 7 9 Pyrogram The Pyrogram is the graph resulting from a sequencing reaction performed using Pyrosequencing technology Incorporated nucleotides are shown as peaks in the Pyrogram PyroMark Q48 Autoprep User Manual 10 2015 83 7 9 1 AQ SNP and CpG assays B4 TAYAGATTYGAAGTTTGAGGTATAGGGYGYGGTTYGYGA 2 3 Set Max Y Axis Value gt Reset Max Y Axis Value Copy as Image Show Histogram Show Reference Peaks Pyrogram showing a CpG assay result The following information
107. icons and colors are displayed and used in the Pyrogram pane for an AQ SNP or CpG assay The well name and the sequence to analyze are shown in the upper left corner a 4 The analysis result allele frequencies in AQ mode ses genotype in SNP mode T T or methylation percentage 23 or average methylation percentage 22222 25 in CpG mode or CpN mode is displayed above each variable position The background color shows the quality assessment of the analysis result see Quality colors in section 7 15 3 If a quality assessment has been edited by the user this is displayed by a border around the analysis result for example Note in white indicates that a site was deselected by the user 3 in white indicates that the software does not support the analysis e g analysis of SNP in the CoG mode ma in red indicates that the analysis was not possible due to lack of data e Variable regions are highlighted with a blue gray background color When showing reference peaks blue diamonds are displayed above the reference peaks A filled blue diamond indicates the reference peak is enabled and a hollow blue diamond indicates the reference peak is disabled Bisulfite treatment controls are highlighted with a light orange background color When showing reference peaks orange diamonds are displayed above the bisulfite treatment controls CpG assays only To view the height of a peak posit
108. iggered only the most serious ones are displayed in the Well Information area e SEQ assays Affects the quality assessment for either the whole sequence or from a specific dispensation and forward All warnings triggered within the quality control window are displayed in the Well Information area Clicking the Warning Info button Warning Info provides a short description and recommendation for each shown warning For some of the warnings the criteria for occurrence and the effect on the quality assessment can be modified by the user in the Analysis Parameters tab see section 7 15 4 Note For other warnings please refer to section 9 or contact QIAGEN Technical Service Quality assessments The quality assessments of the analysis results are displayed by Quality bars in the disc overview see Get an overview of the results this section The background color of the analysis results allele frequencies the methylation percentages 96 or genotype in the Pyrogram for example or the base called sequence The peaks in the compensated Pyrogram are colored according to their quality assessments SEQ assays only PyroMark Q48 Autoprep User Manual 10 2015 114 Quality colors e Blue Passed Yellow Check Red Failed White Not analyzed Either analysis is not supported by the software e g analyzing single nucleotide polymorphisms in CoG mode or the variable position has been deselected
109. in section 7 11 6 To apply changes made in the Pyrogram click the green button which is enabled if a change has been made In the Apply Analysis Setup dialog box apply the changes to all or the selected wells To 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 dialog box click To All To apply the changes to the selected wells i e the white wells that are selected in the Apply Analysis Setup dialog box click To Selected During the analysis a progress dialog box is shown The dialog box 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 To save the changes click lel Note If analysis parameters quality assessments or base called sequence for SEQ results have been edited by the user the well is marked with a warning icon A in the Overview tab Note All changes are logged To view the analysis log for a selected well select Analysis Log from the Tools menu Use modified assay in other runs Changes made in the Analysis Setup tab will not be saved in the original assay file To use the modified assay in other runs 1 Select a well that is using the modified assay and click Save Assay The Save Assay As dialog box opens PyroMark Q48 Autoprep User Manual 10 2015 118 2 Save the changes to the original file
110. ing in 18 minutes PyroMark Q48 Autoprep User Manual 10 2015 56 If a disc has not been inserted the instrument will warn the user with an audible beep and display the following message Disc Check No disc was detected A 48 well disc is required for this run Check sample disc is correctly inserted Insert the disc and select the Retry button If the cartridge lid is left open the instrument will display the following message Pressure Failure Pump failed to reach pressure Check 1 All cartridge lids are closed and locked 2 All cartridges are correctly inserted If problems persist contact your support representative Close the injector lid and select the Retry button 6 4 1 Manual sequence primer loading The instrument will notify the user that the sequence primer is required to be loaded with an audible beep and by displaying the following message Select OK to open lid and add primers 1 Begin manual sequence primer loading by selecting the OK button The chamber lid will automatically open allowing the user to load the primers Selecting Cancel will bring up a confirmation dialogue box Do you wish to cancel Selecting Yes will terminate the run 2 Manually pipette 2 pl of the required sequence primer 4 yM into the designated sample The software will display the required sequence primer as the given assay name next to the sample well The final concentration of primer in the 10 pl react
111. inside instrument after extreme storage conditions W13 CAUTION Condensed humidity inside the instrument may cause personal injuries or A damage to the system when connecting the instrument to the power mains In order to avoid this let the instrument adjust to the new ambient conditions for at least 2 hours prior to connecting the power cable 7 Insert the Power cable into the back of the PyroMark Q48 Autoprep and connect it to a power main Turn on the instrument The instrument will take a few seconds to initialize During the first startup of the PyroMark Q48 Autoprep the software will display an installation screen on the instrument touchscreen Current Device Time 26 11 2014 11 02 36 Change System Time 8 Select the Unlock button to open the instrument lid The lid will automatically slide open Note If the lid is impeded it will stop and the software will inform the user Remove any obstacle impeding the lid and select Retry 9 Change the system time and time zone and select Save Set the system date and time Day Month Year Hour ug February 2013 UE March 2014 09 April 2015 10 May 2016 11 June 2017 12 Time Zone GMT 10 00 Brisbane T PyroMark Q48 Autoprep User Manual 10 2015 10 Select the Continue button 11 The touch screen should now display the Home screen with three available options Sequence Clean and Tools The time will be displayed in the upper right corner of the screen
112. into the reservoir If priming problems persist 1 Perform the Injector Diagnostics Test 2 Contact your support representative PyroMark Q48 Autoprep User Manual 10 2015 6 2 4 Inserting the PyroMark Q48 Disc into the instrument The PyroMark Q48 Disc may be loaded with template and bead mix after insertion into the instrument Alternatively the disc can be loaded outside the instrument and inserted just before the run To insert a disc into the instrument follow these steps 1 Insert a PyroMark Q48 Disc into the instrument Hold the disc using the disc grips position the disc index hole outer hole near well B1 over the disc index pin on the rotor hub and insert The orientation of the disc grips should match up to those on the rotor The disc detection system will recognize and warn the user if a disc is not properly inserted 2 Lock the disc into position by screwing down the lock nut Important The disc must be locked into position to avoid spilling the contents of the disc into the chamber PyroMark Q48 Autoprep User Manual 10 2015 52 6 2 5 Loading template and beads into the PyroMark Q48 Disc The Pyrosequencing template Biotinylated PCR product and PyroMark Q48 Magnetic Beads are loaded into a disc manually according to the following instructions Note When running the P4 extra protocol 5 pl PyroMark Binding Buffer must be manually pipetted into the disc wells along with the Pyrosequencing temp
113. ion Missing peaks in cycle starting at dispensation PyroMark Q48 Autoprep User Manual 10 2015 Comments and suggestions Signal intensity too low Check PCR yield Ensure that the correct amount of beads is pipetted into wells of the PyroMark Q48 disc and correct concentration of sequence primer is used Verify that the enzyme and substrate mixtures are not expired are stored correctly and are completely dissolved Signal intensity too low Check PCR yield Ensure that the correct amount of beads is pipetted into wells of the PyroMark Q48 disc and correct concentration of sequence primer is used Verify that the enzyme and substrate mixtures are not expired are stored correctly and are completely dissolved Peak heights deviate from expected levels around the indicated dispensation Check for unknown SNPs Peaks deviate from expected level Check annealing procedure Ensure there is no nucleotide degradation contamination Check for unknown SNPs Too high increase or decrease in baseline Verify that the environmental temperature did not change e g air conditioner cycling on or off and remains under 32 C during the run Ensure that reagents have been adapted to room temperature prior fo the run Desired nucleotide not properly dispensed Clean the cartridge properly see section 8 3 3 and rerun If problem persists perform injector test Wide peaks indicate bad chemistry Check reagent storage conditions Check sh
114. ion steps are performed 7 After this process the samples are prepared for primer annealing which can be automatically performed by the instrument for up to four different sequencing primers If more sequencing primers are used the primers can be manually added to the single stranded DNA samples 8 Enzyme mixture and then substrate mixture are dispensed into all wells used 9 Nucleotides are dispensed into two waste wells before being dispensed into the wells Nucleotides are added in a predefined order and 60 seconds elapses between the additions of each nucleotide to ensure all enzymatic reactions are completed 10 The instrument collects data from all wells using a PMT located above the inserted disc Data are stored on the instrument 11 After the run data are automatically transferred to the USB stick or the network drive If the USB stick has been removed during a run data can be retrieved manually from the instrument PyroMark Q48 Autoprep User Manual 10 2015 3 4 PyroMark Q48 Autoprep instrument PyroMark Q48 Autoprep front open view 1 USB port 5 For import and export of run files 2 User interface 6ABC Wizard style touch interface to control the instrument 3 Transport lock screw 7 Prevents the chamber lid from moving during transportation must be removed during installation 4 Chamber 8 Contains the disc optical detection system rotor disc heater and an absorber strip cavity Lid can also
115. ion the mouse pointer over the top of the peak A tooltip displays the height PyroMark Q48 Autoprep User Manual 10 2015 84 When showing the histogram the histogram is displayed in gray over the peaks It is best viewed when zoomed in Note By right clicking the Pyrogram pane it is possible to toggle between viewing and hiding the histogram and reference peaks The Pyrogram Y axis scale may be manually adjusted by right clicking on it and selecting Set Max Y Axis Value The value is limited to three digits and must be confirmed by pressing the Enter button or by clicking the mouse button 7 9 2 SEQ assays CACCCATTGCAGTTGIBRICAAATAGTAAACAAGGGTACCACAGGATGCTTATTCCTGTCATCTTGGA 7 Set Max Y Axis Value Reset Max Y Ass Value Copy a Image Show Basecalled Sequence a Show Histogram Show Known Bases Show Peak Levels CTGACTGACTGACTGAC TGACTGACTGACTGACTGACTGACTGACTGAC TGAC TGACTGACTGACTGAC TGACTGACTGACTGAC TGAC TGAC TGAC TGA 5 10 15 20 pi 30 35 40 45 a0 35 60 65 0 fo 20 BS g 95 LOL f When a base is selected in the base called sequence the corresponding peak is highlighted in both the upper and lower areas Within the Pyrogram pane and vice versa The following information and colors are displayed and used in the Pyrogram pane for an SEQ assay The well name is shown in the upper left corner To view the height of a peak position the mouse pointer over the top of the peak A tooltip di
116. ion will be 800 nM Note Sequence primer concentration can be optimized to suit a particular assay PyroMark Q48 Autoprep User Manual 10 2015 57 3 After all of the sequencing primers are loaded into the samples select the Next button to proceed The chamber lid will close automatically and a countdown timer will display the time until sequencing No further action is required from the user until the completion of the run 6 4 2 Automatic sequence primer loading The instrument will automatically load the required sequencing primer located in injector positions Pl P2 and or P3 into the sample well programmed within the PyroMark Q48 Autoprep Software No action is required from the user during this stage or until the completion of the run Note When running the P4 extra protocol a fourth injector position P4 BB can be used for sequencing primers Two microliters 2 pl of sequencing primer 4 pM will be loaded into an 8 pl volume of annealing buffer per well to give a final concentration of 800 nM Multiple primers can be added into one injector to increase assay throughput Ensure that each of the primers is at the correct concentration 4 pM Furthermore ensure that the primers will not cross react 6 4 3 Automatic gain adjustment After the addition of enzyme and substrate the software determines the optimum gain value to provide a similar baseline for all samples within the run to ensure instrument is working i
117. is Parameters tab to their default values click Revert to Default Enable or disable reference peaks and bisulfite treatment controls Nonvariable 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 references when calculating the single peak height level and as internal controls when PyroMark Q48 Autoprep User Manual 10 2015 97 assessing the quality For the best possible quality assessment of the results we recommend that the reference peaks generated by the software are kept enabled By left clicking a reference peak diamond in the histogram the peak is either enabled or disabled as a reference peak depending on the previous status The diamond displays the status e Filled blue diamond Enabled as a reference peak Hollow blue diamond Disabled as a reference peak By left 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 status The diamond displays the status Filled orange diamond Enabled both as a bisulfite treatment control and a reference peak 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 the mouse pointer over the
118. istet werden kann muss das Ger t gegen unbefugte oder unabsichtliche Benutzung gesichert werden Kontaktieren Sie anschlie end den Technischen Service von QIAGEN Die elektrische Sicherheit des Ger ts ist nicht mehr gegeben wenn das Netzkabel besch digt ist e das Ger t l ngere Zeit unter ung nstigen Bedingungen d h unter anderen Bedingungen als in Anhang A angegeben gelagert wurde e das Ger t unsachgem transportiert worden ist Fl ssigkeiten in das Ger t gelangt sind PyroMark Q48 Autoprep User Manual 10 2015 180 16 4 Biologische Sicherheit Wenden Sie beim Umgang mit biologischen Materialien nur sichere Laborverfahren an wie sie z B in Ver ffentlichungen wie Biosafety in Microbiological and Biomedical Laboratories HHS www cdc gov od ohs biosfty biosfty htm beschrieben werden ARNUNG Biologische Materialien we Gehen Sie beim Umgang mit biologischen Materialien mit der gr tm glichen Vorsicht und gem den erforderlichen Sicherheitsbestimmungen vor Tragen Sie immer eine Schutzbrille zwei Paar Laborhandschuhe und einen Laborkittel Die verantwortliche Person z B der Laborleiter muss alle erforderlichen Vorsichtsma nahmen treffen um sicherzustellen dass die unmittelbare Umgebung des Arbeitsplatzes sicher ist und die Bediener des Ger ts ausreichend geschult sind Au erdem d rfen die Grenzwerte in Bezug auf infekti se Erreger die in den entsprechenden Sicherheitsdatenbl ttern
119. it must be taken to an approved treatment facility or to a designated collection point for recycling according to local laws and regulations The separate collection and recycling of waste electronic equipment at the time of disposal helps to conserve natural resources and ensures that the product is recycled in a manner that protects human health and the environment PyroMark Q48 Autoprep User Manual 10 2015 164 Recycling can be provided by QIAGEN upon request at additional cost In the European Union in accordance with the specific WEEE recycling requirements and where a replacement product is being supplied by QIAGEN free recycling of its WEEE marked electronic equipment is provided To recycle electronic equipment contact your local QIAGEN sales office for the required return form Once the form is submitted you will be contacted by QIAGEN either to request follow up information for scheduling collection of the electronic waste or to provide you with an individual quote FCC Declaration The United States Federal Communications Commission USFCC in 47 CRF 15 105 declared that the users of this product must be informed of the following facts and circumstances This device complies with part 15 of the FCC Operation is subject to the following two conditions 1 This device may not cause harmful interference and 2 this device must accept any interference received including interference that may cause undesired
120. ited by the user the affected wells are listed at the top of the report 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 PyroMark Q48 Autoprep User Manual 10 2015 120 Report options aa AQ Analysis Statistics Report Selection All wells Selected wells Group By Assay Assay and sample ID Include Results with the Following Quality Assessments V Passed Check Preview save Cancel In the Analysis Statistics Report dialog box there are the following options Option Description All wells selected wells The wells to be included in the report Assay Assay and sample The analysis results statistics can be grouped according to ID Assay Wells with the same assay will be grouped e Assay and sample ID Wells with the same assay and sample ID will be grouped Can be useful when experiments with replicates are performed Passed Check The analysis results to be included The calculations can be performed on results with Passed and or Check quality assessment Note If all Passed and Check results are to be included in the report you can exclude results within this group by turning off the Analyze option f
121. k box 2 Lock the cartridge into place using the previously removed thumb screws 3 Turn on the instrument The instrument will recognize the type of cartridge inserted along with all the calibrated volumes In case a cartridge is placed into the wrong position or installed incorrectly the user will be notified G Avoid removing the cartridges repeatedly Continual insertion and removal may result in failure of the sealed connection with the air inlet pin 8 3 3 Cleaning cartridges The cartridges must be cleaned regularly to ensure proper function When necessary clean the cartridges externally and the surrounding with high purity water and lintfree cloths After the last run of the day perform a cleaning using the corresponding function in the instrument software see section 6 5 2 If required perform a Pyrophosphate clean as described in section 8 6 8 3 4 Exchanging cartridges Annual servicing of the cartridges is recommended to ensure they are within specification for dispensed volumes Servicing of the cartridges can be organized through QIAGEN Technical Services 8 4 Lid movement test The lid movement test ensures the chamber lid is operating correctly The lid will be homed and then moved to the open closed all forward and rear injector positions and then end in the open position PyroMark Q48 Autoprep User Manual 10 2015 133 8 5 Injector test The injector test performs a diagnostic test of the inject
122. k drive and processed on the instrument directly see section 5 7 Optional If desired enter the Reagent ID i e the lot number for PyroMark Q48 Autoprep Reagents a Disc ID of the PyroMark Q48 Disc and a Run Note in the run file 6 2 Preparing templates and reagents At the beginning of the run the instrument will guide you through run preparation including absorber strip insertion injector loading disc insertion and bead and template loading PyroMark Q48 Autoprep User Manual 10 2015 44 To start a sequencing run select Sequence on the home screen and load the run either via USB stick or network connection selectable on the left side of the screen Start the run setup by clicking on its name and the arrow appearing behind the run name Samples to be analyzed using the PyroMark Q48 Autoprep should be prepared according to the instructions below The following equipment and reagents are required for template preparation All reagents and solutions should be at room temperature 15 25 C before starting All steps are performed at room temperature unless otherwise stated Equipment and reagents to be supplied by the user PyroMark Q48 Disc Absorber strip PyroMark Q48 Magnetic Beads Sequencing primer diluted to 4 pM with PyroMark Annealing Buffer or high purity water PyroMark Q48 Advanced Reagents or PyroMark Q48 Advanced CpG Reagents reconstitute the reagents according to the handbook supplied with the
123. late and PyroMark Q48 Magnetic Beads Note When running the Standard protocol the PyroMark Binding Buffer is automatically injected into the sample wells by the instrument injector so there is no need to manually pipet it into the wells Note The two wells on the disc marked by the symbol are waste wells it is not necessary to load beads into these wells PyroMark Q48 Magnetic Beads PyroMark Q48 Magnetic Beads clump very quickly after mixing due to their large size Please follow these guidelines when pipetting the beads Failure to do so will result in inconsistent bead amounts and poor sequencing results Use the supplied electronic multi dispense pipet when dispensing beads into a larger number of wells gt 5 When using the multi dispense pipet never dispense more than 5 samples at a time as the beads will clump inside the pipet PyroMark Q48 Autoprep User Manual 10 2015 Always use the reverse pipetting function to ensure sufficient volume for the last dispensation Ensure that the bead slurry is mixed before taking the next aliquot Always centrifuge any residual bead slurry from the top of the stock tube before returning to storage The consistency of the slurry can change over time if residual solution is left at the top of the stock tube Note See manual of the supplied multi dispense pipet for detailed information on programming Vortex the bead slurry to homogeneity before dispensing
124. lengthen the sequence to analyze Two or more variable sites share dispensations and can therefore not be distinguished If possible use recommended dispensation order 145 Message text Analysis messages in AQ mode Uncertain due to high peak height deviation in variable position or Failed due to high peak height deviation in variable position Missing peaks in variable site Not analyzable due to lack of data Assay setup messages in CoG mode Cannot resolve sequence direction Deselected by user Sequence uncertain due to lack of terminal sequence information Last variable position not analyzable due to lack of terminal sequence information Sequence not in phase at the end of dispensations PyroMark Q48 Autoprep User Manual 10 2015 Comments and suggestions Peak heights deviate from expected levels Even at a variable position the software expects a certain pattern e g a certain sum of the peak heights Check sequence to analyze Check for unknown SNPs The software has detected that all four nucleotides have been dispensed but no signal was detected Clean the cartridge see section 8 3 3 in case there was a dispensation error If problem persists perform injector test see section 8 5 No peaks detected Check PCR yield Check sequencing primer Clean Cartridges Check reagents Check correct usage of buffers A forward CpG assay cannot contain a C after bisulfite treatment A reve
125. les op rateurs de l appareil sont convenablement form s et ne sont pas expos s des niveaux dangereux d agents infectieux comme cela est d fini dans les fiches de donn es de s curit FDS ou dans les documents de l OSHA de l ACGIHT ou du COSHH applicables Pour plus d informations voir le site www qiagen com safety l vacuation des vapeurs et la mise au rebut des d chets doivent s effectuer conform ment toutes les r glementations et lois nationales r gionales et locales relatives la sant et la s curit 15 5 Produits chimiques AVERTISSEMENT Produits chimiques dangereux W7 A La solution de d naturation contient de l hydroxyde de sodium qui est irritant pour les yeux et la peau Toujours porter des lunettes de protection des gants et une blouse de laboratoire La personne responsable par exemple le directeur du laboratoire doit prendre les pr cautions n cessaires afin de garantir que le lieu de travail environnant est s r et que les op rateurs de l appareil ne sont pas expos s des niveaux dangereux de substances chimiques ou biologiques toxiques comme cela est d fini dans les fiches de donn es de s curit FDS ou dans les documents de l OSHA de ACGIHT ou du COSHH applicables Pour plus d informations voir le site www qiagen com safety l vacuation des vapeurs et la mise au rebut des d chets doivent s effectuer conform ment toutes les r glementations et lois nationales r
126. ly WARNING Moving parts A The chamber lid of the PyroMark Q48 Autoprep opens and closes automatically during operation to prevent moisture buildup inside the instrument CAUTION Disc insertion C4 A The PyroMark Q48 Disc must be locked into position to avoid spilling the contents of the disc into the instrument chamber Ensure the disc is locked into position by screwing down the lock nut CAUTION Chamber lid operation C5 A The lid operation is driven by a motor controlled by the software Insufficient space at the rear of the instrument less than 20 cm or 7 9 in to operate the chamber lid may result in damage to the lid upon opening 2 6 Heat hazards WARNING Hot surface W10 A The disc heater within the PyroMark Q48 Autoprep instrument can reach temperatures of up to 95 C 203 F Avoid touching it when it is hot CAUTION Risk of overheating C6 A To ensure proper ventilation maintain a minimum clearance of 5 cm 2 in at the sides and 20 cm 7 9 in at the rear of the PyroMark Q48 Autoprep Slits and openings that ensure the ventilation of the PyroMark Q48 Autoprep must not be covered PyroMark Q48 Autoprep User Manual 10 2015 17 2 7 Consumables CAUTION A Unsupported consumables C7 Do not connect or use any consumables accessories or external equipment other than that specified 2 8 Waste disposal CAUTION A CAUTION A Removal of absorber strip C8 Upon completion
127. lysis is finished or by clicking Stop Note When a well has been analyzed the well color changes to light blue Note If the analysis of a well resulted in an error the well is marked with a red cross PyroMark Q48 Autoprep User Manual 10 2015 Analysis modes PyroMark Q48 Autoprep Software has four analysis modes AQ SNP CpG and SEQ To toggle between the modes select AQ SNP CpG or SEQ in the toolbar A disc can include wells that must be analyzed in different modes To complete all analyses 1 With the run file open in the Analysis view toggle to the desired analysis mode 2 After selecting the analysis mode all wells with an Assay Setup corresponding to the analysis mode will be automatically analyzed and the run will be automatically saved in the background after the analysis is complete If none of the wells has a valid Assay Setup for the chosen analysis mode the analysis is not performed AQ SNP and CpG assays can be analyzed in any analysis mode without modifying analysis parameters To do so select the relevant wells and click Analyze selected Multiple single nucleotide polymorphisms in a variable region can be analyzed in SNP mode but not in AQ mode These variable regions will be automatically disabled in AQ mode InDels can be analyzed in AQ and SNP mode but not in CoG mode Analyzing assays in CoG mode requires that the sequence to analyze be a valid CpG sequence If necessary the appropriate s
128. n Sie die Netzanschlusskabel aus den Steckdosen bevor Sie Wartungsarbeiten am Ger t vornehmen WARNUNG Gefahr durch Stromschlag W5 A Jede Unterbrechung des Schutzleiters Erdungs bzw Masseleiter im Ger t oder auBerhalb des Gerdts und jede Abtrennung des Schutzleiters am Anschluss der Netzleitung erh ht die Gefahr eines Stromschlags Eine absichtliche Unterbrechung der Schutzleiter verbindung ist verboten Gef hrliche Spannung im Ger t Wenn das Ger t an die Stromversorgung angeschlossen ist sind die Anschlussstellen spannungsf hrend Durch das ffnen der Abdeckungen oder das Entfernen von Geh useteilen k nnen spannungsf hrende Komponenten freigelegt werden Vermeiden Sie dass Fl ssigkeiten auf oder in das Ger t gelangen Trennen Sie das Ger t unverz glich von der Stromversorgung falls Fl ssigkeiten auf das Ger t versch ttet wurden Um einen zufriedenstellenden und sicheren Betrieb des PyroMark Q24 Instrument zu gew hrleisten befolgen Sie bitte die nachstehenden Hinweise Die Ger te Netzkabel m ssen an Wechselstrom Steckdosen mit Schutzleiter Erdungs Masseleiter angeschlossen werden Sorgen Sie daf r dass die Netzstecker jederzeit frei zug nglich sind falls das Ger t einmal schnell vom Stromnetz getrennt werden muss Benutzen Sie nur Netzanschl sse und kabel die mit dem Ger t geliefert werden Falls die elektrische Sicherheit bei der Bedienung des Ger ts nicht mehr gew hrle
129. n place en vissant l crou de verrouillage Fonctionnement du capot de la chambre C5 Le fonctionnement du capot est gouvern par un moteur contr l par le logiciel Un espace insuffisant l arri re de l instrument inf rieur 20 cm pour faire fonctionner le capot de la chambre pourrait endommager le capot au moment de l ouverture 15 7 Dangers li s la chaleur AVERTISSEMENT A AVERTISSEMENT A Surface chaude w10 Le chauffe disque l int rieur de l instrument PyroMark Q48 Autoprep peut atteindre des temp ratures de l ordre de 90 C viter de le toucher lorsqu il est chaud Risque de surchauffe C6 Pour garantir une bonne ventilation laisser un espace d gag d au minimum 5 cm sur les c t s et 20 cm l arri re du PyroMark Q48 Autoprep Les fentes et les ouvertures qui assurent la ventilation de l appareil ne doivent pas tre obstru es PyroMark Q48 Autoprep User Manual 10 2015 175 15 8 Consommables ATTENTION A Consommables incompatibles C7 Ne pas relier ni utiliser de consommables d accessoires ni d quipement externe autres que ceux sp cifi s 15 9 Traitement des d chets ATTENTION A ATTENTION A Retrait de la bandelette absorbante C8 Une fois l analyse termin e la bandelette absorbante contiendra la solution de d naturation laquelle renferme de l hydroxyde de sodium Cette substance peut tre irritante pour les yeux et la peau Porter toujours
130. n the maximum dynamic range Automatic gain adjustment may take a few minutes to complete 6 4 4 Viewing a Pyrogram during sequencing During sequencing the Pyrogram of an individual sample is displayed in real time To change the displayed sample touch the Sample Table to bring up the sample list Scroll to locate the sample of interest and then touch it to display that sample Pyrogram PyroMark Q48 Autoprep User Manual 10 2015 58 Time remaining 17 minutes A countdown timer is located at the bottom right corner indicating the time left to run completion The run name is located in the top left hand corner 6 4 5 Aborting a run A run can be aborted at any time during the sequencing step by selecting the Abort button in the top right corner The message Abort Run will appear Select Yes to continue the abort The run will transfer the data to the USB flash drive or network drive Once transferred the user will be notified Run Aborted The transferred run file will end with Aborted 1 Multiple aborted runs will be in sequential order to avoid confusion with other aborted runs with the same name The original run setup will remain available to start again This negates the need for the user to go back to their PC to setup another run Aborting a run during primer annealing To abort the run during primer annealing select the Abort button in the top right corner A confirmation message will appea
131. nalyze the InDel will give a high signal Quantification may be uncertain Check results carefully The dispensations used to analyze the genotype will give a high signal Genotyping may be uncertain Check results carefully The last dispensation is the terminal base of the sequence 147 Message text terminal sequence information Last variable position not analyzable due to lack of terminal sequence information Sequence not in phase at the end of dispensations Analysis messages in SNP mode Failed genotype determination Uncertain due to high peak height deviation in variable position or Failed due to high peak height deviation in variable position Missing peaks in variable site Not analyzable due to lack of data Messages in SEQ mode Basecalling not consistent with entered known bases PyroMark Q48 Autoprep User Manual 10 2015 Comments and suggestions to analyze Add at least one more base at the end of the sequence to analyze so that one or more additional base dispensations are generated The added base must be different from the base currently at the end of the sequence to analyze The last dispensation is included in terminal variable position Add at least one more base at the end of the sequence to analyze so that one or more additional base dispensations are generated The added base must be different from the base currently at the end of the sequence to analyze One allele is seq
132. name sample ID and well note and Pyrograms for all or selected wells see section 7 7 If analysis parameters quality assessments or base called sequence for SEQ results have been edited by the user this is stated in the report The following information icons and colors are displayed and used in the AQ SNP and CpG reports The well name and the sequence to analyze The analysis result allele frequencies AQ report genotypes SNP report or methylation percentages and methylation averages CpG report is displayed above each variable position for example ar se InDel and 28 The background color shows the quality assessment of the analysis result see Quality colors in section 7 15 3 Note in white indicates that a site was deselected by the user na in white indicates that the software does not support the analysis e g analysis of SNP in the CoG mode EEE in red indicates that the analysis was not possible due to lack of data If desired the variable positions are highlighted with a blue gray background color PyroMark Q48 Autoprep User Manual 10 2015 124 Bisulfite treatment controls are highlighted with a light orange background color CpG report only The following information and colors are displayed and used in the SEQ report The well name The base called sequence The background color of a base in the sequence is according to its quality assessment see
133. ncertain due to possible dispensation error at dispensation or Failed due to possible dispensation error at dispensation Uncertain due to high peak height deviation at more than 5 dispensations or Failed due to high peak height deviation at more than 5 dispensations Uncertain due to high noise level or Failed due to high noise level Missing peaks between dispensations Uncertain due to high peak height deviation in surrounding sequence or Failed due to high peak height deviation in surrounding sequence Uncertain reference sequence pattern overall or Failed reference sequence pattern overall Bad Suspect drop detected on A C G T at dispensation PyroMark Q48 Autoprep User Manual 10 2015 Comments and suggestions Desired nucleotide not properly dispensed Clean cartridge properly see section 8 3 3 and rerun If problem persists perform injector test see section 8 5 Peak heights deviate from expected levels at more than 5 dispensations Ensure there is no nucleotide degradation contamination Check sequence to analyze Check for unknown SNPs The noise of the baseline generated by the instrument is too high Check instrument lid Contact QIAGEN Technical Services The software has detected that all four nucleotides have been dispensed but no signal was detected Clean the cartridge see section 8 3 3 in case there was a dispensation error If problem persists perform injector
134. nd of dispensations to fully empty this injector PyroMark Q48 Autoprep User Manual 10 2015 62 If the clean is aborted during the purge the status of each injector that was selected for a clean is updated to a red cross If the user exits the clean protocol a message will appear Some injectors are not clean Exit clean Selecting Yes will return the user to the Home screen Even though the clean has been aborted the clean process must be completed in full at another time Not cleaned injectors will be flagged before any new run begins Pipet another 200 pl of high purity water into the reservoirs of the injectors selected to be cleaned Select the Start button to begin the second clean Confirm Empty Injectors by selecting Yes During the second clean the injectors will be dispensed over the back half of the absorber strip A pause step is introduced after the injectors are loaded to ensure that any leftover reagent is sufficiently dissolved into the water The injectors are then emptied after the pause Successfully cleaned injectors will be displayed with a green tick Failed injectors will be displayed with a red cross and a message indicating what to do next Clean Injectors Step 7 Clean completed Press the home button to exit clean v y y vw y wi y 200u 200p 200p 200pl 200p 200u 200 1 A Cc G T DS E S Unlock the cartridge lids and leave them only slightly open This hel
135. note layout file above Using the paste sample layout feature You can for example generate and copy layouts from 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 PyroMark Q48 Autoprep User Manual 10 2015 108 To paste a sample layout into an existing run file 1 Copy all the information in the source file 2 Right click a well in the Disc Setup and select Paste Sample Layout from the context menu The software will paste the sample IDs into the disc starting at well Al Well notes that have been entered into the wells are kept Edit Format View Help 1323 324 325 326 327 An example of a sample layout created in Microsoft Notepad Qc Setup The result when copying and pasting the sample layout created in Microsoft Notepad 7 13 6 Review the disc setup The Well Information area shows the following information about a well selected in the Disc Setup e Well name Type of assay AQ CpG SNP or SEQ e Assay name Sample ID if entered Sequence to analyze if entered AQ CpG and SNP assays Dispensation order e Well note if entered PyroMark Q48 Autoprep User Manual 10 2015 109 If several wells are selected in the Disc Setup the information for the first selected well is shown If the wells
136. ns Air temperature Relative humidity Environmental class 15 C to 30 C 59 F to 86 F in manufacturer s packaging Max 85 noncondensing 1K2 IEC 60721 3 1 1M2 IEC 60721 3 1 Unless otherwise specified herein PyroMark Q48 Autoprep User Manual 10 2015 163 User software Instrument computer Integrated PC with 7 in touchscreen Analysis computer See section 3 5 File transfer Via USB storage device or network connection Interface Wizard driven user interface with comprehensive information for loading and running instrument Compatibility Linked to PyroMark Q48 Autoprep Software General Workflow Automated template binding template denaturation primer annealing and all associated wash cycles needed for a sequencing reaction Input template 0 5 2 picomoles of biotinylated PCR product Sequencing primer Optional automated sequence primer loading 4 injector positions available Applications Simultaneous running of multiple assays and assay types including SNP AQ CpG and SQA to achieve methylation analysis de novo sequencing mutation characterization including In Dels speciation quantitative allele sequencing and SNP genotyping Waste Electrical and Electronic Equipment WEEE This section provides information about disposal of waste electrical and electronic equipment by Users The crossed out wheeled bin symbol see below indicates that this product must not be disposed of with other waste
137. nsufficient the run files are deleted in chronological order regardless of whether files have been already copied onto an USB stick or not Copy run files as follows 1 Click Tools and select Run Files to access run files Click on the run you want to copy Click the arrow that appears in the right corner E Alternatively all run files can be saved at once using the Copy All button 5 3 Viewing software and hardware version View the PyroMark Q48 Autoprep software and hardware versions by clicking Tools and selecting System 5 4 Upgrading the instrument software If you have received a software upgrade from QIAGEN upgrade the software as follows Download the PyroMark Q48 Autoprep Embedded software from the QIAGEN website Transfer the file to a USB flash drive Note Ensure that only one version and copy of the PyroMark Q48 Autoprep Embedded software is on the USB flash drive 1 Insert the USB flash drive into the PyroMark Q48 Autoprep instrument 2 Select the Tools option on the Home screen and then select the System folder PyroMark Q48 Autoprep User Manual 10 2015 36 3 Install the latest software version by selecting the Update Software button The upgrade version of software will automatically be detected Select Yes to install the software It will take a few seconds to install the new software Once installed select Yes to confirm and restart the software The software will return the user to the Home s
138. nt to inspection at the factory or in the field by an authorized factory representative provided that such defect manifested under normal and proper use Limitation of warranties and remedies THE FOREGOING WARRANTY IS QIAGEN S SOLE AND EXCLUSIVE WARRANTY AND REPAIR OR REPLACEMENT OF DEFECTIVE PARTS IS THE SOLE AND EXCLUSIVE REMEDY THERE ARE NO OTHER WARRANTIES OR GUARANTEES EXPRESS OR IMPLIED THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE EXPRESSLY EXCLUDED TO THE FULLEST EXTENT PERMITTED BY LAW NOTE SOME STATES DO NOT PERMIT DISCLAIMERS OF IMPLIED WARRANTIES SO THIS LIMITATION MAY NOT APPLY TO YOU WITH THE EXCEPTION OF THE ABOVE REFERENCED REPAIR OR REPLACEMENT REMEDY QIAGEN SHALL HAVE NO OBLIGATION OR LIABILITY OF ANY NATURE WHATSOEVER WITH RESPECT TO THE QIAGEN INSTRUMENTATION WHETHER ARISING IN CONTRACT TORT STRICT LIABILITY OR OTHERWISE INCLUDING BUT NOT LIMITED TO LIABILITY FOR INDIRECT CONSEQUENTIAL INCIDENTAL AND OR SPECIAL PUNITIVE MULTIPLE AND OR EXEMPLARY DAMAGES AND OR OTHER LOSSES INCLUDING LOSS OF USE LOST REVENUES LOST PROFITS AND DAMAGE TO REPUTATION EVEN IF SUCH DAMAGES WERE FORESEEN OR FORSEEABLE OR WERE BROUGHT TO QIAGEN S ATTENTION IN NO EVENT SHALL QIAGEN S LIABILITY TO YOU EXCEED THE PURCHASE PRICE OF THE PRODUCT Liability clause QIAGEN shall be released from all obligations under its warranty in the event repairs or modifications are made by persons o
139. oad reagents as indicated Xe XA X 4 XA XA X 100 70 7041 70 wl 580p 3201 32041 2680 pl A c G T DS E S AB x 320 ul BB i 2 Select injectors to prime by touching on the injector box Selected injectors will display a droplet symbol All injectors requiring mandatory injector priming will be displayed with a red cross and drop symbol You cannot override these selected injectors Note Any injectors not being used in the run will be left blank and cannot be selected for injector priming Injectors with a green tick X indicate that they have been primed and do not require injector priming However the user may elect to perform another prime by touching on the injector box to display a drop symbol Injectors with a question mark suggest discretionary injector priming These injectors were previously primed but may require another prime as they have been idle for an extended period of time 3 Select if this is the last run of the day If there is more than one run per day the user can enable the software by deselecting this option to calculate and add the extra volume required for the next run without requiring further injector priming Note If the option last run of the day is selected the software will calculate the minimum volumes required to achieve one run only A mandatory clean will be required after the last run of the day 4 Pipet each reagent into the designated injector acco
140. om Pyrogram 117 Analyze a Run Analyze selected wells 111 Analyze Run Edit analysis parameters 117 Assay design and validation 155 Amplicon length 155 Analysis results 161 Assay setup 158 PCR 155 PCR optimization 157 PCR primers 155 PCR setup 156 PyroMark Q48 Autoprep User Manual 10 2015 Pyrosequencing analysis 158 Sequencing primer 156 Template preparation 158 Validation of a new assay 160 Assay setup 41 Assay Setup view 77 Bead loading 53 Cartridge care 131 Cautions 13 Chamber 27 Cleaning 130 Light detector 131 Consignes de s curit 171 Copying run files 35 Date and time 35 Disc Disc insertion 52 DNA amplification 45 FCC Declaration 165 File menu and toolbars 74 Finishing work and shutting down 60 Cleaning the injectors 61 Removing the disc 60 Replacing the absorber strip 64 Gain adjustment 58 Glossary 152 Grounding requirements 30 Help Menu 77 Histogram 82 Injector cartridge 24 25 29 Care 131 Cleaning 61 133 Exchange 133 Installation 133 Loading 47 Priming 47 Removal 132 Installation 29 Analysis software 33 Delivery 29 Grounding requirements 30 Power requirements 30 Site 29 Unpacking and installation 30 Instrument administration 35 Instrument related errors 150 Intended use 11 185 Legal Manufacturer 166 Light detector maintenance 131 Main menu and toolbars 74 Maintenance 130 Cartridge care 131 cleaning 130 Injector test 134 Lid mo
141. on Note When using QIAGEN kits use the settings stated in the kit handbooks Note All saved changes are logged To view a change log for an assay open the assay file and click Show Change Log Edit analysis parameters in the Analysis Parameters tab The following analysis parameters can be edited in the Analysis Parameters tab Parameter Description Unsuccessful bisulfite These parameters state the highest acceptable percentage of treatment CpG assays unconverted sequence to achieve Passed quality assessment and only 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 The highest acceptable percentage of unconverted sequence to for passed quality achieve Passed quality assessment for the CpG sites Above this value the warning Uncertain bisulfite conversion at dispensation number s is triggered during the analysis and a Check quality assessment is assigned The default value is 5 Note The value cannot be higher than the allowed percentage value for check quality see below PyroMark Q48 Autoprep User Manual 10 2015 96 Parameter Allowed percentage for check quality Peak height threshold Required peak height for passed quality Required peak height for check quality Parameters A peak reduction factor Description The highest acceptable percentage of unc
142. on interference caused by unauthorized modifications of this equipment or the substitution or attachment of connection cables and equipment other than those specified by QIAGEN GmbH Germany The correction of interference caused by such unauthorized modification substitution or attachment will be the responsibility of the user Legal Manufacturer QIAGEN GmbH QIAGEN Strasse 1 40724 Hilden GERMANY Tel 49 2103 29 0 PyroMark Q48 Autoprep User Manual 10 2015 166 13 Appendix C PyroMark Q48 Autoprep Accessories Ordering Information Product Contents Cat no Instrument software and disposables PyroMark Q48 Autoprep PyroMark Q48 Discs 50 PyroMark Q48 Absorber Strips 100 Services PyroMark Q48 Autoprep Cartridge Replacement Software and licences PyroMark Q48 Software License 1 PyroMark Q48 Software License 5 PyroMark Assay Design SW 2 0 PyroMark IdentiFire SW 1 0 Reagents and consumables PyroMark Q48 Autoprep Starter Kit PyroMark Q48 Advanced Reagents 4 x 48 PyroMark Q48 Advanced CpG Reagents 4 x 48 PyroMark Q48 Autoprep User Manual 10 2015 Instrument software and pipet 50 discs for running PyroMark Q48 Autoprep reactions 100 absorber strips for running PyroMark Q48 Autoprep reactions Replacement of all 3 PyroMark Q48 Autoprep Cartridges 1 additional license for PyroMark Q48 Software Only valid together with PyroMark Q48 Autoprep Software 5 additional licen
143. on the first time an assay is run Redesign the assay Dispensation error Perform the injector test see section 8 5 If the problem remains contact QIAGEN Technical Services Unknown SNP in sample Insert the SNP in the sequence to analyze and regenerate the dispensation order Rerun the sample with the new dispensation order Plus shift Change the dispensation order Minus shift Ensure that homopolymers are followed by an extra dispensation 9 4 Analysis software related errors Error or error message Comments and suggestions Red cross over wells in the The analysis of the well resulted in an error Contact Overview tab during analysis QIAGEN Technical Services Exception dialog box appears Save the error report and send to QIAGEN Technical Services for information Click Continue to proceed with analysis If the dialog box remains click Quit and restart the software Could not create assay from Ensure a valid assay file type AQ CpG or SNP is being specitied PyroMark Assay Design imported Software file Assay missing dispensation order Add a dispensation order to the assay setup Cannot be added to well Failed to save file Access denied The file being saved is open in another application Save the file with a different name PyroMark Q48 Autoprep User Manual 10 2015 141 Error or error message The assay setup for well AT is locked and cannot be transferred to a new analysis mode No well has a valid assa
144. one by right clicking the folder containing the file in the shortcut browser and selecting Explore from the context menu 3 In Windows Explorer right click the file and select Properties from the context menu 4 When the Properties dialog box opens turn on M the Read only attribute and click OK backup should be performed frequently 7 18 5 Protection of analysis results It is not possible to edit the analysis parameters or results for a locked assay Pa To lock an assay open the assay file and click the Lock Assay button at the bottom of the assay setup window Lock the assay before adding it to the disc PyroMark Q48 Autoprep User Manual 10 2015 129 8 Maintenance Procedures The only components requiring general user care during the working life of the instrument are the cartridges and the chamber waste area Cleaning of both is mandatory to ensure proper function of the instrument We highly recommend exchanging the cartridges annually 8 1 Cleaning the PyroMark Q48 Autoprep Important Switch the instrument off and disconnect the line power cord from the power outlet before cleaning WARNING Risk of electric shock W12 CAUTION Do not open any panels on the PyroMark Q48 Autoprep A Only perform maintenance that is specifically described in this user manual CAUTION Damage to the instrument C10 A Do not use solvents or reagents containing acids alkalis or abrasives to clean the PyroMark Q48 Autoprep
145. onverted sequence to achieve Check quality assessment for the CpG sites Above this value the warning Failed bisulfite conversion at dispensation number s is triggered during the analysis and a Failed quality assessment is assigned The default value is 7 Note The value cannot be lower than the allowed percentage value for passed quality see above These parameters define the lower intensity limit for the single peak height level of the Pyrogram The minimum signal value for a peak to achieve Passed quality assessment for the variable positions Below this value the warning Uncertain due to low peak height is triggered during the analysis and a Check quality assessment is assigned The default value is 20 Note The value cannot be lower than the required peak height value for check quality see below The minimum signal value for a peak to achieve Check quality assessment for the variable positions Below this value the warning Failed due to low peak height is triggered during the analysis and a Failed quality assessment is assigned The default value is 10 Note The value cannot be higher than the required peak height value for passed quality see above The factor by which the A peak intensities are multiplied to account for the fact that A peaks are higher than other peaks The default value is 0 90 To reset the parameters in the Variable Positions tab and the Analys
146. operation This Class B digital apparatus complies with Canadian ICES 003 The following statement applies to the products covered in this manual unless otherwise specified herein The statement for other products will appear in the accompanying documentation Note This equipment has been tested and found to comply with the limits for a Class B digital device pursuant to part 15 of the FCC Rules These limits are designed to provide reasonable protection against harmful interference in a residential installation This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instructions may cause harmful interference to radio communications However there is no guarantee that interference will not occur in a particular installation If this equipment does cause harmful interference to radio or television reception which can be determined by turning the PyroMark Q48 Autoprep User Manual 10 2015 165 equipment off and on the user is encouraged to try to correct the interference by one or more of the following measures Reorient or relocate the receiving antenna Increase the separation between the equipment and receiver Connect the equipment into an outlet on a circuit different from that to which the receiver is connected Consult the dealer or an experienced radio TV technician for help QIAGEN GmbH Germany is not responsible for any radio televisi
147. or priming has completed successfully the software will progress to the next step with the chamber lid automatically opening Important Navigating to Home following injector priming will require performing injector test shots in the following run Injectors that fail the test will have additional priming and testing to recheck Further failure will mean the injectors are displayed with a red cross gt If any of the injectors has failed the prime and test protocol a Failed Prime warning will appear indicating which injectors have failed Failure to pass injector priming could be due to insufficient reagent being loaded The user has the option to Retry the priming and testing or Skip to the next part PyroMark Q48 Autoprep User Manual 10 2015 Note If priming fails and more than one priming step must be performed the volume in the failed injector is reduced and is not sufficient for dispensations during the run If this occurs the display will inform the user to load an additional 50 pl into the indicated injector to ensure that it contains the required volume only the final volumes of the failed injectors are displayed 4 520 pl ps E If priming problems persist the user can perform the injector diagnostic test see section 8 5 Important Using injectors that have failed priming and testing may result in poor data Prime Failed A No drops detected on injectors E S Check that sufficient reagents are loaded
148. 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 7 15 5 Edit base called sequences To edit a base called sequence right click it and select the desired option It is also possible to copy segments or the complete base called sequence Click and drag the mouse across the segment to be copied selected segment will be highlighted in blue and press while holding down the Ctrl key to copy the selection to the clipboard It is also possible to select a segment of base called sequence by using the Shift and arrow keys _ _ _ as A CG ICT T CT ACGCAATAAACT GAGA GCAAGGGC UAE Increase Selected p Insert After b Insert A 754 Delete Selected Note All changes are logged To view the analysis log for a selected well select Analysis Log from the Tools menu Note If editing a base called sequence note that the quality assessments are still based on the original sequence the sequence called by the software Note It is not possible to edit the base called sequences for a locked assay A 7 16 Viewing printing and saving Analysis Reports Window Help CpG Analysis Statistics CpG Analysis Results Window Help SEQ Analysis Results SEQ Pyrogram Report SEQ Full Report CpG Pyrogram Report CpG Full Report Reports for AQ and CpG runs Reports for SNP and SEQ runs
149. or system including the drop sensors 1 Select injectors to test The selected injectors will display a drop symbol To continue select the Next button esse Injector Test Diagnostic lt A Step 1 Select injectors to test v4 Ve v v a Ve v v A c G T DS S AB VA Ve va v P1 P2 P3WB P4 BB Next P 2 Add 200 pl of clean water to selected injectors 3 Check that an absorber strip is inserted and check that a disc is inserted Note The absorber strip will be dispensed into during injector priming and at the end of the test to empty the injectors The disc will be dispensed into as part of the test 4 Start test Confirm Start full injector diagnostic test by selecting Yes During the prime the test will determine if the drop sensors are working In the second part of the test the selected injectors will dispense a number of times into the first 12 corresponding disc wells e g dCTP into well A2 At the end of the test the remaining liquid is emptied into the absorber strip 5 Record the test result Visually determine if drops are present inside the indicated wells displayed on screen If drops are present click the red cross The red cross will change to a green tick Any failed drop sensors is indicated by a red cross on the displayed screen PyroMark Q48 Autoprep User Manual 10 2015 134 6 If any of the injectors have failed copy the results and send to QIAGEN Technical Servic
150. or these positions in the Analysis Setup tab see section 7 11 5 To view the report before saving or printing it click Preview PyroMark Q48 Autoprep User Manual 10 2015 121 7 16 2 Analysis results report Reports Window Help AQ Analysis Statistics Reports Window Help AQ Pyrogram Report SNP Pyrogram Report AQ Full Report SNP Full Report Reports Window Help CpG Analysis Statistics Cpo Analysis Results CpG Pyrogram Report CpG Full Report Reports Window Help SEQ Analysis Results SEQ Pyrogram Report SEQ Full Report The Analysis Results report includes the following information for all or selected wells see section 7 7 Well information well name assay name and sample ID The allele frequencies AQ report genotypes SNP report methylation percentages CpG report or base called sequences SEQ report and the quality assessments The mean methylation percentage and the standard deviation of all passed CpG sites or CpN sites if CoN mode was used in a well CpG report only The highest and lowest methylation percentage in a well CpG report only Information on whether the analysis parameters quality assessments and analysis results SEQ report only have been edited by the user or not Optional The analysis version well notes and analysis warnings In the AQ and CpG reports it is also possible to include the names and the original an
151. ote It is not possible to add assays created with PyroMark Q24 Software or PyroMark Q96 Software Note If an assay is locked the well is marked with the A icon Disc Setup I i 7 er I a En x r x ELELEE EEEE saviniiniansaniansae 1 fiir tira RSI N e E iil til certeiery Peer ee ee enti A Lisidmiqaiil Carrer ierrer itr eau E AE LEI INT AL A ER RE TRE Te ENT Kirk SE 1 SE 1 sea 1 well is colored according to the assay type loaded to the well Wells with AQ assays are yellow wells with SNP assays are red wells with CpG assays are green wells with SEQ assays are purple Different shades of each color indicate wells with different assays of the same type PyroMark Q48 Autoprep User Manual 10 2015 104 7 13 4 Print or export disc setup as image The Disc Setup can be printed or copied as an image to the clipboard by right clicking the disc and selecting Print or Copy as Image from the context menu The image can be pasted into applications that support Enhanced Metafile EMF images 7 13 5 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 information available in existing documentation Copy or delete contents from cells To cut the contents of a cell to the clipboard rightclick the cell and select Cut from the context menu To copy the contents of a cell to the clipboa
152. ou have any questions or experience any difficulties regarding the PyroMark Q48 Autoprep or QIAGEN products in general 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 helpful 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 goto TechSupporiCenter or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www qiagen com 1 2 2 Policy statement It is the policy of QIAGEN to improve products as new techniques and components become available QIAGEN reserves the right to change specifications at any time In an effort to produce useful and appropriate documentation we appreciate your comments on this user manual Please contact QIAGEN Technical Services 1 2 3 Version management This document is the PyroMark Q48 Autoprep User Manual version 1 1 3 Intended use of the PyroMark Q48 Autoprep PyroMark Q48 Autoprep is designed to detect changes in specified variable positions in or base calling of DNA prepared from biological samples in molecular biology applications PyroMark Q48 Autoprep is intended to be used only in combination with QIAGEN
153. ow Note column Warnings column Analysis version column Position name column Quality column PyroMark Q48 Autoprep User Manual 10 2015 Description The wells to be included in the report The sorting order of the wells The bases in the base called sequences to be included in the report This option is only available for the SEQ report If this option is checked a column with well notes is included If this option is checked a column with analysis warnings is included If this option is checked a column with the analysis version is included If this option is checked a column with the names of the variable positions is included This option is not available for the SEQ report If this option is checked a column with the current quality assessments is included 123 Option Description Original quality columns If this option is checked a column with the original quality assessments is included This option is not available for the SEQ report To view the report before saving or printing it click Preview 7 16 3 Pyrogram report Reports Window Help AQ Analysis Statistics Reports Window Help AQ Analysis Results SNP Analysis Results AQ Full Report SNP Full Report Reports Window Help CpG Analysis Statistics Reports Window Help CpG Analysis Results SEQ Analysis Results CpG Full Report SEQ Full Report The Pyrogram report includes well information well name assay
154. peak height for check quality Parameters A peak reduction factor Known bases To reset the parameters in the Settings tab and the Analysis Parameters tab to their default values click Revert to Default Description These parameters define the lower intensity limit for the single peak height level at the beginning of the Pyrogram The minimum signal value for a peak to achieve Passed quality assessment in the base called sequence Below this value the warning Uncertain due to low peak height is triggered during the analysis and a Check quality assessment is assigned The default value is 4 Note The value cannot be lower than the required peak height value for check quality see below The minimum signal value for a peak to achieve Check quality assessment in the base called sequence Below this value the warning Failed due to low peak height is triggered during the analysis and the Failed quality assessment is assigned The default value is 2 Note The value cannot be higher than the required peak height value for passed quality see above The factor by which the A peak intensities are multiplied to account for the fact that A peaks are higher than other peaks The default value is 0 90 If there are any known bases in the dispensation order we recommend that these are entered as this can improve the analysis 1 Left click the relevant dispensation and either enter the heigh
155. plification is to compare the peak heights from a heterozygote If the SNP is represented by single base incorporations e g AAC TGG the two alleles C and T peaks should give peaks of equal height An InDel heterozygote should give 50 deletion Template preparation Use 10 pl of a 25 pl PCR for immobilization to PyroMark Q48 Magnetic Beads according to the instructions in section 6 2 5 Pyrosequencing analysis Use default settings in the software for all assay setups if not otherwise stated Assay setup AQ SNP and CpG assays When creating an AQ SNP or CpG assay the sequence to analyze should contain a sufficient number of bases to generate at least five nonvariable reference peaks For InDels a few reference peaks should be included before the variable position If the sequencing primer is placed adjacent to the position to analyze include part of the sequence following the variable position in the Sequence to Analyze text box Ensure that the last base in the text box is not part of the variable position Blank dispensations are automatically generated by the software and serve as built in quality controls for the assay Never exclude the blank dispensations as they function as excellent indicators of unspecific nucleotide incorporation When manually generating a dispensation order include an appropriate number of blank dispensations If possible start the dispensation order with one blank dispensation and have at least
156. 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 marked red in the dispensation order Dispensation Order UCTGAICTTAISC TC id The entered dispensation order is not complete Expanded Dispensation Onder CTGACTGACTGACTTAGCTC The error The entered dispensation order is not complete is caused by a missing or incorrect positioned parenthesis In this example a closing parenthesis is missing 7 12 3 Edit analysis parameters The default analysis settings have been set to give optimal analysis results for most assays If applicable during the assay validation the results may be improved by editing the analysis parameters The Quality Control Window setting in the Settings tab is by default set to 20 If more or less bases are required change accordingly e Edit analysis parameters in the Analysis Parameters tab Ensure changes are validated see Appendix A Assay design and validation Note When using QIAGEN kits use the settings stated in the kit handbook Note All saved changes are logged To view a change log for an assay open the assay file and click Show Change Log at the bottom of the assay setup window PyroMark Q48 Autoprep User Manual 10 2015 100 Edit analysis parameters in the Analysis Parameters tab Parameter Peak height threshold Required peak height for passed quality Required
157. positive controls aim for Sufficient signal intensities Aim for a single peak height of at least 20 RLU No background in blank dispensations No background in variable positions AQ SNP and CpG Expected reference sequence pattern AQ SNP and CpG e All positions AQ SNP and CpG and quality control window SEQ with quality assessment Passed The quality assessments for AQ SNP and CpG assays are based on the sequence context as well as the results in the analyzed positions Deviations from this built in quality control are shown as warnings in the Well Information area The analysis results for SEQ assays are based on the appearance of the peaks in Pyrogram related to peak height levels estimated by the software The inclusion of known bases in SEQ assays can improve the estimation of peak height level PyroMark Q48 Autoprep User Manual 10 2015 16 12 Appendix B Technical Data QIAGEN reserves the right to change specifications at any time Mechanical data and hardware features Dimensions Weight Sample disc well capacity Rotor speed Rotor mixing Injectors Nucleotide injector Primer injector Reagent injector Chamber temperature Waste collection Width 250 mm 9 8 in Depth 300 mm 11 8 in with chamber lid closed 560 mm 22 in with chamber lid open Height 300 mm 11 8 in with injector cover closed 390 mm 15 4 in with injector cover open 8 5 kg 18 7 lb
158. ps to prevent condensation and minimizes the amount of dust that might settle into the cartridges Important Condensation within the reservoirs over time may result in microbial growth and reagent dilution 10 Select the Home button to return back to the Home screen 11 Ensure that the injector cover is closed to avoid dust buildup in the cartridges Apply a second wash if it is certain that the instrument will not be used for a period of more than 2 weeks PyroMark Q48 Autoprep User Manual 10 2015 63 6 5 3 Replacing the absorber strip The user will be notified to replace the absorber strip refer to section 6 2 2 Once the absorber strip is replaced select OK to return to the Home screen G Replace Absorber Strip A Failure to replace the absorber strip may damage the system Select OK to confirm the absorber strip has been replaced 6 6 Shutting down the instrument 1 To shut down the PyroMark Q48 Autoprep navigate to the main menu on the instrument touchscreen 2 Turn off the power using the switch located at the rear of the instrument 6 7 Analyzing a run Detailed instructions for analyzing the run are available in section 7 If using an USB stick for data transfer move the processed run file from the USB stick to a computer running PyroMark Q48 Autoprep Software see section 5 2 Alternatively the run file can be transferred via a network drive see section 5 7 Open the run file by double clickin
159. py as Image from the context menu The image can be pasted into applications that support Enhanced Metafile EMF images 7 10 Starting the software In the Windows Start menu select All Programs PyroMark PyroMark Q48 Autoprep The information in this section on PyroMark Q48 Autoprep Software can be accessed at any time by pressing the F1 key when in the software PyroMark Q48 Autoprep User Manual 10 2015 86 7 11 Setting up an AQ SNP or CpG Assay 7 11 1 Workflow to set up an AQ SNP or CpG Assay New AQ Assay Ctrl A New SNP Assay Cirl M New CpG Assay Ctrl G New SEQ Assay Ctrl Q 1 Click in the toolbar and select New AQ Assay New SNP Assay or New CpG Assay A new assay file is created For analyzing methylation at CpN sites create a New CpG Assay and then enable the CoN mode by checking the box next to CPN mode enabled Alternatively you can create a new assay file in the shortcut browser by right clicking the folder you wish to place it in and selecting New Assay followed by New AQ Assay New SNP Assay or New CpG Assay from the context menu Enter the filename and press Enter You cannot use the same name as another file saved in the same folder To add a shortcut to a folder or drive click Add Folder Shortcut A ores rome ass ar bees his eme PURES SUIS 8 EN PURISFE D gpispe SE Laas F ae Capa ieee i PyroMark Q48 Autoprep User Manual 10 2015 87 2 Enter the sequence to analyze se
160. r Do you wish to abort the run If the user selects Yes a warning message may appear informing the user that Cooling disc heater to 40 C Currently X X C It is highly recommended that the user wait until the temperature is cooled below 40 C before opening the chamber lid However the user can Override the cool if they wish to gain access to the chamber before the temperature is below 40 C PyroMark Q48 Autoprep User Manual 10 2015 WARNING Hot surface w10 A The disc heater within the PyroMark Q48 Autoprep instrument can reach temperatures of up to 95 C 203 F Avoid touching it when it is hot 6 4 6 Run completion Once the run has completed the lid will automatically open and the data will be transferred onto the USB flash drive or network drive It may take a few minutes to transfer larger data files Once the data is transferred the user will be notified Run Complete Select the Home button to return to the home page to begin another run or to clean the injectors Remove the USB flash drive and analyze the data on the PyroMark Q48 Autoprep Software If another run will follow for which other primers are needed the instrument will inform the user that injectors have been changed since the last run and cleaning of the primer injectors is recommended only in case of automatic primer loading Notification to start the cleaning procedure will automatically appear if the last run of the day wa
161. rd either right click the cell and select Copy Cell from the context menu or select the cell and press Ctrl C To paste the clipboard to a cell or a selection of cells see section 7 7 either right click the cell or the selection and select Paste from the context menu or select the cell s and press Ctrl V 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 Enter sample IDs and notes To enter a sample ID or note select the corresponding cell see image below and enter the text To edit a sample ID or note double click the corresponding cell To import a sample and note layout defined in a text file tsv or csv rightclick a well and select Insert Sample Layout File from the context menu For more information see Using the import insert sample layout file feature in section 7 13 5 PyroMark Q48 Autoprep User Manual 10 2015 105 To paste a sample layout from the clipboard rightclick a well and select Paste Sample Layout from the context menu For more information see Using the paste sample layout feature in section 7 13 5 Note Commas and semicolon are not supported A selected cell is highlighted with a blue background c olor Drag copy the contents of a cell to other wells To drag copy the contents of a cell to other wells 1 Select the cell that yo
162. rding to the volumes shown on the instrument touchscreen Use the volumes displayed to ensure sufficient reagent is available for the initial injector prime as well as for dispensations during the run Changing the response in steps 2 or 3 will change the required reagent volumes Important Ensure that the reservoir capacity of 3 8 ml is not exceeded Note Information about the required sequencing primers can be brought up on screen by selecting the information button on the sequencing primer injectors Note Ensure that the tip of the pipette does not touch the filter at the bottom PyroMark Q48 Autoprep User Manual 10 2015 48 Lid lock Reservoir neck _ Filter Note If loading volumes less than 100 ul ensure that the solution is dispensed from the top of the reservoir neck Note Slide the chamber lid toward the front of the instrument to make it easier to load into the reservoirs Note When loading injectors try to avoid air bubbles in the liquid as they can influence single dispensations Note In contrast to other PyroMark systems PyroMark Wash buffer is not needed and is replaced by PyroMark Annealing buffer 5 Once all the reservoirs have been filled with the required reagent close and lock the cartridge lids using the lid locks Important Remember to store the reagents at 4 C once all reservoirs are loaded to ensure long reagent shelf life 6 Select the Start Button to b
163. re de d brancher rapidement l quipement du secteur Utiliser uniquement les fiches et cordons d alimentation fournis avec le syst me N i l appareil pr sente un danger lectrique emp cher le reste du personnel de s en servir et contacter les Services Techniques de QIAGEN L appareil peut pr senter un danger lectrique dans les cas suivants Le cordon d alimentation pr sente des signes de d t rioration l appareil a t stock pendant une longue p riode dans des conditions non conformes celles nonc es dans l Annexe A l appareil a subi des chocs s v res durant le transport Un liquide a p n tr l int rieur de l appareil PyroMark Q48 Autoprep User Manual 10 2015 173 15 4 S curit biologique Lors de la manipulation de substances biologiques employer des proc dures de laboratoire s res comme d crit dans des publications telles que Biosafety in Microbiological and Biomedical Laboratories HHS www cdc gov od ohs biostty biosfty htm AVERTISSEMENT Substances biologiques W Manipuler les substances biologiques avec la plus grande pr caution et conform ment aux r glementations de s curit en vigueur Toujours porter des lunettes de protection deux paires de gants et une blouse de laboratoire La personne responsable par exemple le directeur du laboratoire doit prendre les pr cautions n cessaires afin de garantir que le lieu de travail environnant est s r et que
164. reate a new Run Setup by one of the following methods e Click E in the toolbar Select New Run from the File menu Press the R key while holding down the Ctrl key Right click a folder in the shortcut browser and select New Run from the context menu Enter a run name and press Enter To add a shortcut to a folder in the shortcut browser click Add Folder Shortcut PyroMark Q48 Autoprep User Manual 10 2015 To base your run on a previous run rightclick the processed run file in the shortcut browser and select Copy and Rerun from the context menu Only the run setup not the run and analysis data will be copied 2 If the new run has not yet been saved click al to enter a run name and save the file in the desired folder 3 Set up the disc in the disc layout of the run file by adding assays to wells and if desired entering the sample ID and note for each used well 4 Add an assay to each well used by Dragging an assay from the shortcut browser to a well or a selection of wells Right clicking one well and selecting Load Assay from the context menu A well is colored according to the assay loaded into the well Wells with AQ assays are yellow wells with SNP assays are red wells with CoG assays are green wells with SEQ assays are purple Disc setup There are several ways to set up a disc For example it is possible to import and paste a sample layout defined in a text file For more information
165. rep User Manual 10 2015 73 7 3 Main menu and toolbars 7 3 1 File menu and toolbars Tools Reports Windo New Assay New Run Ctri R Open Ctrl O Import New AQ Assay Ctrl A ave Ctrl 5 New SNP Assay Ctrl N Save As Ctrl Shiit S Exit New CpG Assay Ctrl G New SEQ Assay Ctrl Q Select New Assay or click in the toolbar and select the desired assay type to create a new assay file To set up the assay see section 7 11 for an AQ SNP or CpG assay or section 7 12 for a SEQ Assay Select New Run or click EM in the toolbar to create a new run file To set up the run see section 7 13 Select Open or click EF in the toolbar to open a saved assay or run file Select Create New Run from Sample Layout File from the Import submenu to create a new run using a disc layout for sample IDs and notes optional defined in a tab or comma delimited text file tsv txt or csv see Using the import insert sample layout file feature in section 7 13 5 Select Create New AQ SNP CpG Assay from Assay Design File from the Import submenu to create a new AQ SNP or CpG assay based on an assay file xml created with PyroMark Assay Design Software The software will import the sequence to analyze and the names of the variable positions Select Save or click lal in the toolbar to save changes in the current file If the file has never been saved select the location and enter the filename in the dialog box that opens Select Sav
166. required 5 7 1 Network share settings The PyroMark Q48 Autoprep can load and save run files to a shared folder that is accessible from the network to which it is connected 5 7 2 Network path This defines the location of the network folder used for run files It must be entered as a UNC path which has the general form lt computer name gt lt share name gt lt optional additional path gt For example myserver labfiles PyroMark Q48 Autoprep runs will look for a folder called PyroMark Q48 Autoprep runs in a share called labfiles on a computer called myserver Any shared folder that is compatible with Microsoft Windows networking protocols may be used 5 7 3 User Name Domain Name and Password These three items define the network credentials that will be used to connect to the specified network path The domain name should be set to the name of the Microsoft Windows Domain if the target computer is part of a domain If the target computer is not part of a domain then enter the name of the target computer here The user name and corresponding password should correspond to a user on the domain or target computer The user must have read and write access to the specified network path This password is stored in the machine and may pose a security risk if the device were to be stolen For this reason it is recommended that a special user be set up on the target computer or domain that is given read and write acce
167. riming ccmmmesessseeenennnennnesennnnnnnnn 47 6 2 4 Inserting the PyroMark Q48 Disc into the instrument 52 6 2 5 Loading template and beads into the PyroMark Q48 Disc 53 PyroMark Q48 Autoprep User Manual 10 2015 0 OL IDEE COR ee nee 55 OA MON NORMES in ne 55 6 4 1 Manual sequence primer loading cccccccceeesseeeeeeeeeeeeeaeeeeeeeeeeesaaeeees 57 6 4 2 Automatic sequence primer loading 58 6 4 3 Automatic gain ad Usimenlen nnn een 58 6 4 4 Viewing a Pyrogram during SEQUENCING eeeeeeeeenneesssseeeennnnnnnnneee nenn 58 GA ABO eM cl 4 res ee ee eee eee E 59 0 4 9 RO CO cate ee ences 60 6 5 Finishing work and shutting down 60 6 5 1 Removing the PyroMark Q48 Disc 60 6 5 2 Cleaning the SCOR een 61 6 5 3 Replacing the absorber Sifip 2 4 vince ondeiweennssdennengeids 64 6 6 Shutting down the instrument 64 O7 AO ARS Re 64 6 8 Viewing the analysis results ccccecccccececeeeseeeeeeeeeeeeueeeeeeeeeeeeessaeeeseeeeeeeeaaas 65 6 8 1 Quality assessments 66 6 8 2 AQ analysis results susanne rennen 67 6 8 3 CpG analysis SOS a 67 684 SEQ Analysis F SUS cessions tea 67 6 89 SNP analysis TSI een 69 0 020 Analys DO ee 69 7 PyroMark Q48 Autoprep Software 70 Zel OS SOS ee ee ee 70 72 OO OV WS ce tats ee eee ede oes 7 73 NON MENU NACRE te nee ae eee ee 74 Zal Fiemencand 108 bais
168. rk Q48 Autoprep und die Kartuschen d rfen nicht in direktem Sonnenlicht oder in unmittelbarer N he von W rme und Vibrationsquellen oder elektrischen St rfeldern platziert werden ACHTUNG Besch digung des Ger ts C2 A Versch tten Sie keine Fl ssigkeiten oder Chemikalien auf dem PyroMark Q48 Autoprep Durch versch ttetes Wasser oder versch ttete Chemikalien verursachte Sch den f hren zum Erl schen der Garantie WARNUNG ACHTUNG N Bewegen Sie den PyroMark Q48 Autoprep auf keinen Fall w hrend des Verletzungsgefahr und Besch digung des Ger ts w2 Betriebs WARNUNG Explosionsf hige Atmosph ren W3 ACHTUNG Der PyroMark Q48 Autoprep ist nicht f r den Gebrauch in explosionsf higen A Atmosph ren vorgesehen Explosionsgefahr W4 A Der PyroMark Q48 Autoprep ist ausschlieBlich mit Reagenzien und Substanzen aus den QIAGEN Kits zu benutzen Die Benutzung von anderen Reagenzien oder Substanzen kann Feuer oder eine Explosion ausl sen ACHTUNG Einsetzen des Absorberstreifens C3 Stellen Sie sicher dass der Absorberstreifen im PyroMark Q48 Autoprep A eingesetzt ist wie in Abschnitt 6 2 2 beschrieben um das Eindringen von Fl ssigkeit in die Kammer zu verhindern Schalten Sie im Notfall den PyroMark Q48 Autoprep aus der Netzschalter befindet sich auf der Ger ter ckseite und ziehen Sie den Netzstecker aus der Steckdose PyroMark Q48 Autoprep User Manual 10 2015 179 16 3 Schutz vor Stromschlag Ziehe
169. rminal The problem may be resolved by either entering more sequence sequence information information or reducing the number of dispensations Sequence not in phase at the end of the The problem may be resolved by adjusting the dispensation order dispensations manually or by clicking 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 dispensation order contains If possible enter more sequence information and increase the number less reference peaks than required of dispensations For the best possible quality assessment of the results five or more reference peaks with the height 1 2 or 3 are recommended Some genotypes will generate the same The sequence to analyze contains multiple variable sites within a sequence patterns and will not be variable region that will generate sequence patterns that are distinguishable indistinguishable between genotypes Review results carefully 7 11 4 Add or remove bisulfite treatment controls CpG assays Histogram CpG assays should contain an 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 template
170. rophosphate clean and injector diagnostic test In the event that you need to send a support package to QIAGEN Technical Services locate and copy the file to a USB flash drive Support packages will be compressed to allow for easy transfer through email Click Tools and select Support on the instrument screen to access the available support packages The last 20 support packages are available and are dated and time stamped with the most recent runs at the top of the list Alternatively all the support packages can be saved at once using the Copy All button Clean Clean 0140807 114555 mp Thy OF Aug 2004 11 45 Rundborted Run borte ayaa 114515 2 Thu OF Aug AMA 11 85 as Clean Network Clesn 20140807 114258 zp Thu OF Aug ANA 11 42 IT Clean Dean 201 4080 112646 zip Thu OF Aug 204 1126 Diagnostics co o No USB device available System PyroMark Q48 Autoprep User Manual 10 2015 139 9 3 Analysis related errors PCR failed due to low DNA quality Poorly optimized PCR Biotinylation is omitted or not added to the correct PCR primer Biotinylation is of poor quality Insufficient amount of template for immobilization PyroMark Q48 Magnetic Beads Too much PCR product depletes substrate leading to missing peaks at the end of the sequence One or several of the compartments in the reagent cartridge were not correctly filled One of the injectors in the reagent cartridge is blocked or damaged
171. rse CoG assay cannot contain a G after bisulfite treatment Variable position was deselected for analysis by the user The last dispensation is the terminal base of the sequence to analyze Add at least one more base at the end of the sequence to analyze so that one or more additional base dispensations is generated The added base must be different from the base currently at the end of the sequence to analyze The last dispensation is included in terminal variable position Add at least one more base at the end of the sequence to analyze so that one or more additional base dispensations are generated The added base must be different from the base currently at the end of the sequence to analyze One allele is sequenced ahead of the other s and the two or more alleles are not in phase with the last dispensation Add more sequence information i e lengthen the sequence to analyze 146 Message text Analysis not supported Comments and suggestions This dispensation order will support the analysis of SNPs Variable positions with common dispensations cannot be analyzed The position is not valid as bisulfite treatment control Analysis messages in CoG mode Uncertain bisulfite conversion at dispensation or Failed bisulfite conversion at dispensation Uncertain due to high peak height deviation in variable position or Failed due to high peak height deviation in variable position Missing peaks in varia
172. rtridges are installed in the wrong order you will be prompted to install the cartridges correctly upon pressing the Sequence button on the Home screen PyroMark Q48 Autoprep User Manual 10 2015 30 Remove the parts box and foam packing to gain access to the PyroMark Q48 Autoprep instrument Carefully remove the PyroMark Q48 Autoprep from the box and place it on a surface suitable for installation see section 4 2 1 Remove the transport lock screw and store in a safe place e g in the screw holder on the back of the instrument in case the instrument needs to be transported Remove the injector cartridges from the boxes and insert them in the following positions as shown in the illustration in section 3 4 Nucleotide A C G and T into the left side e Primer P1 P2 P3 and P4 BB into the middle Reagent DS E S and AB into the right side Slot the cartridges into place ensuring that the air pin and air hole align and the electrical connectors align Make sure the cartridges are connected properly by exerting pressure on the rear part of the cartridge black box Lock the cartridges into place using the thumb screws PyroMark Q48 Autoprep User Manual 10 2015 31 Do not insert the silver cartridge thumb screws into the top plate when no cartridges are installed Without the cartridges in place the screws can protrude too deep and scratch the lower plate WARNING Condensate
173. s including warnings date and time and the Windows user account used to perform the analysis see section 7 18 Text files tsv or csv can be imported into Microsoft Excel or other applications that can handle data that is separated by semicolons or tabs This is useful when doing further calculations on the data PyroMark Q48 Autoprep User Manual 10 2015 75 7 3 3 Reports menu Window Help AQ Analysts Statistics AQ Analysis Results AQ Pyrogram Report AQ Full Report AQ runs Window Help SEQ Analysis Results SEQ Pyrogram Report SEQ Full Report Reports Window Help SNP Analysis Results SNP Pyrogram Report SNP Full Report SNP runs Window Help CpG Analysis Statistics CpG Analysis Results CpG Pyrogram Report CpG Full Report SEQ runs CpG runs The Analysis Statistics report includes analysis statistics for all or selected wells The Analysis Results report includes well information and analysis results for all or selected wells The Pyrogram Report includes well information and a Pyrogram for all or selected wells The Full Report includes run parameters run log well information and analysis results including Pyrogram for all or selected wells The report options are only available for processed runs For more information on the reports see section 7 16 Note To view reports in PDF format a PDF reader must be installed on the computer Adobe Reader can be downloaded at www
174. s selected 6 5 Finishing work and shutting down 6 5 1 Removing the PyroMark Q48 Disc 1 Manually open the chamber lid 2 Unscrew the lock nut 3 Carefully lift the disc out using the disc grips This should minimize finger and hand contact with the absorber strip PyroMark Q48 Autoprep User Manual 10 2015 60 gt 6 5 2 Cleaning the injectors Upon completion of the final run of the day the injectors must be cleaned with high purity water CAUTION Injector cleaning C14 A Injectors must be cleaned within a 12 hour period following the last run to ensure long term injector performance Failure to comply can result in injectors blocking To begin the clean protocol select the Cleaning button on the Home screen If the last run of the day was selected then cleaning of the injectors will be mandatory and the cleaning function will appear automatically 1 Select injectors to be cleaned by touching on the injector square A drop symbol will appear indicating selection Injectors can be cleaned in between runs if desired or if incorrectly pipetting reagents Injectors with a red cross and drop symbol 4 require a clean injectors have been used recently and have not been cleaned Unlike in injector priming red cross injectors can be deselected from cleaning It is highly recommended but not mandatory to clean injectors with a question mark injectors have been cleaned previously but have
175. s 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 We recommend directly entering the sequence before bisulfite treatment because this enables the software to generate the sequence to analyze and automatically select one appropriate bisulfite treatment control If the user directly enters the sequence to analyze or if the software does not find a suitable bisulfite control one bisulfite control will need to be added manually by the user after the dispensation order has been generated A bisulfite treatment control can be added manually by adding a before or after a T in a forward assay or by adding a G before or after an A in a reverse assay in the dispensation PyroMark Q48 Autoprep User Manual 10 2015 23 order This is only possible if it s known that in the sequence before bisulfite treatment the suggested bisulfite treatment controls are Cs converted to Ts read as Gs and As in a reverse assay Only one bisulfite control may be included If additional controls are manually added in the dispensation order they will be displayed as red Cs in the histogram Note For CoG assays with CoN mode enabled if the sequence before bisulfite treatment is entered the software will use an unselected CpN site as a bisulfite treatment control 7 11 5 Set up variable positions The variable positions can be set up in the Variable Positions tab Th
176. ses for PyroMark Q48 Software Only valid together with PyroMark Q48 Autoprep Software Software for convenient design of PCR and sequencing primers optimized for Pyrosequencing analysis Software for comparing and matching Pyrosequencing data to a local database PyroMark Q48 Magnetic Beads 300 PyroMark Q48 Advanced CpG Reagents 4 x 48 PyroMark Control Oligo PyroMark Q48 Discs 50 and PyroMark Q48 Absorber Strips 100 Reagents for 4 x 48 PyroMark Q48 Autoprep standard reactions Reagents for 4 x 48 PyroMark Q48 Autoprep CpG and long read reactions 9002470 974901 974912 9244450 9024325 9024326 9019077 9019087 974230 974002 974022 167 Product PyroMark Q48 Magnetic Beads 300 PyroMark Control Oligo PyroMark PCR Kit 200 PyroMark OneStep RT PCR Kit 50 EpiTect Fast DNA Bisulfite Kit 50 EpiTect Fast FFPE Bisulfite Kit 50 t EpiTect Fast LyseAll Bisulfite Kit 50 Larger kit sizes available t 96 well format available Contents Magnetic streptavidin coated Sepharose beads for running 300 PyroMark Q48 Autoprep reactions For installation check of system PCR Master Mix for PCR reactions optimized for Pyrosequencing analysis OneStep RT PCR Enzyme Mix for RT PCR reactions optimized for Pyrosequencing analysis For 50 preps Bisulfite Solution DNA Protect Buffer MinElute DNA Spin Columns Carrier RNA and Buffers For 50 preps Deparaffinization Solution
177. sifs pour nettoyer le PyroMark Q48 Autoprep D t rioration de l cran tactile et de l ordinateur C11 Ne pas verser ni vaporiser des liquides par ex des d tergents sur le PyroMark Q48 Autoprep Utiliser un chiffon humidifi par de l eau seule pour le nettoyage Entretien du d tecteur de lumi re C12 Utiliser des chiffons non pelucheux pour nettoyer soigneusement la fen tre du photomultiplicateur PMT Ne pas utiliser de mouchoirs en papier Voir la section 8 2 pour plus de d tails D t rioration de l instrument C13 A Ne pas exposer le photomultiplicateur PMT une lumi re forte pendant l entretien ATTENTION Nettoyage de l injecteur C14 A Les injecteurs doivent tre nettoy s au cours des 12 heures suivant la derni re analyse pour assurer de bonnes performances durables de l injecteur Le non respect de cette consigne pourrait provoquer une obstruction des injecteurs PyroMark Q48 Autoprep User Manual 10 2015 177 16 Appendix F Safety Information German DE 16 1 Sicherheitshinweise Lesen Sie dieses Handbuch sorgf ltig durch bevor Sie den QlAgility benutzen Beachten Sie dabei insbesondere die Sicherheitshinweise Die Gebrauchsanweisungen und Sicherheitshinweise im Handbuch m ssen befolgt werden um einen sicheren Betrieb des Ger ts zu gew hrleisten und das Ger t in einem sicheren Zustand zu erhalten In diesem Handbuch werden die folgenden Kategorien von Sicherheitshinweisen verwend
178. splays the height When showing the histogram a compensated Pyrogram is displayed in gray over the peaks in the Pyrogram It is best viewed when zoomed in PyroMark Q48 Autoprep User Manual 10 2015 85 When showing known bases peaks with known bases are marked with blue diamonds in Pyrogram When showing peak levels calculated peak levels are displayed in the Pyrogram Colors used in the Graph area correspond to quality assessments see Quality colors in section 7 15 3 By positioning the mouse pointer on a base in the base called sequence a tooltip displays the position number Note By right clicking the Pyrogram pane it is possible to toggle between viewing and hiding the histogram known bases and peak levels 7 9 3 Zoom Pyrogram It is possible to zoom in on the Pyrogram by selecting a stretch 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 The Pyrogram Y axis scale may also be manually adjusted by right clicking on it and selecting Set Max Y Axis Value The value is limited to three digits and must be confirmed by pressing the Enter button or by clicking the mouse button 7 9 4 Export Pyrogram as an image The Pyrogram can be copied as an image to the clipboard by right clicking the Pyrogram area and selecting Co
179. ss only to the specified network path Please consult with the network administrator PyroMark Q48 Autoprep User Manual 10 2015 39 5 7 4 IP Address Settings The IP address settings control how the PyroMark Q48 Autoprep connects to the network By default the PyroMark Q48 Autoprep will attempt to obtain the settings from the network automatically This will be suitable in most circumstances however the network administrator may require the instrument to have a fixed IP address This can be set by selecting the manual option and entering an IP address subnet mask default gateway and DNS address These settings will be provided by the network administrator The IP address currently assigned to the instrument will be visible at the bottom left of the screen If the instrument has been unable to negotiate an address with the network the words Not Connected will appear They may indicate a problem with the IP address settings or more likely a problem with the physical connection to the network In this case check the Ethernet cable connection at the rear of the instrument to make sure it is correctly connected and that the other end of the cable is connected to the appropriate network equipment It may take up to a minute to connect to the network once you plug in an active cable Network Path serveripath Domain Name DOMAIN User Name user Password e Obtain an IP address automatically O Specify an IP a
180. ssays If creating a CpG assay we recommend entering the sequence before bisulfite treatment into the Sequence Before Bisulfite Treatment text box This enables the software to automatically generate the sequence to analyze and select the most appropriate bisulfite treatment control PyroMark Q48 Autoprep User Manual 10 2015 89 The following rules apply when entering the DNA Sequence Before Bisulfite Treatment in the software The allowed characters for sequence input are A C G and T as well as IUPAC codes Variable positions such as SNPs can be entered using either IUPAC codes or a forward slash as a separator between the two potential bases e g C G The sequence should not include more than 400 characters or 100 variable positions e InDels are not supported If the software detects inconsistencies in the sequence before bisulfite treatment the user is informed by an information symbol next to the Sequence Before Bisulfite Treatment text box The warning text is shown in a tooltip when moving the mouse over the symbol Sequence Before Bisulfite Treatment Note C in variable positions will be converted to T Forward Orientation Reverse Onentation If the sequence before bisulfite treatment is edited such that it becomes invalid the check boxes along with the Select all and Deselect all buttons will be grayed out indicating that they are disabled and no longer selectable The check box
181. st of programs select PyroMark Q48 Autoprep Click Uninstall a ae To Repeat steps 3 and 4 for PyroMark Launcher PyroMark Q48 Autoprep User Manual 10 2015 In the Control Panel click Programs and then click Programs and Features 34 5 Instrument Administration This section describes how to operate PyroMark Q48 Autoprep Before proceeding we recommend reading section 3 4 to familiarize yourself with features of the instrument 5 1 Setting date and time Setting the date and time correctly ensures an accurate date and time stamp in the instrument run logs and the analysis reports The System Time should be updated during instrument installation or after a transport lock Set the date and local time by scrolling through the time and date wheels Set the time zone using the selections available in the drop down menu Set the system date and time Day Month Year Hour February March April May 12 June Time Zone GMT 10 00 Brisbane T 5 2 Copying run files Copies of previous run files are stored on internal memory Up to 12 previous run files are available with the most recent run files at the top PyroMark Q48 Autoprep User Manual 10 2015 Pol Testrunt AQ qrun Tue 25 Nov 2014 09 69 Support Testrund SNP qrun Tue 25 Now 2014 09 59 Testruni SEQ grun 2 Tue 25 Nov 2014 09 59 Testrun2 CpG grun Tet a Mow 20d CES No USB device available Copy Al Note When space becomes i
182. t a protocol for the run Enter the remaining run parameters and an optional note see section 7 13 2 6 Set up the disc in the disc layout of the run file by adding assays to wells and if desired entering a sample ID and note for each used well see section 7 13 5 7 When the run is set up and ready to run on the PyroMark Q48 Autoprep instrument click a to save 8 Print the disc setup by selecting Pre Run Information from the Tools menu and when the report opens clicking Note To print the Pre Run Information report in color turn on the Print background colors and images option in the Internet Explorer Tools Internet Options Advanced Printing PyroMark Q48 Autoprep User Manual 10 2015 102 7 13 2 Enter run parameters The following run parameters are available Parameter Run name Protocol Primer loading Disc ID Bar code Reagent lot number Run note Description The name of the run is given when the file is saved Renaming the file also changes the name of the run The Standard protocol is used as the default The P4 Extra protocol enables the use of the fourth sequence primer injector P4 When using this protocol the binding buffer must be loaded manually by the user The maximum template volume is 10 pl to avoid exceeding the well capacity Select either manual or automatic sequence primer loading For automatic primer loading allocate each primer s to a primer injector Optional Ent
183. t in the text box that opens or increase or decrease the height by using the arrows next to the text box 2 To apply the height press Enter PyroMark Q48 Autoprep User Manual 10 2015 10 7 13 Setting up a Run 7 13 1 Workflow to set up a run 1 Create a new Run Setup by one of the following methods e Click E in the toolbar Select New Run from the File menu Press the R key while holding down the Ctrl key Right click a folder in the shortcut browser and select New Run from the context menu Enter a run name and press Enter To add a shortcut to a folder or drive click Add Folder Shortcut To base your run on a previous run rightclick the processed run file in the shortcut browser and select Copy and Rerun from the context menu Only the run setup not the run and analysis data will be copied 2 If the new run has not yet been saved click el to enter a run name and save the file in the desired folder 3 Select either manual or automatic sequence primer loading If manual primer loading is selected the user will be instructed to manually load the sequencing primer into the wells during template preparation For automatic primer loading the instrument will dispense the primer from one of the dedicated sequence primer injectors Allocate each primer used to a primer injector Multiple primers can be loaded into each injector however please validate multiplexing of primers on the instrument 4 Selec
184. t lock 37 Troubleshooting 138 User requirements 12 Version management View Pyrogram 58 Warnings 13 Waste Electrical and Electronic Equipment WEEE 164 Window menu 76 Workflow views 77 187 Trademarks QIAGEN Sample to Insight QlAamp QlAprep DNeasy EpiTect Pyrogram PyroMark Pyrosequencing RNeasy Q Solution QIAGEN Group Excel Microsoft Windows Microsoft Corporation Pentium Intel Corporation Adobe Reader Adobe Systems Inc Sepharose GE Healthcare Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law 2015 QIAGEN all rights reserved PyroMark Q48 Autoprep User Manual 10 2015 188 This page intentionally left blank PyroMark Q48 Autoprep User Manual 10 2015 189 This page intentionally left blank PyroMark Q48 Autoprep User Manual 10 2015 190 This page intentionally left blank PyroMark Q48 Autoprep User Manual 10 2015 191 www giagen com Technical Support www support qiagen com 1096211 10 2015 HB 1971 002
185. t will first initialize which could take a few seconds The run will then begin depending on the selected protocol For both protocols the lid will open during the heating step to avoid condensation build up PyroMark Q48 Autoprep User Manual 10 2015 55 WARNING A WARNING A WARNING A Moving parts w8 To avoid contact with moving parts during operation of the PyroMark Q48 Autoprep the instrument must be operated with the injector cover closed Do not remove the cover panels since there are no user serviceable parts inside If there is a problem with the PyroMark Q48 Autoprep contact QIAGEN Technical Services immediately Moving parts The chamber lid of the PyroMark Q48 Autoprep opens and closes automatically during operation to prevent moisture buildup inside the instrument Hot surface w10 The disc heater within the PyroMark Q48 Autoprep instrument can reach temperatures of up to 95 C 203 F Avoid touching it when it is hot A countdown timer to either manual sequence primer loading or to sequencing will appear If the user has selected automatic sequence primer addition no further action is required until the end of the run and the countdown time will display Sequence starts in x minutes If the user has selected manual sequence primer addition the software will display the countdown timer as Manual primer loading in x minutes ane SEN Sequence Run Manual primer load
186. tein or RNA working as a catalyst to enhance the speed of a biochemical reaction without altering it In Pyrosequencing technology a mixture of Klenow polymerase sulfurylase luciferase and apyrase is used in the sequencing reaction The theoretical representation of the expected Pyrosequencing peak pattern A stretch of identical bases in DNA In Pyrosequencing technology a stretch of more than two identical bases is regarded as a homopolymer 10 2015 152 Term InDels IUPAC PyroMark Q48 Magnetic Beads Out of phase Polymorphism Pyrogram Quality control window Reference peak RLU SEQ Sequence to analyze Sequencing primer Description Insertion and or deletions International Union of Pure and Applied Chemistry An organization providing recommendations on organic and biochemical nomenclature symbols terminology etc IUPAC codes A Adenine W TorA C Cytosine S CorG G Guanine B C T or G not A T Thymine D A T or G not C R Purine A or G Y Pyrimidine C or T M CorA K TorG H A T or C not G V A C or G not T N Any base A C G or T Magnetic streptavidin coated Sepharose beads used for preparation of biotinylated PCR products When one of the alleles is sequenced ahead of the other Genetic variations broadly encompassing any of the many types of variations in DNA sequences that are found within a given population The graph resulting from a sequ
187. test see section 8 5 Peak heights near the variable position deviate from expected levels Ensure there is no nucleotide degradation contamination Check sequence to analyze Check for unknown SNPs Peak heights deviate from expected levels Ensure there is no nucleotide degradation contamination Check sequence to analyze Check for unknown SNPs Either no drops or drops with unusual shape have been dispensed from the indicated nucleotide injector Ensure correct volume of nucleotide was loaded without any air bubbles If problem persists run injector diagnostics see section 8 5 Ensure injectors are cleaned daily 144 Message text No drops and drops with unusual shape have been dispensed from the indicated injector A number of no drops were detected out of the total number of dispensations of a reagent A number of suspect drops were detected out of the total number of dispensations of a reagent Assay setup messages in AQ mode Deselected by user Sequence uncertain due to lack of terminal sequence information Last variable position not analyzable due to lack of terminal sequence information Sequence not in phase at the end of dispensations Variable positions with common dispensations cannot be analyzed PyroMark Q48 Autoprep User Manual 10 2015 Comments and suggestions Either no drops or drops with unusual shape have been dispensed from the indicated nucleotide inj
188. the same number of blank dispensations as the number of variable positions Pay attention to tips and warnings indicated by the red icon and make suitable modifications when the icon appears PyroMark Q48 Autoprep User Manual 10 2015 158 Bisulfite treatment controls in CpG assays CpG assays should contain an internal control to assess successful bisulfite treatment preferably at the beginning of the sequence Cytosines not followed by a Guanine are usually not methylated and should be fully converted to Thymine 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 As and no Gs in these positions We recommend directly entering the sequence before bisulfite treatment because this enables the software to generate the sequence to analyze and automatically select one appropriate bisulfite treatment control A bisulfite treatment control can be added manually by adding a C before or after a T that was a C before bisulfite treatment in a forward assay or by adding a G before or after an A that was a G before bisulfite treatment in a reverse assay in the dispensation order Refer to section 7 SEQ assays Experience with sequencing large numbers of templates indicates that the dispensation order n CTGA gives on average the best sequencing quality Individual templates may however give better
189. ther than its own personnel except in cases where the PyroMark Q48 Autoprep User Manual 10 2015 169 Company has given its written consent to perform such repairs or modifications All materials replaced under this warranty will be warranted only for the duration of the original warranty period and in no case beyond the original expiration date of original warranty unless authorized in writing by an officer of the Company Read out devices interfacing devices and associated software will be warranted only for the period offered by the original manufacturer of these products Representations and warranties made by any person including representatives of QIAGEN which are inconsistent or in conflict with the conditions in this warranty shall not be binding upon the Company unless produced in writing and approved by an officer of QIAGEN PyroMark Q48 Autoprep User Manual 10 2015 170 15 Appendix E Safety Information French FR 15 1 Consignes de s curit Avant d utiliser le QlAgility il est imp ratif de lire attentivement ce manuel et de porter une attention particuli re aux consignes de s curit Afin de garantir un fonctionnement de l appareil en toute s curit et de maintenir l appareil en bon tat de marche il est imp ratif de suivre les instructions et consignes de s curit fournies dans le pr sent manuel d utilisation Les types d informations de s curit suivants sont fournis tout au long du manuel AVERTIS
190. tied into the absorber strip Empty 1 of 4 e Add 200pL of clean water to selected injectors as Wb A c G T 8 Empty 2 of 4 Manually pipette 200 pl of high purity water again and then select the Start button 9 Empty 3 of 4 Manually pipette 200 pl of high purity water again and then select the Start button 10 Empty 4 of 4 Manually pipette 200 pl of high purity water again and then select the Start button The injectors will be emptied for a last time into the absorber strip 11 At the end of the clean the software will instruct the user to open the cartridge lids to prevent condensation 12 Select the Home button to return to the Home screen 13 Remove and replace the absorber strip refer to section 6 5 3 PyroMark Q48 Autoprep User Manual 10 2015 137 9 Troubleshooting If you need to contact QIAGEN Technical Services about an error note the steps leading to the error and any information given in any dialog boxes This will helo QIAGEN in solving the problem When calling QIAGEN Technical Services about errors please have the following information ready Instrument serial number type and version Date of last maintenance performed Error code if applicable Time point when the error occurred for the first time Frequency of error occurrence i e intermittent or persistent error Photo of error if possible Copy support package Take the following action before contactin
191. to facilitate mixing of the reaction A higher speed is applied during a wash cycle to remove supernatant Disc heater ring Allows for the heating of wells to achieve sequence primer annealing and the immobilization of magnetic beads during a wash cycle Disc detector Optical sensor allows for the determination of the disc type used and if a disc is in place correctly Disc index pin Ensures correct positioning of the disc into the instrument 27 3 5 Analysis software The PyroMark Q48 Autoprep is shipped with PyroMark Q48 Autoprep Software The computer used for setup of runs and data analysis should have the following as minimum specifications e Microsoft Windows 7 English version Operating System Pentium IV processor 3 GHz or higher Minimum 1 GB RAM for 32 bit operating system minimum 2 GB RAM for 64 bit operating system Minimum 100 MB free hard drive capacity Recommended display resolution of 1024 x 768 pixels e USB port e CD ROM Pointer device mouse or similar To view reports generated in PDF format a PDF reader must be installed on the computer Adobe Reader can be downloaded at www adobe com PyroMark Q48 Autoprep User Manual 10 2015 28 A Installation Procedures 4 1 System delivery The following items are delivered PyroMark Q48 Autoprep instrument including USB stick Lock nut Thumb screws Injector cartridges Power cords User manual CD
192. to see if there is sufficient liquid in the cartridge reservoir Potential issue with drop sensor or injector Run injector test see section 8 5 Send support package to QIAGEN Technical Services see section 9 2 Ensure that excess solution in injector reservoirs is removed Potential issue with drop sensor or injector Run injector test see section 8 5 Send support package to QIAGEN Technical Services see section 9 2 Ensure that the well capacity volume 20 ul is not exceeded Input a sample name during PyroMark Q48 Autoprep Software run setup Ensure that the correct disc type is inserted into the chamber Ensure USB flash drive is inserted into the instrument or network connection is working Ensure that the run file is saved to USB flash drive or in a shared folder of the network Note For all other instrument error messages please contact QIAGEN Technical Services PyroMark Q48 Autoprep User Manual 10 2015 151 10 Glossary Term AQ Biotin Bisulfite Bisulfite treatment control CpG CpN Cyclic dispensation order Directed dispensation order Dispensation order Drop off Enzyme Histogram Homopolymer PyroMark Q48 Autoprep User Manual Description Analysis mode used for quantification of different alleles A molecule that can bind very strongly to streptavidin PCR primers can be biotinylated to enable the resulting PCR product to bind to streptavidin coated beads
193. ts 10 Reservoir neck Smaller diameter section of the reservoir that holds volumes less than 100 yl 11 Filter Prevents fine particles from blocking the injector Be careful not to damage the filter with a pipet tip PyroMark Q48 Autoprep User Manual 10 2015 25 There are three available cartridges described below Storage capacity per Minimum loading Typical injection Cartridge name injector volume volumes Reagent positions Nucleotide 3 8 ml 60 pl 100 10 nl A dATPaS C dCTP G dGTP T dTTP Primer 500 10 nl P1 Primer 1 P2 Primer 2 P3 Primer 3 P4 BB Primer 4 Binding Buffer Reagent 500 10 nl DS Denaturation Solution E Enzyme S Substrate AB Annealing Buffer Includes volume required to complete injector priming PyroMark Q48 Autoprep User Manual 10 2015 3 4 2 Chamber 1 Lock nut Screws down to hold disc firmly in place 2 Disc Well capacity of 20 pl typical volume of 10 ul The disc index hole outer is used to orient the disc into place while the disc detection hole inner is used as part of the disc detection system 3 Disc grip Help to hold insert and remove the disc A Absorber strip Absorbent strip to collect liquid waste spun out from the disc wells during a wash cycle and priming cleaning waste PyroMark Q48 Autoprep User Manual 10 2015 Absorber strip cavity Contains the absorber strip inside the chamber Rotor The rotor spins at 60 rpm with vibration
194. u 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 Drag copy of the note fresh sample PyroMark Q48 Autoprep User Manual 10 2015 106 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 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 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 Disc Setup Disc Setup The sample
195. uenced ahead of the other s and the two or more alleles are not in phase with the last dispensation Add more sequence information i e lengthen the sequence to analyze The genotype determination is uncertain because the pattern of peaks is too similar to another genotype Check sequence to analyze Check for unknown SNPs Check PCR performance i e ensure enough starting material with sufficient quality Peak heights deviate from expected levels Even at a variable position the software expects a certain pattern e g a certain sum of the peak heights Check sequence to analyze Check for unknown SNPs The software has detected that all four nucleotides have been dispensed but no signal was detected Clean the cartridge see section 8 3 3 in case there was a dispensation error If problem persists perform injector test see section 8 5 No peaks detected Check PCR yield Check sequencing primer Clean cartridges Check reagents Check correct usage of buffers The known bases entered by the user do not match the result Remove change entered known bases 148 Message text Low peak height from dispensation Very low peak height from dispensation Peak height deviates from the expected peak level around dispensation Peak height deviates from the expected peak levels overall Baseline drift Possible dispensation error at dispensation Wide peaks from dispensation 1 Wide peaks from dispensat
196. ustrial Hygienists Vereinigte Staaten von Amerika t COSHH Control of Substances Hazardous to Health Vereinigtes K nigreich PyroMark Q48 Autoprep User Manual 10 2015 181 16 6 Gefahren durch mechanische Teile WARNUNG A WARNUNG ACHTUNG A ACHTUNG A Sich bewegende Ger teteile w8 Um einen Kontakt mit sich bewegenden Teilen beim Betrieb des PyroMark Q48 Autoprep zu vermeiden darf das Ger t nur mit geschlossenem Injektordeckel betrieben werden Entfernen Sie nicht die Abdeckplatten im Ger teinneren befinden sich keine Bauteile die vom Anwender gewartet werden m ssen Setzen Sie sich umgehend mit dem Technischen Service von QIAGEN in Verbindung falls einmal ein Problem mit dem PyroMark Q48 Autoprep auftreten sollte Sich bewegende Ger teteile w9 Der Kammerdeckel des PyroMark Q48 Autoprep ffnet und schlie t sich im Betrieb automatisch um zu verhindern dass sich Feuchtigkeit im Inneren des Ger ts ansammelt Einsetzen der Disc C4 Die PyroMark Q48 Disc muss arretiert werden damit ihr Inhalt nicht in der Kammer des Ger ts versch ttet wird Stellen Sie sicher dass die Disc durch Festziehen der R ndelmutter arretiert ist Bewegung des Kammerdeckels C5 Die Deckelbewegung wird von einem Motor angetrieben der durch die Software gesteuert wird Zu wenig Platz hinter dem Instrument weniger als 20 cm f r die Bewegung des Kammerdeckels kann zur Besch digung des Deckels beim ffnen f hren 16
197. vement test 133 Pyrophosphate clean 135 Network connection 38 Overview tab 78 PA Extra protocol 44 Policy statement 11 Power requirements 30 Process a run 110 Product symbols 20 Protocols 44 PA Extra 44 Standard 44 Pyrogram 83 PyroMark Q48 Autoprep Analysis software 28 Chamber 17 24 27 150 162 Definitions 21 Injector cartridge 25 Installation 29 Instrument diagram 17 24 29 36 56 60 Principle 23 Protocols 44 Unpacking and installation 30 PyroMark Q48 Autoprep Software 70 Pyrosequencing principle 21 Pyrosequencing template 54 Quality assessments 66 114 Reports menu 76 Reset Hardware 37 Results Analysis resports 69 AQ analysis results 67 CpG analysis results 67 Quality assessments 66 SEQ analysis results 67 SNP analysis results 69 View the analysis results 65 Review the disc setup 109 Run Abort 59 Analysis 64 Completion 60 PyroMark Q48 Autoprep User Manual 10 2015 Monitoring 55 Start 55 Run log 138 Run setup 41 42 Run Setup view 78 Safety information 13 Safety information Biological 16 Chemicals 16 Consumables 18 Electrical 15 Maintenance safety 18 Mechanical hazards 17 Proper use 13 Waste disposal 18 Safety Information Heat hazards 17 Safety Information French FR 171 Safety Information German DE 178 Select wells 81 Sequencing primer Automatic loading 58 Manual loading 57 Set up a Run 102 Set Up an AQ SNP or CpG Assay 87 Set Up
198. verview tab the corresponding Pyrogram and theoretical histogram if an AQ SNP or CpG assay or compensated Pyrogram if an SEQ assay are displayed in the Pyrogram pane SEQ Fr Overnew Anabasa Sup cect swer SNP SEQ 1 4 T Replicate 1 Replienie 2 Replicate 1 Hepheate 3 US Ce ers SNP 2 SNP 2 SEQ 2 SEQ 2 Replicate 1 Replicate 2 Replicate i Replicate 2 es Ss 2Q 3 CoG 3 CpG 3 SNP 3 SNP 3 SEQ 3 SEQ 3 ai Y Rosie 2 alla Rolle 2 Reptile 1 sie Regia Race N md CACCCATTGCAGTTG IRRICAAATAGTAAACAAGGGI ACCACAGGAIGCI FATT CCTGICAICTIGGA ro TI seen lat shat if CTGACTGACTGACTGACTGACTGACTGAC TGAC TGAC TGAC TGACTGAC TGAC TGAC TGACTGAC TGAC TGAC TGACTGAC TGACTGACTGACTGACTGA 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 10 CTGACTGACTGACTGACTGACTGACTGACTGACTGACTGAC TGACTGACTGACTGACTGAC TGACTGACTGACTGACTGACTGACTGACTGACTGAC TGA 5 10 15 20 25 30 35 40 45 50 ss 60 65 76 75 80 85 90 95 100 When a base is selected in the base called sequence the corresponding peak is highlighted in both Pyrogram areas and vice versa Simultaneously view Pyrograms of different wells Pyrograms from 2 or more wells can be viewed simultaneously e g if the user wishes to compare Pyrograms by opening one or more additional Pyrograms of selected wells in the lower area of the Pyrogram pane 1 Highlight the wells see se
199. were selected by clicking while holding down Ctrl the information for the last well selected will be displayed 7 14 Processing a run When a run is set up and ready to run on the PyroMark Q48 Autoprep instrument perform the following steps 1 Insert USB stick into instrument Select the run file Ensure the absorber strip is inserted Load the injectors with required volumes of reagents then prime and test the injectors Load the beads and samples Start the run Optional manually load the sequence primer when instructed oY YS SY When the run has been completed and data transferred to the USB stick remove the USB stick 9 After the last run of the day clean the injectors with water and remove the absorber strip 7 15 Analyzing a run 7 15 1 Workflow to analyze a run 1 Move the processed run file from the USB stick to a computer running PyroMark Q48 Autoprep Software insert the USB stick into the computer s USB port and move the run file to the desired location using Windows Explorer 2 Open the run file in PyroMark Q48 Autoprep Software either by selecting Open in the File menu or double clicking the file 4 in the shortcut browser If several assay types are included select the analysis mode in the dialog box that opens The analysis modes displayed in the dialog box are limited to the assay types on the disc PyroMark Q48 Autoprep User Manual 10 2015 110 Note To update the contents of a folder in the
200. y power supplies and cords supplied with the system If the instrument becomes electrically unsafe prevent other personnel from operating it and contact QIAGEN Technical Services The instrument may be electrically unsafe when o The line power cord appears to be damaged o It has been stored for a prolonged period of time in conditions which are outside of the Storage Conditions outlined in Appendix B Technical Data o It has been subjected to severe transport stresses o Liquid has entered the instrument PyroMark Q48 Autoprep User Manual 10 2015 15 2 3 Biological safety When handling biological material use safe laboratory procedures as outlined in publications such as Biosafety in Microbiological and Biomedical Laboratories HHS www cdc gov od ohs biosfty biosfty htm WARNING Biological materials W6 A Handle biological material with the greatest of care and in accordance with the required safety regulations Always wear safety glasses 2 pairs of gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the instrument operators are not exposed to hazardous levels of infectious agents as defined in the applicable Safety Data Sheets SDSs or OSHA ACGIH or COSHH documents For more information visit www qiagen com safety Venting for fumes and disposal of wastes must be in accordance with all
201. y setup for the current analysis mode Use Analyze Selected Wells or edit the sequence to analyze in the Analysis Setup tab No well has a valid assay setup for the current analysis mode Use Analyze Selected Wells Comments and suggestions Locked assays on a processed run cannot be unlocked A new assay and run setup must be created Warning occurs in AQ SNP or CoG mode when Analyze all is clicked in an analysis mode that does not correspond to the assays in the disc Specific wells can be analyzed in the current analysis mode by selecting the wells and clicking Analyze Selected Wells If the sequence to analyze has not been entered in the assay setup e g in SEQ assays enter the sequence in the Analysis Setup tab Warning occurs in SEQ mode when Analyze alll is clicked and no wells contain SEQ assays Specific wells can be analyzed in the current analysis mode by selecting the wells and clicking Analyze Selected Wells 9 4 1 Messages of the PyroMark Q48 Autoprep Software The following is a list of use warning and error messages that may appear in the PyroMark Q48 Autoprep Software during assay setup assay run and data analysis Messages are organized by analysis mode though general messages can occur in any mode For troubleshooting specific to the PyroMark Q48 Autoprep instrument see section 9 5 Message text General setup messages The dispensation order is very long The InDel position contains

PyroMark® Q48 Autoprep User Manual

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PyroMark® Q48 Autoprep User Manual