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PyroMark™Q24 User Manual

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1. User Manual Biotage PyroMark Q24 User Manual 1 02 Table of Contents LEGAL iniciar ES ES ERE ED ERR ne ec se senere 1 Intended Use United States citada a e ED RA sad 1 Mended Purpose CUPOS nr a 1 Wanay and Lani acarrea aaa 1 Limitation of Warranty and Lliabllai ai 1 Trademsis ana Paren Susini 2 A e o E PO EE OO A 2 COD HON ura osado EEN age 2 SYSTEM DESCRIPTION siii aii er rese 3 PYrOMark O24 TERMER iria 4 Functional Sequence OF EVENTS ri id iaai 4 Process E aa E E EE 5 Di pen mo WI eaaa a a E E a EE 5 Da A o RC EE E E N ETA 6 Power and USA Connect aia 6 ik A O a a ere E E o ae ne reer 7 Spa AE 0 A 7 Seneral AMG and MOS ao 7 Ora SOG aN A A OA io E E A 8 PyroMark Q24 Vacuum Prep Worst oO 9 Reagents Consumables and Accessories inician aa 10 o AA e II 10 Instructions for Reagents and Consumables risas 11 Techhical Species NS nia tien iaareanerearensanenexniessepenianiee 12 PyroMark 024 TASTE ri id 12 PyroMark Q24 Vacuum Prep Worst Nr 12 PyroMark Q24 a a 0 o o E ee a ee eee eee 13 Chemical Recital trasandino gel 13 Performance and Ema don USE rieron 14 Use DOCU apar ne ada 15 Miele ee APA Enf oo A E A 15 Ser 1d re 1 ATT IO PERU 5 Moe O SS SEEGER REESE ENS 15 AUS CSS ITEE asain A o ANI E E E 15 S HS aaa 15 Instructions for Reagents and Consumables arrasar 15 PRINCIPLE OF PYROSEQUENCING TECHNOLOGY c scccesscceesnceesnseeeseeeesnsensaseneans 16 SYSTEM ADMINIS RIAU LON cuac ada 17 Instr
2. Q24 User Manual 1 02 Clean the screen by wiping with a clean non abrasive lint free cloth lightly moistened with water If this does not clean the screen properly then you may apply a small amount of 70 ethanol to the cloth Do not allow ethanol to soak into the gaps around the screen protection If necessary clean the exterior of the instrument using a clean lint free cloth lightly moistened with water If required a small amount of mild soap may also be used After cleaning wipe the surfaces dry with a clean dry non abrasive lint free cloth When you are done reconnect the instrument to the mains power Clean the Heating Block and the Light Guides In case of spillage on the heating block clean the heating block and the light guides underneath the block Required items Cotton swabs ethanol 70 and a clean non abrasive lint free cloth e g a camera lens cloth 1 Re et a When the instrument is not processing select Shutdown in the main menu using the and screen buttons and press Ok When the message It is now safe to turn off the instrument appears turn off O the instrument The power switch is located at the rear of the instrument Disconnect the instrument from the mains power There are two mains plugs Open the instrument lid Open the plate holding frame Clean each well hole light guide carefully using cotton swabs lightly moistened with 70 ethanol see image below Clean t
3. Supporting the resolution of the monitor 1280 x 1024 pixels Mouse or similar USB port and CD ROM The instrument covers and the vacuum prep workstation withstand solutions in the range pH 4 to pH 9 common detergents 0 5 M sodium hydroxide and 70 ethanol 13 PyroMark Q24 User Manual 1 02 Performance and Limitation of Use Pyromark Q24 System meets the requirements of Annex III of the European Directive for In Vitro Diagnostic Medical Devices 98 79 EC in certain European countries PyroMark Q24 System is intended to be used to analyze up to 24 DNA templates in terms of i the level of methylation at multiple individual CpG sites ii the proportion of one or more alleles at one or more variable positions or iii the genotype of one or more variable sites Correct performance can only be ensured if the supplied user documentation is followed in combination with correct reagents Performance characteristics for PyroMark Q24 System have been established in normal room temperature 20 C to 25 C for PyroMark Q24 Validation Oligo PyroMark Q24 Validation Oligo includes an A G variable position read as C T in the software The user is responsible for establishing PyroMark Q24 performance characteristics for other templates This is particularly important if detection or quantitation below the recommended lower limit of 5 is required For more information see www biotagebio com CpG Analysis and All
4. For more information see the online help Context sensitive help is accessed by pressing the F1 key when in a dialog or window in PyroMark Q24 Software A PDF version of the online help is available on the Pyrosequencing Technical Support website but can also be accessed by selecting All Programs Biotage PyroMark Q24 Documentation PyroMark Q24 Online Help pdf in the Windows Start menu Tip If you want to base your run on a previous one right click the processed run file in the shortcut browser and select Copy and Rerun from the context menu The process and analysis data will not be copied 20 PyroMark Q24 User Manual 1 02 Prepare Your Samples Samples to be analyzed using PyroMark Q24 Instrument should be prepared as described in this section using the equipment consumables reagents and solutions listed below Allow all required reagents and solutions to reach room temperature before starting All steps are performed at room temperature unless otherwise stated Required equipment and consumables PyroMark Q24 Vacuum Prep Workstation Mixer for immobilization to beads Heating block PyroMark 0Q24 Plate 24 well PCR plate or strips Strip caps O O O O O Tip A 96 well PCR plate can be cut into four 24 well plates Required reagents and solutions Streptavidin Sepharose High Performance 34 um 5 ml GE Healthcare Sequencing primer High purity water Milli Q 18 2 MQ x cm www millipore com or equ
5. PyroMark Q24 Instrument and PyroMark Q24 Vacuum Prep Workstation New products are continually being developed Please refer to Pyrosequencing website for further details and ordering information www biotagebio com PyroMark Q24 User Manual 1 02 PyroMark Q24 Instrument PyroMark Q24 Instrument is intended for DNA sequencing using Pyrosequencing technique PyroMark Q24 Instrument including instrument software for instrument control Functional Sequence of Events The functional sequence of events during the process of a run is as follows 1 2 5 aS EE 10 11 The plate PyroMark Q24 Plate containing the samples is placed on the heating block in the instrument and the reagent cartridge PyroMark Q24 Cartridge filled with the required reagents is placed in the dispensing unit The USB memory stick containing the run file created in PyroMark Q24 Software is inserted into the USB port at the front of the instrument and the run is started using the buttons underneath the screen The pressure in the dispensing unit the speed of the mixer and the temperatures of the heating block process chamber lid and the coolant liquid are adjusted to their preset levels may take several minutes Enzyme and substrate mixtures are predispensed into the rectangular well in the plate to ensure that the dispensation capillaries are flushed and filled with solution The enzyme mixture is dispensed into all u
6. Sample with the new dispensation order Replace dUTP with dTTP since the A nucleotide used in Pyrosequencing reactions binds less stringent to dUTP A change in the dispensation order might help Ensure that homopolymers are followed by an extra dispensation Analysis Software Related Errors Problem Action Red cross over wells in the Overview tab during analysis Exception dialog appears Could not create assay from specified Pyrosequencing Assay Design Software file Please contact 1 Point Support Please save the error report and send to 1 Point Support for information Click Continue to proceed with your analysis If the dialog remains click Quit and reopen your run Ensure a valid assay file type is being imported 40 Instrument Related Errors Error messages PyroMark Q24 User Manual 1 02 Action There are too many unsaved runs in the instrument Please go to folder Unsaved Runs and save them to USB memory The required value was not reached The run will be stopped Run name is invalid Could not copy file to USB memory Failed to connect to the mixer please restart the instrument Failed to connect to the hardware please restart the instrument Connection to the hardware is lost please restart the instrument No valid upgrade folders files found on USB memory Unit failed Automatic recovery of Run name failed Failed to clea
7. be present in for example buffers The graph resulting from a sequencing reaction performed using Pyrosequencing technology Each incorporated nucleotide is shown as a peak in Pyrogram Non variable peaks i e peaks that are not a part of an SNP or CpG site are referred to as reference peaks Reference peaks are used in the analysis both as references when calculating the single peak height and as internal controls when assessing the quality Relative Light Unit entity used in Pyrosequencing to define peak heights in Pyrogram Streptavidin coated beads can be used for preparation of biotinylated PCR products 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 See also the Sequence to Analyze section in the online help The sequencing primer is annealed to the template during the Sample preparation The 3 end of the sequencing primer serves as the starting point for the extension by the polymerase 49 Signal to noise ratio Single nucleotide polymorphism SNP Streptavidin Substrate Theoretical outcome Variable position PyroMark Q24 User Manual 1 02 Positive 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 w
8. bent needles discard the reagent cartridge according to federal state and local environmental regulations for disposal of laboratory waste Ensure to follow the instructions supplied with PyroMark Q24 Gold Reagents Ensure that the cartridge is inserted according to the instructions on page 25 Clean the heating block and light guides according to instructions on page 33 Clean the light guides and the heating block according to the instructions on page 33 Action Sample not prepared according to recommendations Low quality DNA in PCR Incorrect sequence to analyze Background of contaminating sequence from PCR Sequence signals from self annealed sequencing primer and or biotinylated PCR primer Unspecific annealing of the sequencing primer Sequence signal from self annealed template Ensure to follow the sample preparation instructions on page 21 properly Check the PCR samples using a gel technique to confirm that you have one strong specific band Check typing and your reference sequence Check the PCR samples using a gel technique to confirm that you only have one specific band Follow the recommendations in the Assay Design and Validation section on page 43 the first time you run an assay Follow the recommendations in the Assay Design and Validation section on page 43 the first time you run an assay Redesign sequencing primer Follow the recommendations in the Assay Design a
9. file to run 16 28 Select the run file using the and screen buttons To view the contents of a folder select the folder and press Select To go back to the previous view press Back When the run file is selected press Select to Start the run 25 PyroMark Q24 User Manual 1 02 Monitor the Run The instrument will start dispensing reagents when the pressure in the dispensing unit the speed of the mixer and the temperatures of the heating block process chamber lid and the coolant liquid have reached their preset levels Validation run 080214 16 42 Run name Selected well Current time Pyrogram Warning messages 1 2 3 4 Instrument status 5 6 7 e E Estimated remaining time hh mm Instrument Status The instrument status is displayed in the top right corner Environment Waiting for the pressure in the dispensing unit the speed of the mixer and the temperatures of the heating block process chamber lid and the coolant liquid to reach their preset levels may take several minutes Priming Priming the needles of the reagent cartridge to remove any air bubbles Running The enzyme mix and substrate mix are dispensed to all used wells and then the nucleotides are dispensed to the wells according to their dispensation order which is defined in the assay file Stopped The run has been aborted Saving The run data is transferred to the USB memory stick Leave the memory stick mounted
10. filter and a beaker Two waste filters are supplied with the vacuum prep workstation Filters can be ordered at www millipore com Millipore Millex FGso Filter Unit Catalogue Number SLFGO5010 1 Ensure that no vacuum is applied to the vacuum prep tool i e the vacuum switch is closed OFF and the vacuum pump is turned off O Disconnect the vacuum pump from the mains power 3 Pull off the tubing from the filter fittings and insert the tubing into an empty beaker to empty it of any liquid waste see image 4 Discard the filter Push the tubing onto the fittings of the new filter dl 6 If necessary empty the waste container Note that the cap can be removed without disconnecting the tubing 7 Reconnect the vacuum pump to the mains power 36 PyroMark Q24 User Manual 1 02 Troubleshooting WARNING Before performing any procedures in this chapter please read and A observe the safety requirements in the PyroMark Q24 Installation and Safety document Failure to follow those requirements may result in personal injury and or equipment damage WARNING If the system has been damaged or does not function properly shut down the system and contact 1 Point Support immediately www biotagebio com Before contacting 1 Point Support please take the following actions 1 Check the run log to assess if the system was working properly during the run 2 Consult the troubleshooting guide below 3 Verify proper insta
11. instructions supplied with the used reagents and cartridge 4 Set up the plate a Add an assay to each used well e g drag an assay in the shortcut browser to a well or a selection of wells Plate Setup CoG Assay CoG Assay 2 AQ Assay 1 eal A well is colored according to the assay loaded to the well b To enter a sample ID or note select the cell and enter the text A selected cell is highlighted with a blue background color Click tal in the toolbar 6 Print a list of required volumes of reagents and the plate setup select Pre Run Information from the Tools menu and when the report appears click amp 7 Close the run file and copy it to one of the USB memory sticks supplied with the system 1 To add a shortcut to a folder in the shortcut browser click Add Folder Shortcut 2 The more frequently a file is saved the more information is recovered if there is a power failure or similar problem while the file is open To secure the data a backup of PyroMark Q24 files should be performed frequently Optional If desired enter the Reagent ID i e the lot number for PyroMark Q24 Gold Reagents a Plate ID a Barcode number for the plate and a Run Note More Information There are several ways of setting up a plate It is for example possible to import and paste a sample layout defined in a text file and drag copy and increment a sample ID if the last part of the entered sample ID is a number
12. page 31 Check the Performance Verify that PyroMark Q24 System functions according to specifications by measuring imprecision bias and linearity using PyroMark Q24 Validation Oligo Perform the validation according to the instructions supplied with the product To order PyroMark Q24 Validation Oligo please contact your local sales representative www biotagebio com Maintenance of PyroMark Q24 Instrument Clean the Instrument If the instrument has been contaminated by dust and spillage clean it according to the instructions below Notes e Avoid harsh cleaners and chemicals and getting moisture inside the instrument e The cleaning liquid must be applied to the cloth only e Do not use any organic solvent or detergent other than ethanol when cleaning the screen Required items Ethanol 70 water mild soap if necessary and clean non abrasive lint free cloths 1 When the instrument is not processing select Shutdown in the main menu using the and screen buttons and press Ok 2 When the message It is now safe to turn off the instrument appears turn off O the instrument The power switch is located at the rear of the instrument Disconnect the instrument from the mains power There are two mains plugs 4 Open the instrument lid Clean the area around the dispensing unit the process chamber and the heating block using a clean lint free cloth lightly moistened with 70 ethanol 32 PyroMark
13. replaced after preparation of approximately 100 plates Note Use gloves powder free to avoid contaminating the filter probes Required items Powder free gloves 2 mm Allen key supplied with the system high purity water Milli Q 18 2 MQ x cm www millipore com or equivalent and new filter probes supplied by Pyrosequencing 1 Ensure that no vacuum is applied to the vacuum prep tool i e the vacuum switch is closed OFF and the vacuum pump is turned off O Disconnect the vacuum pump from the mains power Remove the tool from the tubing Loosen the four screws using the 2 mm Allen key supplied with the system Pull out the old filter probes Gently insert new filter probes without pressing on the filter tips When you are done fasten the four screws and reconnect the vacuum pump to the mains power Replace the Rubber Seal If the filter probes are loose and or fall out either the four screws needs to be tighten or the rubber seal needs to be replaced Required items Powder free gloves 2 mm Allen key supplied with the system and a new rubber seal supplied by Pyrosequencing 1 Ensure that no vacuum is applied to the vacuum prep tool i e the vacuum switch is closed OFF and the vacuum pump is turned off O 2 Disconnect the vacuum pump from the mains power 3 Remove the tool from the tubing 4 Remove the four screws using the 2 mm Allen key supplied with the system 5 Gently remove the filter pro
14. the Enter key Type or paste the Sequence to Analyze Click the Generate Dispensation Order button Click Al in the toolbar wes A Before running your samples validate your assay using reference samples see the Assay Design and Validation section on page 43 1 To add a shortcut to a folder in the shortcut browser click Add Folder Shortcut 2 The more frequently a file is saved the more information is recovered if there is a power failure or similar problem while the file is open To secure the data a backup of PyroMark Q24 files should be performed frequently Optional If desired enter a Note about the assay and set up the variable positions at the Variable Positions tab If creating a CpG assay we recommend that you add bisulfite treatment controls by left clicking a bold orange T or A in the histogram Preferable in the beginning of the sequence DE Add Bisulfite Treatment Control Before Dispensation Add Bisulfite Treatment Control After Dispensation Note In the sequence before bisulfite treatment can be entered in the assay check whether the suggested bisulfite controls are Cs converted to Ts read as Gs and As in a reverse assay or not 19 PyroMark Q24 User Manual 1 02 Set Up a Run 1 In the shortcut browser right click the folder you want to place the run file in and select New Run from the context menu 2 Enter the file name and press the Enter key 3 Select Instrument Method see the
15. the defined range 0 HE E Dark green above the defined range Analysis Reports To generate a report select the desired report from the Reports menu For more information about the reports see the online help Reports Window Help Reports Window Help 40 Analysis Statistics CpG Analysis Statistics 40 Analysis Results CpG Analysis Results AQ Program Report AG Full Report Col Pyragram Report CpG Full Report SMP Analysis Results SMP Pyerogram Report SMP Full Report SMP Overview Report More Information In order to view the Full Pyrogram and SNP Overview reports you must have Adobe Acrobat Reader installed on your computer Adobe Acrobat Reader is available on the PyroMark Q24 Software CD but can also be downloaded at www adobe com Context sensitive help is accessed by pressing the F1 key when in a dialog or window in PyroMark Q24 Software A PDF version of the online help is available on the Pyrosequencing Technical Support website but can also be accessed by selecting All Programs Biotage PyroMark Q24 Documentation PyroMark Q24 Online Help pdf in the Windows Start menu 30 PyroMark Q24 User Manual 1 02 When You Have Finished the Day s Work Shut Down the Instrument 1 When the instrument is not processing select Shutdown in the main menu using the and screen buttons and press Ok 2 When the message It is now safe to turn off the instrument appears turn off O the ins
16. the system A printout of the document is supplied with the system A PDF version of the document is available on the PyroMark Q24 Software CD and on the Pyrosequencing Technical Support website but can also be accessed by selecting All Programs Biotage PyroMark Q24 Documentation PyroMark Q24 Installation and Safety pdf in the Windows Start menu User Manual PyroMark Q24 User Manual provides a description of the system technical specifications instructions for operation and maintenance and guides for assay design and validation and troubleshooting The manual is available as a PDF document on the PyroMark Q24 Software CD and on the Pyrosequencing Technical Support website but can also be accessed by selecting All Programs Biotage PyroMark Q24 Documentation PyroMark Q24 User Manual pdf in the Windows Start menu Quick Guides The three quick guides contain short descriptions on how to operate PyroMark Q24 Instrument PyroMark Q24 Software and PyroMark Q24 Vacuum Prep Workstation Printouts of the guides are supplied with the system one printout per guide A PDF version of the guides is available on the PyroMark Q24 Software CD and on the Pyrosequencing Technical Support website but can also be accessed by selecting All Programs Biotage PyroMark Q24 Documentation PyroMark Q24 Quick Guide pdf in the Windows Start menu Online Help The online help included with PyroMark Q24 Software contains use
17. 4 User Manual 1 02 Clean and Test the Reagent Cartridge If the reagent cartridge is to be reused clean it directly after use and ensure that it can be used for analysis It is recommended that the reagent cartridge is used no more than 30 times Notes Handle the needles of the reagent cartridge with care Small particles and fibers may obstruct the needles Observe all federal state and local environmental regulations for disposal of laboratory waste Required items Powder free gloves high purity water Milli Q 18 2 MQ x cm www millipore com or equivalent a beaker if necessary and a lint free tissue To clean and check that the reagent cartridge can be used for analysis 1 2 3 4 Discard remaining solutions Rinse the reagent cartridge four times with high purity water Spray the outside of the needles using high purity water Rinse the needles Fill the compartments completely with high purity water b Hold the reagent cartridge over a sink while pressing firmly on top of each compartment with a finger and e Check that the needle is clear A jet of water should squirt out from the tip of the needle If a needle is blocked e g if the reagent cartridge has been left overnight without cleaning fill the compartments with high purity water and immerse the cartridge in a beaker with high purity water that covers the needles Leave the reagent cartridge in the beaker for one hour rinse it and repeat st
18. DNA Template Preparation Using a Vacuum Filtration Sample Transfer Device BioTechniques 34 862 866 868 2003 PyroMark Q24 User Manual 1 02 Reagents Consumables and Accessories PyroMark Q24 Instrument and Vacuum Prep Workstation should be used with the following Pyrosequencing reagents consumables and accessories e PyroMark 0Q24 Plate a 24 well microtiter plate designed for use with PyroMark Q24 Instrument e PyroMark Q24 Plate Holder e PyroMark Q24 Cartridge e PyroMark Q24 Troughs e PyroMark Q24 Gold Reagents e Vacuum Prep Tool Filter Probes e PyroMark Control Oligo e PyroMark Q24 Validation Oligo e Annealing Buffer e Binding Buffer e Denaturation Solution e Washing Buffer 10x Storage The consumables and accessories should be stored as follows Item Storage Comment temperature All unopened reagent vials 4 C PyroMark Q24 Gold Reagents Reconstituted Enzyme and Substrate 4 C Use the same day mixtures PyroMark Q24 Gold Can be stored in 20 C for Reagents longer storage Opened dNTP vials 4 C Use within 1 month PyroMark Q24 Gold Reagents Do not freeze Sample preparation solutions 4 C Annealing Binding and Washing Buffer and Denaturation Solution PyroMark Control Oligo and 20 C PyroMark Q24 Validation Oligo PyroMark Q24 Plate PyroMark Q24 RT Reagent cartridges can be Plate Holder reused if cleaned and kept PyroMark Q24 Cartridge p
19. EFUINION sica aaa 25 PyroMark Q24 Software sesers 19 Pr ES te RED Er RNERETe 25 Status Instrument saciacossnssicaosiciorentare 26 A RENSEDE ERE SS LEDERNE KENDES RER 10 12 System AGIMIMISUALIOM arranco cds 17 SSC Ollariic A 3 T Technical Specifications seceeeeee 12 Test Filter POBRES 455 annen 34 Reagent Cartridge ccccceeeeeeseess 28 Trademarks and Patents c eeeseeeees 2 Troubleshooting sc5 teccesitteaceuscepiacionnees 37 ANAIS ari 38 Analysis Software csi illa 40 MSM oscense antes 41 Pei LOG E E E ia 37 Vacuum Prep Workstati0nN 42 Verify Installation and Operation 42 U A Geaiaweatcaansant nd 27 Upgrade Instrument Software 18 User Documentation mosurariianiosirarian 15 Installation and Safety 1 0 15 Online Helado 15 QUICK GUIS nasa rca 15 Reagents and Consumables 15 User Manual rin arkade 15 Using The SS iinciinncariinna cs 19 V Vacuum Prep Workstation b o risana 9 o nirst een ket 34 Prepare Samples sisas 21 Specifications sad 12 Troubleshooting irske 42 Validation of ASSAY isiiisttescreniianacsesees 43 Verify Installation and Operation 42 View Acknowledgment cccceceeceees 18 Analysis Reports siii rar 30 Analysis Results asii 29 Instrument Software and Hardware WEPSION S cti 18 W Warranty and Liability Waste PyroMark Q24 User Manual 1 02 Empty Container and Troughs 31 Replace Waste Filter cccee
20. 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 Q24 User Manual 1 02 Protect Your Files e If you want to protect a file from being edited by another user save the file in a folder that can only be accessed by you contact your system administrator for more information e If you want to protect a file from being accidentally overwritten by you or another user set the read only attribute for the file using Windows Explorer 1 Close the file in PyroMark Q24 Software 2 Open Windows Explorer and locate the file Tip This can be done by right clicking the folder containing the file in the shortcut browser 3 In Windows Explorer right click the file and select Properties from the context menu 4 When the Properties dialog appears turn on HI the Read only attribute and click OK e A backup should be performed frequently see the Backup PyroMark Q24 Files section on page 18 The more frequently a file is saved the more information is recovered if there is a power failure or similar problem while the file is open To save a file click al in the toolbar If the file has never been saved select location and name of the file in the dialog that appears More Information PyroMark Q24 Software is described in detail in the online help Contex
21. HANTABILITY FITNESS FOR PURPOSE OR NON INFRINGEMENT EXCEPT FOR THE IMPLIED WARRANTY OF TITLE ARE HEREBY EXPRESSLY EXCLUDED PYROSEQUENCING AB SHALL IN NO EVENT BE LIABLE FOR ANY INDIRECT OR CONSEQUENTIAL OR PUNITIVE DAMAGES OF ANY KIND FROM ANY CAUSE ARISING OUT OF THE SALE USE OR INABILITY TO USE ANY SOFTWARE INCLUDING WITHOUT LIMITATION LOSS OF PROFITS GOODWILL OR BUSINESS INTERRUPTION EVEN IF PYROSEQUENCING AB HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES PyroMark Q24 User Manual 1 02 Trademarks and Patents Biotage 1 Point Support Pyrosequencing Pyrogram PyroMark and E are trademarks owned by Biotage AB Pyrosequencing technology is covered by patents including patents US 5405746 US 6210891 US 6258568 US 6828100 US 7078168 EP 0932700 EP 0946752 EP 1495137 JP 3510272 and JP 3533223 and patent applications owned by Biotage AB In view of the risk of trademark degeneration authors intending to use the trademark designations are respectfully requested to acknowledge the trademark status of the products at least once in each article Other Trademarks Other product and company names mentioned herein may be trademarks or registered trademarks and or service marks of their respective owners and are used only for explanation and to the owners benefit without intent to infringe Copyright Pyrosequencing AB reserves the right to make changes to the information contained herein without prior notice No p
22. 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 PCR Optimization The quality of DNA for PCR optimization should be as high as possible Bisulfite treated DNA is usually heavily degraded after the conversion Ensure that bisulfite treatment gives reproducible amounts of converted DNA that are sufficient for robust amplification Several commercial kits for conversion are available for example from ZymoResearch www zymoresearch com and Qiagen www giagen com Evaluate conditions for the PCR carefully The optimal ranges for annealing temperature and MgCl concentration may be very narrow especially for a CpG assay We recommend testing at least two MgCl concentrations e g 1 5 mM and 3 mM The annealing temperature gradient should span 15 C for both magnesium concentrations at intervals of approximately 3 degrees Celsius For all PCR optimization tests analyze 1 10 of the PCR on an agarose gel and aim for one strong specific band with minimal excess of primers Equal Amplification of Both Alleles Reliable results in quantification assays depend on equal amplification of both alleles and this has to be carefully tested To ensure equal amplification in a CpG assay non methylated DNA can be mixed with increasing proportions of completely methylated DNA These should include a minimum of five mixtures eq
23. Set Up a Run section on page 20 Note that the total volume per well should be 80 ul e Seal the PCR plate or the strips using strip caps Ensure that no leakage is possible between the wells f Agitate the PCR plate or the strips constantly for 5 10 minutes using a mixer 1400 rpm Note Sepharose beads sediment quickly and capturing of beads must take place within one minute of the agitation being terminated Tip During immobilization prepare the vacuum prep workstation for the sample preparation steps 3a through 3h on page 22 21 PyroMark Q24 User Manual 1 02 3 Separate the DNA Strands and Release the Samples in PyroMark Q24 Plate workstation contains sodium hydroxide that is irritating to eyes and skin Use adequate ventilation Use impermeable gloves chemical safety goggles and protective clothing MSDS can be downloaded at www biotagebio com Xx WARNING Irritant The Denaturation Solution which is used with the a Ensure that the vacuum prep workstation has been assembled correctly and securely see the PyroMark Q24 Installation and Safety document The mains plug shall be easily accessible in case of the need of quickly disconnecting the vacuum pump from the mains power Notes e To ensure that the filter probes are working properly perform the function test on page 34 All probes should be replaced after preparation of approximately 100 plates see the instructions on page 34 e If the wa
24. a mild detergent Do not touch the tips of the filter probes 4 Wipe the worktable and the tool except for the filter probes dry using a clean lint free cloth 5 When you are done reconnect the vacuum pump to the mains power Replace and Test the Filter Probes Perform Function Test Filter Probes To check that the filter probes are working properly perform the function test below Required items A PCR plate a trough and high purity water Milli Q 18 2 MQ x cm www millipore com or equivalent 1 Add 100 ul of high purity water to each well of a PCR plate 2 Fill a trough with 70 ml of high purity water 3 Start the vacuum pump I 4 Apply vacuum to the vacuum prep tool by opening the vacuum switch 5 Lower the filter probes into the trough with high purity water and hold them there for approximately 20 seconds Ensure that the water is transported to the waste container i e that vacuum is applied If not check the connections 6 Lower the filter probes into the PCR plate and check that the water is aspirated evenly in all wells and that all wells are empty after a maximum of 10 seconds 7 If the wells are not empty after 10 seconds repeat the procedure from step 1 If the test fails replace the failing filter probes 34 PyroMark Q24 User Manual 1 02 Replace Filter Probes The filter probes are individually replaceable To ensure proper flow rate trough the filter probes all probes should be
25. ac ii 31 A AA E OI une RE SERENE ES NE TE TTT 31 MEN ENN EET Eee eee eler 32 check che o A eo or SE EE Tn iene senesced mendda adit 32 Maintenance of PyroMark Q24 Instrument taa 32 ceon Cie ISTMO Escalada cos 32 Clean the Heating Block and the Light Guides assi 33 Maintenance of PyroMark Q24 Vacuum Prep WorkstatiOn cccccceeeee cece eeeeeeneeeeees 34 Clean Cie Won tac citando bil dai id ESTERE 34 Replace and Pest the Filter ProDES ems ticas 34 PyroMark Q24 User Manual 1 02 Replace THE RUDD Seal areas needed 35 Pee Pace ie A o E st ececese iecesemianereceuteds 36 Replace the Waste Plain pia 36 TROUBLESHOOTING css siccstscasinetancedensdintansannscicndtencssesennisiesacaiiaes inceceeedsbenteneaeisianes 37 Ce ere Te RUN LOD sete cetera ne 37 WORMS Rooting CUE siere ina dodo sialsco asia apa dos becaloa 37 Analysis Relatcd EIO iracion 8 NM 38 Analysis Software Related ENF ONS id 40 Rigs eS so A A A 41 Vacuum Prep Workstation Related Errors sssssssssssssssnrsnnnssnensnenrnnsnenrensrenrennn 42 Verify Proper Installation and Operation na 42 Ge aie oem ese ge 6 8 010 asocio 42 ASSAY DESIGN AND VALIDATION ccccccssccceescceeseeeeseseeseneeesneeesaseesaneeesaeensaneneanes 43 A A ee eer 43 E A cage A E EEE POA A E E A AT E IT EAT E PT T 43 essa ela ANAYSSI A or o A o aera eee ae ery 43 DetalSd Desc r are a a iranere 44 aaa De ari rl ea iii 44 Pe Ao o o EE coesetecrcceatseceestiteeaenaaecen 45 PER ODUMI
26. and by the DNA polymerase Each incorporation event is accompanied by release of pyrophosphate PPi in a quantity equimolar to the amount of incorporated nucleotides Step 3 ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5 phosphosulfate This ATP drives the luciferase mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP The light produced in the luciferase catalyzed reaction is detected by charge coupled devices CCDs and seen as a peak in Pyrogram Each light signal is proportional to the number of nucleotides incorporated Step 4 Apyrase a nucleotide degrading enzyme continuously degrades unincorporated nucleotides and excess ATP When degradation is complete another nucleotide is added Step 5 Addition of nucleotides is performed one at a time It Should be noted that deoxyadenosine alfa thio triphosphate dATPaS is used as a substitute for the natural deoxyadenosine triphosphate dATP since it is efficiently used by the DNA polymerase but not recognized by the luciferase As the process continues the complementary DNA Strand is built up and the nucleotide sequence is determined from the signal peak in Pyrogram Polymerase D NA PPi n 1 DNA dNTP Sulfurylase light p APS PPi ATP luciferin oxyluciferin Luciferase AT z nucleotide incorporation generate
27. art of this manual may be reproduced or transmitted in any form or by any means electronic or mechanical for any purpose without the expressed written permission of Pyrosequencing AB Copyright 2008 Pyrosequencing AB All rights reserved Pyrosequencing AB Kungsgatan 76 SE 753 18 Uppsala SWEDEN Phone 46 18 56 59 00 Fax 46 18 59 19 22 E mail pyrosequencing biotage com Website www biotagebio com Pyrosequencing AB is a wholly owned subsidiary of Biotage AB Biotage AB is listed on the OMX Nordic Exchange ticker BIOT www biotage com PyroMark Q24 User Manual 1 02 System Description PyroMark Q24 System is a system for detecting changes in specified variable positions in human DNA that may have clinical relevance using Pyrosequencing technique The instrument can analyze 24 samples simultaneously and an easy to use protocol is used to prepare the samples PyroMark Q24 System consists of e PyroMark Q24 Instrument including instrument software for instrument control PyroMark Q24 Software for run setup and analysis e PyroMark Q24 Vacuum Prep Workstation for preparation of samples e PyroMark Q 24 Plate which is a special microtiter plate designed for use with PyroMark Q24 Instrument e PyroMark Q24 Troughs e PyroMark Q24 Cartridge e PyroMark Q24 Gold Reagents e Sample preparation solutions Annealing Buffer Binding Buffer Denaturation Solution and Washing Buffer 10x ks 7
28. bes Avoid contaminating the filter probes 6 Remove the metal plate and replace the rubber seal Reassemble the tool and reconnect the vacuum pump to the mains power 8 Check that the filter probes are working properly by performing the function test on page 34 35 PyroMark Q24 User Manual 1 02 Replace the Tubing If the tubing is broken or distorted replace it Note Observe all federal state and local environmental regulations for disposal of laboratory waste Required items New tubing supplied by Pyrosequencing and a beaker 1 Ensure that no vacuum is applied to the vacuum prep tool i e the vacuum switch is closed OFF and the vacuum pump is turned off O 2 Disconnect the vacuum pump from the mains power Pull off one end of the broken tubing and empty it of any liquid waste using an empty beaker see image 4 Pull off the other end of the tubing and discard the tubing and any liquid waste 5 Cut the new vacuum tubing into three pieces and assemble it Ensure that the tubing is connected to the pump s Vacuum fitting 6 When you are done reconnect the vacuum pump to the mains power Vacuum fitting Replace the Waste Filter If the waste filter is wet due to e g the waste container is full no vacuum is attained and the filter has to be replaced Note Observe all federal state and local environmental regulations for disposal of laboratory waste Required items New waste
29. bisulfite treatment The preferable controls are those dispensations that are located in the beginning of the sequence and or represent single base incorporations Note In the sequence before bisulfite treatment check whether the suggested bisulfite controls are Cs converted to Ts read as Gs and As in a reverse assay or not Validation of a New Assay Controls Use a reference DNA sample when testing a new assay and ensure that appropriate analysis parameters in PyroMark Q24 Software are used Interactions between primers or loops formed on single stranded DNA can serve as priming sites for base incorporation by DNA polymerase A few important controls should be included when an assay is analyzed for the first time 46 PyroMark Q24 User Manual 1 02 e A PCR without template DNA shows if the primers interact to give a background signal in Pyrosequencing reactions e A PCR with template DNA but with no sequencing primer added at sample preparation shows if the template can loop back on itself and give a background Signal in Pyrosequencing reactions e A sequencing primer without any PCR product will show if the sequencing primer can form duplexes or hairpins and give background signal in Pyrosequencing reactions Pyrogram traces from these controls should not show any peak after any nucleotide addition Quality Assessment The user is warned if something in the assay might reduce the quality of the result given by the analy
30. d Safety document see page 15 Backup PyroMark Q24 Files The data generated by PyroMark Q24 Software is stored on your computer in files with the following suffixes e pyrorun run files displayed with the and Y icons e pyrosetup assay files displayed with the icon To secure the data a backup should be performed frequently This can be done by copying PyroMark 0Q24 files pyrorun and pyrosetup to another location This alternative location is preferably on another physical drive or on a permanent medium For more information about backup contact your system administrator 18 PyroMark Q24 User Manual 1 02 Using the System WARNING Before performing any procedures in this chapter please read and A observe the safety requirements in the PyroMark Q24 Installation and Safety document Failure to follow those requirements may result in personal injury and or equipment damage Set Up a Run Note Detailed instructions are available in the online help press the Fi key when in a dialog or window in the software Start PyroMark Q24 Software In the Windows Start menu select All Programs Biotage PyroMark Q 24 Context sensitive help can be accessed at any time by pressing the F1 key Set Up an Assay 1 In the shortcut browser right click the folder you want to place the assay file in and select New Assay and then AQ Assay or CpG Assay from the context 1 menu Enter the file name and press
31. e 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 the same number of blank dispensations as the number of variable positions Pay attention to tips and warnings indicated by the red icon Y Variable Positions If possible variable positions should not include homopolymers a stretch of two or more nucleotides of the same kind If the variable position is preceded by a homopolymeric stretch place the sequencing primer adjacent to the position to analyze If the variable position is followed by a homopolymeric stretch change the orientation of the assay C G and T are the preferred bases in variable positions A peaks are slightly higher than the other three base peaks due to the chemistry of the modified A nucleotide dATPaS In exceptional cases the heights of the A peaks may vary between assays This can be compensated by changing the A peak reduction factor at the Analysis Parameters tab CpG Assays When creating a CpG assay the software indicates possible dispensations that can be controls for completion of
32. e tool and move to beyond 90 vertical for a few seconds see image q Close the vacuum switch on the tool OFF r Release the beads in the plate filled with 0 3 UM sequencing primer in 25 ul Annealing Buffer in each well by shaking the tool from side to side while allowing the filter probes to rest on the bottom of the wells s Move the tool to the trough containing high purity water trough 4 and agitate the tool for 10 seconds t Wash the filter probes by lowering the probes into high purity water trough 5 and applying vacuum to the tool Let approximately 70 ml of water flush through the filter probes i e empty the trough u Move the tool to beyond 90 vertical for a few seconds v Close the vacuum switch on the tool OFF and place the tool in the Parking position w If preparing more than one plate refill the troughs to the levels stated in step b and repeat the procedure from step h x Turn off the vacuum pump O y At the end of a working day liquid waste and any solutions left in the troughs Should be discarded and the vacuum prep workstation checked for dust and spillage according to the instructions on page 31 4 Anneal the Samples to a Sequencing Primer WARNING Hot surface Do not touch the hot surfaces on the plate holder and the heating block a Heat the plate PyroMark Q24 Plate with the samples at 80 C for 2 minutes using the plate holder supplied with the vacuum prep workstatio
33. ele Quantification PyroMark Q24 System has been shown to give the following performance when tested using i PyroMark Q24 Validation Oligo in accordance with the Instructions for Use supplied with the product and ii PyroMark Q24 Validation Oligo giving a signal of 30 120 Relative Light Units for single peaks e Quantitation limits of 5 lower limit and 95 upper limit e The method has been demonstrated to be linear from 5 C to 95 C with an allowable non linearity of 3 percentage units Clinical and Laboratory Standards Institute Guideline EP6 A e Repeatability measured as standard deviation for 8 replicates better than 3 percentage units in the range 5 C to 95 C e Bias less than 5 percentage units for a mean of 8 replicates in the range 5 C to 95 C Quantitation limit This is the lowest or highest value of C that can be measured quantitatively with a specified measurement uncertainty Repeatability Precision of successive measurements of C carried out under essentially unchanged conditions in this case replicates Bias Difference between the measured value of C and the true value 14 PyroMark Q24 User Manual 1 02 User Documentation User documentation in other languages can be downloaded at the Pyrosequencing Technical Support website Installation and Safety PyroMark Q24 Installation and Safety contains safety requirements and instructions for installing moving and re installing
34. ell at the Overview tab and the following information is shown e Well information Assay name sample ID note and any analysis warnings are listed in the Well Information area e Pyrogram The analysis results the allele frequencies or the methylation percentages are displayed above the variable positions in Pyrogram for example 36 A5 YGGATAGYGATTTTTAAYGYGTAAGYGTATA 400 A asc A SES ESS O RR cso ooa aooaa 300 A A ir Er E A A E o a ao 200 A A o BE o A ere GACTATGTCGATTGA TCA 5 10 15 20 25 LU MU Variable positions are highlighted with a blue gray background color and bisulfite treatment controls with a light yellow background color 29 PyroMark Q24 User Manual 1 02 Quality Assessments A41489FS The quality assessments for the variable positions are displayed by Pio e Quality bars in the plate overview at the Overview tab Ci 44 e The background color of the analysis results in Pyrogram T ses 17 L Off white not analyzed L Blue passed 1 Yellow check IM Red failed Either analysis is not supported by the software e g SNP when in the CpG mode or the variable position has been deselected by the user Methylation Levels When in the CpG mode a methylation bar at the Overview tab shows the methylation level for each CpG site in the well None Assay Sample ID Note Quality L Light green below the defined range Mean IN Green within
35. ep 4 e Check that the jet of water is straight in the direction of the needle not angled If angled refill the compartment with water and try again If the test fails discard the reagent cartridge When all needles have been rinsed and tested with satisfying results discard the water and leave the reagent cartridge to dry on the side on a lint free tissue When the reagent cartridge is dry store the cartridge in a dust free place between uses 28 PyroMark Q24 User Manual 1 02 Analyze Your Run Note Detailed instructions are available in the online help press the F1 key when in a dialog or window in the software Analyze the Run 1 Move the processed run file from the USB memory stick to a computer running PyroMark Q24 Software 2 Open the run file by double clicking the run file Y in the shortcut browser At the Overviev tab either analyze all wells or a selection of wells with a valid analysis setup for the selected analysis mode Analyze All Wells Analyze Selected Wells 1 To add a shortcut to a folder in the shortcut browser click Add Folder Shortcut Analysis Modes AQ assays are analyzed in the AQ mode and CpG assays are analyzed in the CpG mode SNP genotyping can be accessed trough the AQ mode To toggle between the modes select AQ or CpG in the toolbar Note How the analysis is performed can be modified at the Analysis Setup tab View the Analysis Results Select an analyzed w
36. he lid is opened when it is not safe WARNING Sharp needles Do not touch the needles at the bottom of the reagent cartridge Start the Instrument 1 Before turning on the instrument ensure that o The mains plugs are connected to properly grounded earthed mains outlets with the correct mains voltage and frequency see page 12 o The mains plugs are easily accessible in case of the need of quickly disconnecting the instrument from the mains power 2 Turn on I the instrument The power switch is located at the rear of the instrument Start the Run Load the reagent cartridge and the plate 1 When the instrument is not processing open the instrument lid An audible warning signal will alert you if the lid is opened when it is not safe Open the cartridge gate and insert the filled reagent cartridge with the label facing you Push the cartridge the whole way in and then down Ensure the line is visible in front of the cartridge and close the gate Open the plate holding frame and place the plate on the heating block 5 Close the plate holding frame and the instrument lid Select run file and start the run 6 Plug the USB memory stick containing the run Pyromarkgz 16 28 file into the USB port at the front of the instrument Leave it mounted until the run is finished Select Run in the instrument s main menu using EJ Directory the and screen buttons and press Ok EERE Ere ate Palen
37. he space between the heating block and the light guide block by carefully inserting a clean non abrasive lint free cloth lightly moistened with 70 ethanol and then moving it from side to side a couple of times see image below Caution Do not use regular paper tissues Use for example a camera lens cloth When you are done close the plate holding frame and the instrument lid and reconnect the instrument to the mains power 33 PyroMark Q24 User Manual 1 02 Maintenance of PyroMark Q24 Vacuum Prep Workstation workstation contains sodium hydroxide that is irritating to eyes and skin Use adequate ventilation Use impermeable gloves chemical safety goggles and protective clothing MSDS can be downloaded at www biotagebio com Xx WARNING Irritant The Denaturation Solution which is used with the Clean the Vacuum Prep Workstation If the vacuum prep workstation needs to be cleaned to remove dust and spillage follow the instructions below Required items Powder free gloves high purity water Milli Q 18 2 MQ x cm www millipore com or equivalent a mild detergent if necessary and clean lint free cloths 1 Ensure that no vacuum is applied to the vacuum prep tool i e the vacuum switch is closed OFF and the vacuum pump is turned off O 2 Disconnect the vacuum pump from the mains power Clean the worktable and the tool except for the filter probes using a clean lint free cloth moistened with water or
38. height the warning Failed Uncertain surrounding reference sequence pattern appears Both warnings with the second being much more severe are indications that the assay needs further optimization in terms of design or PCR conditions Deviation in Sum or Pattern in Variable Position Unexpected peak height in the variable position results in the warning Failed Uncertain due to high sum deviation in variable position Failed Uncertain due to high pattern deviation in variable position or Failed Uncertain due to high peak height deviation at dispensation N For AQ assays both homozygotes should be analyzed and it is important that there is no background incorporation from the other homozygote For a CpG assay ideally both completely unmethylated and fully methylated samples should be analyzed It might however be difficult to obtain both 0 and 100 methylated material If several sites are included in an assay and one of these warnings occurs for just one position and no other warnings appear this position can simply be excluded from the analysis 47 Glossary Biotin Bisulfite Bisulfite treatment control Cyclic dispensation order Directed dispensation order Dispensation order Drop off Enzyme Histogram Homopolymer Instrument methods PyroMark Q24 User Manual 1 02 A molecule that can bind very strongly to streptavidin PCR primers can be biotinylated to enable the resulting PCR product to bind to st
39. here there is a positive reaction with the added nucleotide light is emitted giving rise to a peak in Pyrogram To enable fast mixing of samples and reagents in the plate the heating block inside the process chamber is constantly vibrated during the run The heating block maintains the correct temperature of the plate and its contents Dispensing Unit A reagent cartridge PyroMark Q24 Cartridge filled with the required volumes of reagents is inserted into the dispensing unit The instrument will start dispensing the reagents when the pressure in the dispensing unit the speed of the mixer and the temperatures of the heating block process chamber lid and the coolant liquid have reached their preset levels may take several minutes During the run the reagent cartridge is positioned over each well in the plate and the reagents are dispensed in a zigzag fashion by a pneumatic The reagent cartridge is inserted into the dispensing unit PyroMark Q24 User Manual 1 02 Instrument Software The instrument is controlled via the six buttons underneath the screen Runs are started and monitored through the software provided with the instrument During the processing of a run the software displays the following information Validation run 080214 16 42 Run name Selected well Current time KR W N e Instrument status For more information see page 26 5 Pyrogram To select another well use the and screen button
40. ices and Distributors Please visit www biotagebio com for contact information Technical Support Europe 1 pointsupport eu biotage com U S amp Canada 1 pointsupport biotage com Rest of the world Please contact your local distributor For phone numbers and more information please visit www biotagebio com Revision History Version Revision date 1 01 March 2008 1 02 April 2008 51 Index A ADORA Urdaibai ias 26 ACCOSSOUIOS carne 10 Administration Backup PyroMark Q24 Files 18 INSTA ais crisi 17 Analysis ye AA Een seer 29 ROOTS ias cod ESS 30 SOU no at 7 TOUDIGSNOOUING sccestcsccsecventcedssudeens 38 Analysis Software Shortcut BFOWSEN vicecaractasceaiiacweasdanes 7 SPSCHICA ONS oser nende 13 SURE A A 19 WPOUBICSINOOUING costaneros 40 ANa ZE RU aaa andas 29 Assay Design and Validation 43 A eo 19 B Backup PyroMark Q24 Files 18 C Check Filter Probes manera cis ri 34 INSTRUMENT diteanteeticanteederstdanevaasaaned 31 Performance lacada 32 Reagent Cartridge sssesese0ee 28 A pees ceceoeireiense a 37 Chemical Resistance ccccceseeueeueenees 13 Clean Heating Block and Light Guides 33 INSEE errre anaa 32 Reagent Cartridge sssssssssssssssenns 28 Vacuum Prep Workstation 34 Colors Methylation Levels ccccseeeeueenseuees 30 Quality Assessments cccceeeeeeeeees 30 COMMCCHIONS eirinen 6 CONSUMAbDIES road cial
41. ill be sequenced ahead of the rest of template sequences Negative 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 The possible genotypes for a specific polymorphic position are calculated on the basis of the sequence to analyze and the dispensation order A region in the dispensation order where the sequence varies at one or more variable bases In Pyrosequencing software the variable positions are highlighted with a blue gray background color in the histogram and Pyrogram 50 PyroMark Q24 User Manual 1 02 Contact Information mn Pyrosequencing AB Kungsgatan 76 SE 753 18 Uppsala SWEDEN Phone 46 18 56 59 00 Fax 46 18 59 19 22 E mail pyrosequencing biotage com Website www biotagebio com Sales Off
42. ing the plate i Place the PCR plate or the strips and PyroMark Q24 Plate on the worktable see image above Ensure that the plate is in the same direction as when you loaded your samples see image for guidance j Apply vacuum to the tool by opening the vacuum switch 22 PyroMark Q24 User Manual 1 02 k Capture the beads containing immobilized templates on the filter probes by carefully lowering the probes into the PCR plate or the strips and holding them there for approximately 15 seconds Be careful when you pick up the tool Note Sepharose beads sediment quickly If more than one minute has passed since the plate or the strips was agitated agitate it for approximately one minute before capturing the beads Ensure that all liquid has been aspirated from all wells and that all beads have been captured onto the filter probe tips If the wells are not empty or white beads remain the filter probes might have to be cleaned or replaced see the instructions on page 34 m Move the tool to the trough containing 70 ethanol trough 1 and let the ethanol flush through the filter probes for 5 seconds n Move the tool to the Denaturation Solution trough 2 and let the solution flush through the filter probes for 5 seconds o Move the tool to the Washing Buffer trough 3 and let the buffer flush through the filter probes for 10 seconds p To allow liquid to completely drain from the filter probes pick up th
43. is 10 Contact 1 Point SUPPONE acord 42 Contact Information sdcicna as ciorodgera 51 Copy EO Pie A eetaues 18 Recently Saved RUMNS ccccceeenenees 17 Unsaved RUNS sirvio teen 17 CODI nE 2 D Design and Validation of Assay 43 PyroMark Q24 User Manual 1 02 Dispensing Unit sotornarica acia 5 DOCUIMENLAUON anar cru ral 15 Installation and Safety 006 15 Online Help jrancheteesdevinisiuaweseanacuisin 15 Quick Guides 42 bosoner sees 15 Reagents and Consumables 15 User Manual aaa 15 E Empty Waste Container and Troughs 31 Errors AMES rr 38 Analysis Software aia ad 40 TVS Criada bre ere 41 Vacuum Prep Workstati0N 42 Extract Damaged RUNS cccccccccccnocnnoss 18 G General Hints and TipS occocccnnnnnonnnoos 7 El a y E o 48 I Instrument Administration siria 17 Check Coolant Level ooooooooo 31 CONNECLIONS amning 6 PEDER AA 5 Functional Sequence of Events 4 Process Chamber asada 5 Pyrogram and WarningS ssss0s 26 SHU DOWN dra 31 Ola ea 6 Specifications sata dad 12 Sanar 25 EUS sido por dit 26 Troubleshooting sssirrsraaacnares 41 Upgrade Software nia 18 Intended Purpose Europe ceeees 1 Intended Use United States 1 L A E TE renere E ERR 1 Limitation MSS aaa inn O 0 E E 14 Warranty and Liability erario 1 M MaM ONC O aaa 32 INS MENE griner 32 Vacuum Prep Workstati0N 34 52 Methylati
44. ivalent Ethanol 70 Binding Buffer Pyrosequencing Denaturation Solution Pyrosequencing Washing Buffer Pyrosequencing Annealing Buffer Pyrosequencing s K 101 Solution Annealing Butt Shing Buffer 10 Binding Buff amp gt ns Seems y Sosa 55 40 0033 mome Sven Fm aream ae 200 me ny re nam y on me tay hr ne REE me mo per a a ham ae T pc O O O O O 0 0 1 Amplify the DNA by PCR with One of the Primers Biotinylated To receive valid analyze data follow our recommendations in the Assay Design and Validation section on page 43 2 Immobilize the PCR Product to Beads Biotinylated PCR products are immobilized on streptavidin coated Sepharose beads Streptavidin Sepharose High Performance GE Healthcare a Gently shake the bottle with streptavidin coated Sepharose beads from side to side until a homogenous solution is obtained b Mix the total required amount of streptavidin coated Sepharose beads 2 ul per Sample and Binding Buffer 40 ul per sample in a tube Add 18 28 ul high purity water to make the total volume 80 ul per well Example If using 15 ul of PCR product 2 ul of beads and 40 ul of Binding Buffer you need to add 23 ul of high purity water Add the solution prepared in step b above to the PCR plate or the strips d Add 10 20 ul of a well optimized biotinylated PCR product to each well of a 24 well PCR plate or strips according to the plate setup see the
45. l testing laboratories All operations must be performed according to instructions provided through dialogs appearing on the screen the manuals instructions for use and by Pyrosequencing AB s technical support staff and within limits set by the technical specifications x For more information see www biotagebio com Warranty and Liability Pyrosequencing AB warrants for a period of twelve 12 months from the date of shipment Warranty Period that PyroMark Q24 System i meets the published specifications and ii is free from defects in material and workmanship under normal use and service and when used in compliance with the applicable operating instructions Pyrosequencing AB s sole liability and Buyer s exclusive remedy for a breach of this warranty is limited to replacement repair or refund at the sole option of Pyrosequencing AB This warranty does not apply to any consumable items included in PyroMark Q24 System such as but not limited to filter probes tubing fittings o rings and gaskets Limitation of Warranty and Liability PYROSEQUENCING AB HEREBY EXPRESSLY DISCLAIMS AND BUYER HEREBY EXPRESSLY WAIVES ANY WARRANTY REGARDING RESULTS OBTAINED THROUGH THE USE OF THE SOFTWARE INCLUDING WITHOUT LIMITATION ANY CLAIM OF INACCURATE INVALID OR INCOMPLETE RESULTS ALL OTHER WARRANTIES REPRESENTATIONS TERMS AND CONDITIONS STATUTORY EXPRESS IMPLIED OR OTHERWISE AS TO QUALITY CONDITION DESCRIPTION MERC
46. lems Action The instrument is making Check that the cartridge is inserted correctly see unexpected noise when starting instructions on page 25 No contact with the USB memory The used USB memory stick is damaged or not Stick compatible with the system We recommend using USB memory sticks supplied by Pyrosequencing USB memory stick can not be Broken USB contact please contact 1 Point inserted Support Vacuum Prep Workstation Related Errors Problem Action No vacuum is received Turn off the vacuum pump and open the cap to the waste container to release any pressure Close the cap and start the pump again If the waste container is full empty it as described on page 31 Ensure that the tubing is connected properly see image on page 36 and that there is no leakage The waste filter might be wet and needs to be replaced see instructions on page 36 Liquid left in some wells in the Replace corresponding filter probe see immobilization plate instructions on page 34 White remains Sepharose beads Do not leave the plate for longer than one minute in the plate for immobilization after mixing is finished If necessary mix for an extra minute Verify Proper Installation and Operation PyroMark Control Oligo is sold together with the PyroMark Q24 System to verify proper installation and operation of your system The Control Oligo consists of one wobbled base measured as C single bases of all four nucleotides and h
47. llation and operation of your system using PyroMark Control Oligo Check the Run Log It is advisable to check the run log to assess if the system was working properly during the run 1 Open the run file 2 Select Run Information from the Tools menu The Run Information report is opened Check the run log at the end of the report for any problems during the run 4 If deviations from the preset block temperature pressure and or mixer values are noticed several times during a run and for longer time periods or in repeated runs please contact 1 Point Support If requested to send an Environment Data report a Select Export Environment Data from the Tools menu b Select the destination folder from the Save in drop down list c Enter the file name in the File name text box and click Save Troubleshooting Guide The troubleshooting guide suggests corrective actions for a number of analysis and instrument problems e Analysis related errors on page 38 e Analysis Software related errors on page 40 e Instrument related errors on page 41 e Vacuum Prep Workstation related errors on page 42 37 PyroMark Q24 User Manual 1 02 Analysis Related Errors Low or Missing Peaks in Pyrogram Possible cause Action PCR failed due to low DNA quality Poorly optimized PCR The biotinylation is omitted or not added to the right PCR primer Biotinylation is of poor quality Insufficient amount of template f
48. n PyroMark Q24 Plate Holder and a heating block b Let the samples cool to room temperature for at least 5 minutes The plate can now be processed in PyroMark Q24 Instrument 23 PyroMark Q24 User Manual 1 02 Prepare PyroMark Q24 Gold Reagents WARNING Sharp needles Do not touch the needles at the bottom of the reagent cartridge 1 Open the PyroMark Q24 Gold Reagents box and take out the vials containing freeze dried enzyme and substrate mixtures and the vials with nucleotides Reconstitute the required volumes of reagents and fill a reagent cartridge PyroMark Q24 Cartridge according to the instructions supplied with the reagents The required volumes of reagents for your plate are listed in the Pre Run Information report see the Set Up a Run section on page 20 Jul O08 1 la Label Note If you re use a reagent cartridge ensure that it is cleaned according to the instructions on page 28 It is recommended that the reagent cartridge is used no more than 30 times If the reagent cartridge has not been used for a while has been stored check that it can be used for analysis by performing the function test on page 28 steps 4 through 6 24 PyroMark Q24 User Manual 1 02 Process Your Run on PyroMark Q24 Instrument A WARNING Pinch and impact hazards due to moving parts Keep the instrument lid closed while the instrument is processing An audible warning signal will alert you if t
49. n the main menu using the and screen buttons and press Ok Select Copy Log Files and press Ok 4 When the instrument confirms that the log files have been saved to the memory stick press Close Remove the memory stick 6 To send the files to 1 Point Support see contact details on page 51 Extract Damaged Runs If you have damaged runs e g due to the instrument was turned off during the run follow the instructions below 1 When the instrument is not processing plug one of the USB memory sticks supplied with the system into the USB port at the front of the instrument 2 Select Administration in the main menu using the and screen buttons and press Ok Select Extract Damaged Runs and press Ok 4 When the instrument confirms that the runs have been saved to the memory stick press Close Remove the memory stick 6 For more information contact 1 Point Support see contact details on page 51 View Acknowledgments Versions Legal and Contact Info If you want to view acknowledgments legal information software and hardware versions and or contact information follow the instructions below 1 Select About in the main menu using the and screen buttons and press Ok 2 Select the information you want to view and press Ok Upgrade the Instrument Software If you have received a software upgrade from Pyrosequencing upgrade your instrument as described in the PyroMark Q24 Installation an
50. nd Validation section on page 43 the first time you run an assay Redesign non biotinylated primer or add NNN in the 5 end 39 Contaminated sample has led to unusually high consumption of substrate at substrate addition which is noticed as a high pre Sequencing signal Reagents incorrectly diluted or incorrectly stored Crosstalk when light from one well appears in the neighboring well Dispensation error Unknown SNP in sample dUTP is used in the PCR reaction Plus shift see the Glossary section on page 48 for an explanation Minus shift see the Glossary section on page 48 for an explanation PyroMark Q24 User Manual 1 02 Ensure to follow the sample preparation instructions on page 21 properly Change buffers and do not use any other buffers than those supplied by Pyrosequencing Check if any peaks have been generated using the zoom in function select a stretch of Pyrogram with the left mouse button Ensure to follow the instructions supplied with PyroMark Q24 Gold Reagents Do NOT store the nucleotides in 20 C Include an empty well only Annealing Buffer in your run setup to check if background peaks are coming from the nucleotides Avoid placing assays with high signals close to assays with low signals Replace the reagent cartridge If the problem remains contact 1 Point Support Insert the SNP in the sequence to analyze and regenerate the dispensation order Rerun your
51. nsure that the filter probes are working properly see instructions on page 34 Ensure that the tubing is connected properly and there is no leakage see image on page 36 The waste filter might be wet and needs to be replaced see instructions on page 36 Check that you loaded the plate for immobilization and PyroMark Q24 Plate according to the plate setup Ensure to add enough reagents open the run setup and select Pre Run Information from the Tools menu Ensure to follow the instructions for use supplied with the products 38 One of the nucleotide needles in the reagent cartridge the one that has not been dispensed is blocked or damaged No enzyme or substrate is added to the well which is noticed as a missing pre sequencing signal and no peaks in Pyrogram Obstructed or damaged reagent cartridge needles Reagents incorrectly diluted or incorrectly stored The reagent cartridge is not inserted correctly into the instrument The instrument has been started without a plate inserted Low signal due to dirty light guides Poor or Faulty Sequence Possible cause PyroMark Q24 User Manual 1 02 Clean the reagent cartridge and check that it is working properly see instructions on page 28 Clean the reagent cartridge and check that it is working properly see instructions on page 28 Clean the reagent cartridge and check that it is working properly see instructions on page 28 In case of
52. o 25 C for optimal performance 10 C to 40 C 420 x 390 x 525 mm with the lid closed Approx 700 x 700 x 600 mm 28 kg One USB port 2 0 1 24 samples run 24 well microtiter plate PyroMark Q24 Plate Individual for each well 28 C 1 Depending on the number of dispensations for example 20 dispensations take 24 minutes PyroMark Q24 Vacuum Prep Workstation Input voltage and current Power consumption Laboratory environment Storage temperature 100 VAC 50 60 Hz 1 7 1 4 A or 115 VAC 60 Hz 1 5 A or 230 VAC 50 Hz 0 6A Maximum 25 Watt Temperature 15 C to 32 C Humidity 20 to 90 relative humidity Normal laboratory conditions Use adequate ventilation 10 C to 40 C 12 Dimensions HxWxD Clearance space HxWxD Weight Batch size Process time PyroMark Q24 Software PyroMark Q24 User Manual 1 02 The worktable is 68 x 295 x 353 mm Approx 400 x 350 x 700 or 400 x 700 x 350 mm 11 kg including filled troughs and a full waste container 1 24 samples Less than 5 minutes for up to 24 samples in parallel The office computer used to set up runs and analyze data should be a personal computer with the following minimum specifications Operating system Processor RAM Free hard disk space Graphics card Monitor Pointer device Interfaces Chemical Resistance Microsoft Windows XP or Windows Vista English versions Intel Pentium 4 3 GHz 1 GB 100 MB
53. o AAA eE o E iaai 45 Sample Propa TACO irrita 46 O O eee teem Er SEE 46 Valla cOn a NEW ASS as 46 AOS eS tls arnes 47 GLOSSARY risa 48 CONTACT INFORMATION LEE Eee 51 sales Oces and D Shr ONS eaten eli nen meditate 51 H S gt pp gt ODO ici pias 51 REVISION HISTORY a A A Eee rs 51 INDEX osito 52 PyroMark Q24 User Manual 1 02 Legal Intended Use United States PyroMark Q24 System is designed for Laboratory Use Only which means it may be used for either research purposes or by high complexity CLIA certified laboratories All operations must be performed according to instructions provided through dialogs appearing on the screen the manuals instructions for use and by Pyrosequencing AB s technical support staff and within limits set by the technical specifications Intended Purpose Europe PyroMark Q24 System is a system for detecting changes in specified variable positions in human DNA that may have clinical relevance PyroMark Q24 System is available for research and in certain European countries for in vitro diagnostic applications PyroMark Q24 System meets the requirements of Annex III of the European Directive for In Vitro Diagnostic Medical Devices 98 79 EC For in vitro diagnostic medical use PyroMark Q24 System may only be operated i by personnel who have received special education and training with regard to procedures utilizing in vitro diagnostic medical devices and ii accredited medica
54. oid homopolymers in variable positions if possible e Use C G or T in variable positions if possible Controls in first run of new assay e PCR without DNA e Sequencing primer alone e Template without sequencing primer Analysis e Ensure you use appropriate analysis parameters For samples and positive controls aim for e Sufficient signal intensities Aim for a single peak height of at least 20 RLU e No background in blank dispensations e No background in variable positions e Expected reference sequence pattern e All positions with quality assessment Passed 43 PyroMark Q24 User Manual 1 02 Detailed Description Assay Design Primers should be designed with Pyrosequencing 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 analysis PCR Primers PCR primers should be 18 to 24 bases in length with annealing temperatures that are similar and typically in the range 60 C to 70 C nearest neighbor method One of the primers must be biotin labeled to enable immobilization to streptavidin coated Sepharose 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 The primers should not form strong hairpin loops or dimers wi
55. omopolymers of two and three bases For information on how to use the Control Oligo please see the instructions supplied with the product Contact 1 Point Support When contacting 1 Point Support see contact details on page 51 please have the following information ready e Your name address telephone fax numbers and e mail address e Serial number of your PyroMark Q24 Instrument see the rear of the instrument e Lot number of the regents PyroMark Q24 Gold Reagents you are using e Description of the problem encountered including the run log see page 37 e Details of a run that has been carried out if this helps clarify the problem 42 PyroMark Q24 User Manual 1 02 Assay Design and Validation Summary PCR e Use Pyrosequencing Assay Design Software for primer design e Ensure oligonucleotides are of a high quality including HPLC purified biotinylated primer e For CpG assays use fresh reagents or commercial kits for bisulfite treatment e Use hotstart DNA polymerase in PCR e Ensure there is sufficient DNA in PCR e Aim for PCR products that give a single strong band on an agarose gel with no excess of primers e Test for equal amplification of both alleles Pyrosequencing Analysis When creating an assay e Include at least five reference peaks e Include blank dispensations they are automatically generated by the software e Make suitable modifications when the warning icon appears fl e Av
56. on LevelsS ccccsseceeeeuseueeeens 30 A chtecanucs 26 P Patents and TrademarkS ccssseeeeeens 2 PCR ODIO sisas 45 co URE e OPONE PE 5 ee EER E ES SETE ene 45 Performance and Limitation of Use 14 Prepare PyroMark Q24 Gold Reagents 24 Salles drid 21 Process Chamber 2 ormene herre 5 Protect Your Files arrasar 8 PyroMark Control Oligo scscusntescwangaasss 42 PyroM rk Q24 Arve 3 Analysis Software cccccesseeueeeeusauees 7 Sree aia 4 Reagents Consumables and Accessories vu cccsecccncsnnsenuncecnsenunnse 10 Vacuum Prep Workstation sses 9 Pyrosequencing Technology 16 Q Quality Assessments ccceeeeeees 30 47 R Reagent Cartridge clean and Testa 28 Poc etl rninn e 10 Replace Filter PrODOS ias 39 Rubber coda oeiead 35 e AP eee sassesenerneneciase 36 Waste FIICEI sana 36 NODO tada bia a 30 REVISION HISTORY uses lerede 51 Run ADD iio 26 Analysis REPO Siria 30 ANAIYZE A dose sienien 29 A RRESEEEEEE MESS SENERE ER 26 O A A ene 20 Sr e 25 MA A 27 View Analysis Results ceceee ees 29 S Sample Preparation aria aos 46 Set Date and TiM ooccoccccnnnnncnnnnnnno 17 Set Up MOS y COOP PEL 19 PO ro paa 45 RUI antro ENE EGER A TE 20 PyroMark Q24 User Manual 1 02 SMORECUL BIOW SSC iaa 7 Shut Down Instrument sii 31 Software INSTUMeN Casada ipnid 6 Setup and Analysis essa ia 7 Upgrade Instrument Software 18 Specificatii anios 12 Start NS
57. or immobilization to Sepharose beads Not enough enzyme or substrate for all wells Too much PCR product depleats substrate leading to missing peaks at the end of the sequence Samples not prepared according to recommendations The filter probes are not working properly Vacuum lost during sample preparation The wells marked in the run setup do not agree with sample placement in the plate for immobilization or PyroMark Q24 Plate One or several of the nucleotide compartments in the reagent cartridge are not correctly filled with reagents or nucleotides Check the PCR samples using a gel technique to confirm that you have one strong specific band If not rerun PCR with high quality DNA Check the PCR samples using a gel technique to confirm that you have one strong specific band If not re optimize PCR see the Assay Design and Validation section on page 43 Check your assay design see the Assay Design and Validation section on page 43 Use a well recommended primer supplier Ensure the biotinylated primer is HPLC purified or similar Ensure to follow the recommendations for amount of template see the Assay Design and Validation section on page 43 Ensure to fill the reagent cartridge according to the Pre Run Information report open the run setup and select Pre Run Information from the Tools menu Use less PCR product Ensure to follow the sample preparation instructions on page 21 properly E
58. or CpG assay design can include e Look for the region of most interest and select it as the target e Place PCR primers without covering more than one CpG site e Ifa primer needs to be located over a CpG site ensure the CpG site is in the 5 part of the primer e Look for warnings and check for analysis parameters that can be improved Tm for primers mispriming duplexes between biotinylated primer and sequencing primer amplicon length etc e Try to improve the final score by changing the length of the primers and by moving them back and forth 44 PyroMark Q24 User Manual 1 02 PCR Setup PCR reactions of 25 ul are set up using a hot start polymerase Ensure that you follow the instructions provided with the PCR reagents One of the primers must be labeled with biotin for immobilization to Streptavidin Sepharose High Performance beads GE Healthcare during the sample preparation step PCR Mixture e PCR buffer e 1 5 3 mM MgCl e 5 pmol forward PCR primer 200 uM e 5 pmol reverse PCR primer 200 uM e 0 2 mM of each dNTP e Units DNA polymerase according to supplier e 10 ng genomic DNA e High purity water to 25 ul t Milli Q 18 2 MQ x cm www millipore com or equivalent filtered through 0 22 um filter The annealing temperature is usually in the range 55 C to 60 C Run the PCR at the optimal annealing temperature for 45 cycles Using fewer cycles might give insufficient yield and cause background problems in
59. r instructions for PyroMark Q24 Software Context sensitive help is accessed by pressing the F1 key when in a dialog or window in PyroMark Q24 Software A PDF version of the online help is available on the Pyrosequencing Technical Support website but can also be accessed by selecting All Programs Biotage PyroMark Q24 Documentation PyroMark Q24 Online Help pdf in the Windows Start menu Instructions for Reagents and Consumables Detailed instructions for use are included with PyroMark Q24 Gold Reagents PyroMark Q24 Cartridge PyroMark Control Oligo and PyroMark Q24 Validation Oligo MSDS for PyroMark Q24 Gold Reagents PyroMark Control Oligo PyroMark Q24 Validation Oligo and sample preparation solutions can be downloaded at the Pyrosequencing Technical Support website 15 PyroMark Q24 User Manual 1 02 Principle of Pyrosequencing Technology Pyrosequencing is sequencing by synthesis an easy to use technique for accurate and quantitative analysis of DNA sequences Step 1 A sequencing primer is hybridized to a single stranded PCR amplified DNA template The template is incubated with the enzymes DNA polymerase ATP sulfurylase luciferase and apyrase and the substrates adenosine 5 phosphosulfate APS and luciferin Step 2 The first of four nucleotides is added to the reaction If the nucleotide is complementary to the base in the template strand it will be incorporated into the DNA str
60. r temporary file storage Failed to create log files Failed to create run file file name Failed to extract damaged runs Failed to set time and date Internal memory full Upgrade failed Transferred unsaved runs to a USB memory stick see instructions on page 17 Restart the run If the room temperature is high and a temperature problem remains 1 Ensure the cooling device is receiving power a light indicator at the rear is lit If not check your connections see page 6 2 Check the coolant level see instructions on page 31 Ensure that the run file is created in PyroMark Q24 Software Try another USB memory stick We recommend using USB memory sticks supplied by Pyrosequencing If the problem remains after restart of the instrument please contact 1 Point Support If the problem remains after restart of the instrument please contact 1 Point Support If the problem remains after restart of the instrument please contact 1 Point Support Ensure your upgrade installation files are located in a folder called Upgrade at the root of the memory stick Please contact 1 Point Support Please contact 1 Point Support Please contact 1 Point Support Please contact 1 Point Support Please contact 1 Point Support Please contact 1 Point Support Please contact 1 Point Support Please contact 1 Point Support Please contact 1 Point Support 41 PyroMark Q24 User Manual 1 02 Other prob
61. reptavidin coated Sepharose beads HSO 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 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 signal height normally seen in Pyrogram A protein or RNA working as a catalyst to enhance the speed of a biochemical reaction without altering it In Py
62. roperly see instructions PyroMark Q24 Troughs on page 28 Vacuum Prep Tool Filter Probes l RT room temperature Please refer to the Pyrosequencing website www biotagebio com or contact your local sales representative for the latest information on accessories and consumables 10 PyroMark Q24 User Manual 1 02 Instructions for Reagents and Consumables Detailed instructions for use are included with PyroMark Q24 Gold Reagents PyroMark Q24 Cartridge PyroMark Control Oligo and PyroMark Q24 Validation Oligo MSDS for PyroMark Q24 Gold Reagents PyroMark Control Oligo PyroMark Q24 Validation Oligo and sample preparation solutions can be downloaded at the Pyrosequencing Technical Support website 11 PyroMark Q24 User Manual 1 02 Technical Specifications PyroMark Q24 Instrument Input voltage and current Power consumption Laboratory environment Storage temperature Dimensions HxWxD Clearance space HxWxD Weight Connections Capacity Batch format Dispensation order Process temperature Process time 100 240 VAC 47 63 Hz 1 1 0 45 A grounded From the external power supplies to the instrument 12 VDC and 24 VDC nominal Maximum 160 Watt Temperature 15 C to 32 C Humidity 20 to 90 relative humidity Draft free location not close to window Keep the instrument out of direct sunlight We recommend that the instrument is run in normal room temperature 20 C t
63. rosequencing 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 A method that describes physical settings for the instrument such as mixer frequency block temperature and pulse time settings 48 PyroMark Q24 User Manual 1 02 International Union of Pure and Applied Chemistry An organization providing recommendations on organic and biochemical nomenclature symbols terminology etc IUPAC Codes A Adenine K TorG C Cytosine TorA Guanine CorG Thymine C T or G not A Uracil A T or G not C Purine A or G A T or C not G Pyrimidine C or T or G not T CorA N Any base A C G or T Out of phase When one of the alleles is sequenced ahead of the other Polymorphism Pre sequencing Signal Pyrogram Reference peak Sepharose beads Sequence to analyze Sequencing primer Genetic variations broadly encompassing any of the many types of variations in DNA sequences that are found within a given population As the substrate is dispensed into a well indicated by a S contaminants such as ATP or PPi will be converted to light Too high a substrate peak indicates that high levels of a contaminant might
64. s 6 Warning messages only the three latest For suggested actions see the Troubleshooting section on page 37 7 Estimated remaining time hh mm Power and USB Connections The instrument shall be connected to properly grounded earthed mains outlets using the two power supplies 12 VDC and 24 VDC supplied with the system A light indicator at the rear of the instrument is lit when the cooling device is receiving power The instrument is turned on by pressing the power switch at the rear of the instrument A light indicator in the left corner of the LCD display is lit when the instrument is turned on Note that the screen is blank during start up which may take up to one minute Run files are transferred to and from the instrument using the USB memory sticks supplied with the system and the USB port at the front of the instrument WARNING The power switch does not disconnect the instrument completely from mains power Always disconnect the instrument from the mains power before any maintenance is performed PyroMark Q24 User Manual 1 02 PyroMark Q24 Software Runs can be set up and analyzed on an office computer ee Rone running PyroMark Q24 Software The software has two analysis modes es New Cph Assay Oo AQ A variety of quantification studies and SNP analysis e CpG Methylation analysis in multiple consecutive CpG sites AQ assays and CpG assays can be performed on the same PyroMark Q24 Pla
65. s light seen as a peak in Pyrogram time Apyrase dNTP y dNDP dNMP phosphate Apyrase ATP gt ADP AMP phosphate nucleotide sequence G A GG GE T _ _ _ eee _ Y gt nucleotide added Ronaghi M et al A Sequencing Method Based on Real Time Pyrophosphate Science 281 363 365 1998 PyroMark Q24 User Manual 1 02 System Administration Instrument Administration Administration 11 19 Set Date and Time co EOS MUA Copy Recently Saved Runs l Copy Log Files Setting the date and time correctly ensures an accurate TPO a eg RRE date and time stamp in the instrument and run logs and Set Date and Time the analysis reports Upgrade Software 1 When the instrument is not processing select Administration in the main menu using the aand screen buttons and press Ok ok Select Set Date and Time and press Ok Select the parameter that you want to edit using the and buttons Edit the selected parameter using the and w buttons If you want to edit several parameters repeat steps 3 through 4 ow Ss A To save the change s press Set Copy Unsaved Runs If the USB memory stick is removed before the run is finished the run data has to be retrieved from the instrument as described below 1 When the instrument is not processing plug the USB memory stick into the USB port at the front of the instrument 2 Select Administration in the main menu using the and
66. sed wells The substrate mixture is dispensed into all used wells The pressure in the dispensing unit is increased The nucleotides are predispensed into the rectangular well in the plate The nucleotides are dispensed into all used wells according to their respective dispensations order Hence the first nucleotide in the dispensation order of each well is dispensed then the second and so on A period of 65 seconds cycle time is allowed between the addition of each nucleotide in a sequence to ensure that all enzymatic reactions are completed The instrument collects data using 24 CCDs underneath the heating block from all the wells simultaneously during the whole process In wells where there is a positive reaction with the added nucleotide light is emitted giving rise to a peak in Pyrogram The data is continuously stored on the instrument When the run has been completed the run data is stored on the USB memory stick If the memory stick is removed during the run the run data has to be manually retrieved from the instrument PyroMark Q24 User Manual 1 02 Process Chamber The process chamber includes a heating block that maintains the correct temperature of the plate PyroMark Q24 Plate and its contents If the room temperature is too high the heating block is cooled by PyroMark Q24 Cooling Device pre installed Data is collected from all the wells simultaneously by 24 CCDs underneath the heating block In wells w
67. sis software The default settings for the analysis parameters are chosen to indicate the seriousness of the problem by giving a Check or Failed quality message The ultimate goal for a well optimized assay is that all positions have the quality assessment Passed when using default or more stringent analysis parameters Such results will be shown as an entirely blue quality bar in the well after analysis Results of lower quality are indicated as Check yellow or Failed red together with error messages Analysis Results The analysis results for an assay are based on the sequence context as well as the results in the analyzed position Deviations from this built in control are shown as warnings in the Well Information area Deviations in Reference Sequence Pattern Reliable results depend on correct peaks with sufficient signal intensities The default setting for required single peak height is 20 RLU Blank dispensations are automatically included in the assay design by the software and these serve as controls for unspecific incorporation If peaks of a certain height are generated at these positions the warning Failed Uncertain reference sequence pattern at dispensation N is shown in the Well Information area The warning Failed Uncertain reference sequence pattern at dispensation N also appears if incorporation at a certain dispensation results in a peak that deviates from the expected height If several peaks deviate from the expected
68. ste container needs to be emptied see the instructions on page 31 b Fill five troughs supplied with the vacuum prep workstation o One with approx 50 ml of 70 ethanol 1 o One with approx 40 ml of Denaturation Solution 2 co 3 Plate o One with approx 50 ml of Washing Buffer 3 3 PA k o One with approx 50 ml of high purity water 4 02 a B e all 4 o One with approx 70 ml of high purity water 5 me A suggested setup is shown in the image above Refill the troughs to approximately these levels whenever needed c Start the vacuum pump I d Apply vacuum to the tool by opening the vacuum switch e Wash the filter probes by lowering the probes into high purity water trough 5 Let approximately 70 ml of water flush through the filter probes i e empty the trough Ensure that the water is being transferred to the waste container If not e Ensure that the tubing is connected properly and there is no leakage To replace broken tubing see the instructions on page 36 e The waste filter might be wet and needs to be replaced see instructions on page 36 f Refill trough 5 with 70 ml of high purity water g Close the vacuum switch on the tool OFF and place the tool in the Parking P position h Fill a plate PyroMark Q24 Plate with 0 3 UM sequencing primer in 25 ul Annealing Buffer in each well to be used Tip Use one of the supplied PyroMark Q24 Plate Holder as support when preparing and mov
69. t sensitive help is accessed by pressing the F1 key when in a dialog or window in the software A PDF version of the online help is available on the Pyrosequencing Technical Support website but can also be accessed by selecting All Programs Biotage PyroMark Q24 Documentation PyroMark Q24 Online Help pdf in the Windows Start menu PyroMark Q24 User Manual 1 02 PyroMark Q24 Vacuum Prep Workstation Samples to be analyzed using PyroMark Q24 Instrument should be prepared as described on page 21 using PyroMark Q24 Vacuum Prep Workstation In order to perform DNA analysis using Pyrosequencing technology PCR products have to be processed to yield single stranded DNA to which a sequencing primer can be annealed A simple and robust sample preparation process to produce high quality DNA from PCR reactions without prior purification is outlined below Prol amp 1 PCR product 100 300 bp one primer biotinylated ox De ie i gt 4 2 Immobilize onto streptavidin coated Sepharose Or R A K beads N 3 Separate strands by denaturation in NaOH Oe lies K OFr Lu x 4 Wash neutralize the immobilized strand M 5 Anneal sequencing primer Oer Aly a x om 1 0 x Ronaghi M et al Real Time DNA Sequencing Using Detection of Pyrophosphate Release Anal Biochem 242 84 89 1996 Ronaghi M et al Analyses of Secondary Structures in DNA by Pyrosequencing Anal Biochem 267 65 71 1999 Dunker J et a Parallel
70. t the i coolant level is visible in the window see image If not please contact 1 E Point Support see contact details on page 51 a i ai 31 PyroMark Q24 User Manual 1 02 Maintenance WARNING Before performing any procedures in this chapter please read and MA observe the safety requirements in the PyroMark Q24 Installation and Safety document Failure to follow those requirements may result in personal injury and or equipment damage WARNING Always shut down and disconnect the equipment from the mains power before any maintenance is performed WARNING Do not remove the cover panels there are no user serviceable parts inside If you believe there is a problem with your system please contact 1 Point Support immediately www biotagebio com WARNING If the system has been damaged or does not function properly shut down the system and contact 1 Point Support immediately www biotagebio com CAUTION In order to maintain compliance only consumables and accessories approved or supplied by Pyrosequencing must be used in the equipment Please refer to the Pyrosequencing website www biotagebio com or contact your local sales representative for the latest information on consumables and accessories Notes e Instructions on how to clean and test a reagent cartridge are available on page 28 e Instructions on how to empty the waste container and troughs used with the vacuum prep workstation are available on
71. te To toggle between the analysis modes in the analysis view select AQ or CpG in the toolbar Shortcut Browser PyroMark Q24 The integrated shortcut browser provides a quick and easy File Tools Help way to access folders contents and commonly used files A The following icons are used to display information about l HJE Example Files the files S Lagerholm 071012 1 i Gone 3 AQ assay file 071012 3 hl AQ Assay 1 la AQ Assay 2 bic CpG assay file 2 CpG Assay 2 Refresh New Assay seg Explore a A run file that has not been processed A run file that has been processed New Run Remove Shortcut 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 Properties x Tip If you position the mouse pointer over 1 an assay file a tooltip displays the assay note if entered and 2 a porro ie run file a tooltip displays the plate ID if entered General Hints and Tips Log On to Windows Using Your Own User Account All saved analyses performed are logged with used analysis settings analysis mode AQ or CpG and analysis version results including any warnings date and time of the analysis and who performed it For the 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
72. th themselves or with the other primers The biotinylated primer should be carefully checked for hairpin loops and duplexes with the sequencing primer as excess biotinylated primer might cause background in Pyrosequencing assays If possible avoid placing primers over polymorphic positions 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 streptavidin coated Sepharose beads Amplicon Length The optimal amplicon length is between 80 and 200 bp although products up to 500 bp might work well Sequencing Primer The sequencing primer should be 15 to 20 bases long and have an annealing temperature in the range 45 C to 55 C Ideally the sequencing primer should differ from the PCR primer by at least one additional specific base at the 3 end Specific Considerations for CpG Assays In CpG assays the optimal length of the primers is slightly increased to function on bisulfite treated DNA which has a high proportion of A and T PCR primers should usually be 22 30 bases and sequencing primers should be 18 23 bases long Since bisulfite treatment gives low quality DNA the amplicons for CpG assays should ideally be shorter than 200 bp If sequences have densely packed CpG sites the design may require manual intervention However the penalties and scores given by the software are still very informative Manual operations f
73. trument The power switch is located at the rear of the instrument Empty the Waste Container and Troughs VPW At the end of a working day liquid waste and any solutions left in the troughs should be discarded and the vacuum prep workstation checked for dust and spillage WARNING Irritant The Denaturation Solution which is used with the workstation contains sodium hydroxide that is irritating to eyes and skin Use adequate ventilation Use impermeable gloves chemical safety goggles and protective clothing MSDS can be downloaded at www biotagebio com Note Observe all federal state and local environmental regulations for disposal of laboratory waste Required item High purity water Milli Q 18 2 MQ x cm www millipore com or equivalent 1 Ensure that no vacuum is applied to the vacuum prep tool i e the vacuum switch is closed OFF and the vacuum pump is turned off O 2 Discard any solutions left in the troughs 3 Rinse the troughs with high purity water or if necessary replace them 4 Empty the waste container Note that the cap can be removed without disconnecting the tubing 5 If the vacuum prep workstation needs to be cleaned from dust and spillage see the instructions on page 34 Check the Instrument im a Check the instrument for dust and spillage If the instrument needs to be e cleaned see the instructions on page 32 MM At the rear of the instrument press the light button and check tha
74. ually distributed in the range 5 to 95 preferably in triplicate Regression analysis of the frequency of one allele measured in PyroMark Q24 as a function of the input expected allele should give an R value greater than 0 9 Methylated DNA is commercially available or can be obtained by in vitro methylation using the enzyme SsslI For an AQ assay the allelic variants being part of the variable position can be mixed at different ratios similar to the procedure for a CpG assay If the variable position in an AQ 45 PyroMark Q24 User Manual 1 02 assay is an SNP the easiest way to test for equal amplification 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 Sample Preparation Use 10 20 ul of a 25 ul PCR for immobilization to Streptavidin Sepharose High Performance GE Healthcare according to instructions in the manual This will be equivalent to 1 4 picomoles of PCR product providing the recommended PCR protocol has been used to develop a well optimized assay Assay Setup The sequence to analyze should contain a sufficient number of bases to generate at least five non variable reference peaks 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 th
75. ueeuees 36 When a Day s Work is Finished 31 When Run is Finished s0eceeeeees 27 54 Biotage
76. ument AR IE Oia ita od 17 eG Date T names aca L7 PyroMark Q24 User Manual 1 02 E sain E ERE A E T 17 Copy Recently Saved RUNS solaris 17 FEER OG FS ua or arar 18 EXC Dada uo a albo Te Tee 18 View Acknowledgments Versions Legal and Contact Info ccc cece eee e eee ees 18 Upgrade the Instrument Software croacia vr e 18 Ba RUD PyroMark O24 FES iaa ds oa radios 18 USING THE SYSTEM sonas aia in 19 A e A e A 19 Start Py oMark FO A o e 19 Seb UD ail Assay A e ea 19 A e nase ES ERE SEEGER SEES E 20 Mere toa ancestro ere eee 20 Prepare YOUR Samples rra 21 1 Amplify the DNA by PCR with One of the Primers Biotinylated cccceeee ees 21 2 Imamobilize the PCR Product to Beads auroras Di anA 21 3 Separate the DNA Strands and Release the Samples in PyroMark 0Q24 Plate 22 4 Anneal the Samples to a Sequencing Primer c cee eee ee eerseeeenneeeeeesseeusaanneeeegs 23 Prepare PyroMark Q24 Gold Readers 24 Process Your Run on PyroMark Q24 INSTUMeR Lisandro ea 25 BEES e Ea AAA o o RR 25 o A o SETE STER ENERET T ENE SENE EEN 25 ei A e E E PROS CE 5 EVER 26 Wher tne RUN I FINE NEU iaa ii ie dios 27 Anah ze 1OUl RUN di 29 Analyze Ce Ar cad 29 View the Anos Sc ton ee nr ee a eT 29 Analysis EDO Sarabia arras 30 Pe TO yan de q vic xcs pose anc cnwntss cused Meee A A 30 When You Have Finished the Day s Work aia 31 SMe DOV pep Alp Agea aeaa red A 31 Empty the Waste Container and Troughs CV PW isa
77. until the instrument confirms that the run file has been saved to the memory stick Finished The run has been completed and the run data has been transferred to the USB memory stick Pyrogram and Warnings The run name and the selected well are displayed in the top left corner To select another well use the a and screen buttons Any instrument warnings are displayed below the Pyrogram area only the three latest For suggested actions see the Troubleshooting section on page 37 For information on how to contact 1 Point Support see contact details on page 51 Abort the Run To abort the run press the Stop button 26 PyroMark Q24 User Manual 1 02 When the Run Is Finished Note Observe all federal state and local environmental regulations for disposal of laboratory waste 1 ae et ee When the instrument confirms that the run file has been saved to the USB memory stick press Close Remove the memory stick Open the instrument lid Open the cartridge gate and remove the reagent cartridge by lifting it up and pulling it out Close the gate Open the plate holding frame and remove the plate from the heating block Close the plate holding frame and the instrument lid Discard the plate Clean the reagent cartridge according to the instructions on page 28 or discard it If this was the last run for the day see the When You Have Finished the Day s Work section on page 31 27 PyroMark Q2
78. vw screen buttons and press Ok Select Copy Unsaved Runs and press Ok 4 Select the run file you want to retrieve using the and buttons and press Select 5 When the instrument confirms that the run file has been saved to the memory stick press Close 6 Remove the memory stick Copy Recently Saved Runs Copies of run files are kept on the instrument as long as there is enough free space on its internal memory When the storage capacity becomes insufficient the run files are deleted in chronological order Files that have never been saved to a USB memory stick will not be deleted 1 When the instrument is not processing plug one of the USB memory sticks supplied with the system into the USB port at the front of the instrument 2 Select Administration in the main menu using the and screen buttons and press Ok Select Copy Recently Saved Runs and press Ok 4 Select the run file you want to retrieve using the and buttons and press Select 5 When the instrument confirms that the run file has been saved to the memory stick press Close 6 Remove the memory stick 17 PyroMark Q24 User Manual 1 02 Copy Log Files If you are requested to send the log files to 1 Point Support follow the instructions below 1 When the instrument is not processing plug one of the USB memory sticks supplied with the system into the USB port at the front of the instrument 2 Select Administration i

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