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PSQ 96 SNP Software User Manual

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1. 35 Guidelines for defining 36 Disposal of plates and cartridges 57 E Entering an SNP 2224488 4d cue dude cued dd 31 33 Evaluating results Administration menu 60 Administration menu User administration 61 Algorithm sje ins sue rem nets NEN ERE ser 74 Analyze SNP tab parameters Analyze 67 Analyze SNP tab parameters Criteria 66 Analyze SNP tab parameters Export 62 Analyze SNP tab parameters Print 73 Analyze SNP tab parameters Set category 62 Displaying overall results 67 Displaying well results 70 Selecting runs EE 63 64 9 PYROSEQUENCING 9 PYROSEQUENCING SNP Analysis results 64 SNP Analysis results Editing 71 Starting evaluation module 59 Starting module es i ss ate ere Fes hr X de eee 59 Exporting data 53 62 External equipment for connection to PSQ 96 Instrument 3 G Guidelines for defining a dispensation order Longer extension 2322s ey NR as ee a me S 37 Negative controls 25e da dues de patte aie ce abla ames 37 Out of phase analysis 37 Guidelines for entering data
2. 34 H Hardware requirements 9 I Important User Information 1 Declaration of Conformity 3 Warranty and Liability 4 Installation Changing IP addresses 15 Items required but not provided 11 Multiple PSQ 96 Instruments 13 Stand alone 4 lore per pex pen mate ed 11 TesBnB eu duae big Sark sane rie ae EVER ae 22 Installing PSQ 96 SNP Software 11 Installing PSQ 96 SNP Software Office version 12 IPaddresses 9 dons ok Erbe RE a nee pr RA ES 15 L Local area network requirements 10 M Methods s ode yw xVePHU USE Ge PI Dane ae A 1 Monitoring fun issues deb RR ERR RR RET H 52 Multiple instrument set up 13 O Opening SNP Entry 27 28 Operating system 10 Index P Preferences izle eu akse e EAR RR E ALE Geena Se 19 Product family EE soi b Qe pA rte 7 PSQ 96 Instrument Control Closing down 56 Exporting data i salse e e a mee te eske en 53 Menu Dates Lie eser mare cr eis Satie Globe Ex LN EN 41 Monitoringarun 52 Performing an SNP analysis 45 Process Identification inform
3. 3 If no sequencing primer has been entered a negative control is suggested according to C T G In this case also the first base in the sequence to be analyzed is considered according to the rules above 4 Directly after the SNP another negative control is added according to the following priority C gt T gt G gt A The bases in the SNP or the base immediately following the negative control position is never used 3 5 2 Secondary rules The following rules are activated when a user has entered a sequence that differs from the recommendations CG PYROSEQUENCING 3 36 Chapter 3 PSQ 96 SNP Entry 1 If the entered sequence contains information about less than five non polymorphic bases PSQ 96 SNP Entry will use the available information 2 If the user wants to add more bases than those suggested manual modification of the dispensation order is necessary If the dispensation order created will generate more than eight additions of the same base per well PSQ 96 SNP Entry will warn the user of the risk of running out of nucleotides This might be the case if a whole plate is run with this dispensation order 3 A warning message will be shown if the sequence to analyze includes a homopolymeric stretch equal to or longer than four A bases or six bases of any of G C or T 4 A warning message will be generated if there are homopolymeric stretches on both sides of the polymorphic position 5 A warning mess
4. Figure 4 1 PSQ 96 Instrument power switch Note PSQ 96 Instrument requires a warm up time of about 90 minutes for the output from the CCD camera to stabilize If PSQ 96 Instrument has not been allowed to warm up 9 PYROSEQUENCING 4 40 Chapter 4 Running PSO 96 Instrument sufficiently before use the baseline values will fluctuate making the results less reliable 4 2 2 Description of indicator lights on PSQ 96 Instru ment power hd busy message Figure 4 2 Indicator lights on PSQ 96 Instrument There are three indicator lights on the front of PSQ 96 Instrument Power this indicates that PSQ 96 Instrument has been connected to the mains electricity supply and that the instrument is receiving power Busy when this light is illuminated or flashes a run is underway and the lid should not be opened Message when the message light is illuminated during a run an entry will appear in the Event Log Such messages can include a warning that the instrument lid is not correctly closed including an audible warning During start up of the PSQ 96 Instrument all the lights will illuminate When the instrument software in PSQ 96 Instrument has been loaded 1 3 minutes the Busy light will turn off and the Message light will flash This indicates that the instrument is ready to receive instructions from the operator s computer When an application has been selected and the connection between
5. Stop button Stops a run that is underway and saves the data obtained When clicked on a window is opened asking the user to confirm the termination of the run When confirmed the run is stopped immediately and cannot be restarted Status texts These status texts appear to the right of the Stop button 1 Ifthere is no text to the right of the Stop button no application has been selected and no connection with the PSQ 96 Instrument has been established 2 No connection No connection can be established between the operator s computer and the PSQ 96 Instrument Make sure that the instrument is switched on and that the power light is illuminated and the message light flashing Check that the Ethernet cable is properly connected to the instrument and operator s computer 3 Idle PSQ 96 Instrument can be started once new run information has been entered 4 Preparing for run PSQ 96 Instrument has been set to run and the start up process is underway During this process the heating block heats up pressure in the system builds up etc 5 Adding reagents Enzymes and substrates are being dispensed into the PSQ 96 Plate CG PYROSEQUENCING 4 44 Chapter 4 Running PSO 96 Instrument 6 Running The run is underway 7 Pause The Pause button has been clicked on To continue the run click on the Run button 8 Service Is shown when the System Information application is running Process progress scale At the right
6. 4 Start PSQ 96 The PSQ 96 Instrument Control Instrument main screen will appear When Control see contact is established between the section 4 2 3 operator s computer and the Starting the PSQ instrument the Status Bar will be 96 Instrument visible showing the instrument name Control and the Application button Click on the Application button 5 Log on Log on in the User field as Administrator No password is necessary 9 PYROSEQUENCING 2 24 9 PYROSEQUENCING Chapter 2 System Installation and Administration Click on SNP Analysis button Process Identification tab will open with the following text in the Status bar Idle The red light will go out Ifthe text No connection appears in the Status Bar and the red light on the instrument is still flashing check that the Ethernet cable is properly connected to the instrument and operator s computer Fill in the following fields mandatory Run ID Kit ID Plate ID Use the default setting Full plate Choose Process Parameters tab The Process Parameters tab will open Choose Method SNP method Choose Collection PSQ 96 SNP Choose SNP name BE4PN Click in well A 1 hold down the left mouse button and drag the pointer to copy the SNP to all wells in the plate Chapter 2 System Installation and Administration 2 25 Put in an empty PSQ 96 Plate and close the holder Pu
7. Electrical contact pins in the heat ing block are corroded Remove the heating block and clean the lens array as described see the chapter on Maintenance in the PSQ 96 Reference Manual If the contact pins look dis colored clean them carefully with fine grade emery paper If the disturbances continue the contact pins should be changed see the chapter on Maintenance in the PSQ 96 Instrument Reference Man ual See accessory list on Pyrosequencing s website www pyrosequencing com 9 PYROSEQUENCING 6 84 9 PYROSEQUENCING Chapter 6 Troubleshooting If none of these actions correct the problem please contact Pyrosequencing AB contact details are shown on page 5 When contacting Pyrosequencing AB please have the following information ready 1 Your name address telephone and fax numbers e mail address 2 Serial number of PSQ 96 Instrument see back of instrument or use the Report function described on page 77 Lot number of the PSQ 96 SNP Reagent Kit reagents you are using Description of the problem encountered Details of a run that has been carried out if this helps to clarify the problem s a registered user you can also request technical support via the support section on Pyrosequencing s web page http www pyrosequencing com Appendix Appendix A 1 1 Appendix 1 1 Defining Methods There is usually no reason to alter the parameters in the methods that are
8. Note Unless you fully understand the consequences of making changes in the following dialogs do not make any changes Select Data Source x File Data Source Machine Data Source Look in Data Sources v amp dBASE Files not sharable dsn Excel Files not sharable dsn FoxPro Files not sharable dsn MS Access 97 Database not sharable dsn Test Files not sharable dsn DSNName New Select the file data source that describes the driver that you wish to connect to You can use any file data source that refers to an ODBC driver which is installed on your machine con te Figure 2 7 Select Data Source dialog 2 Click on the Machine Data Source tab The dialog will open Select Data Source 21x File Data Source Machine Data Source Data Source Name Type Description System MSMQ Information Server Database System System Ver 1_1b7_PSO96db System New Machine Data Source is specific to this machine and cannot be shared User data sources are specific to a user on this machine System data sources can be used by all users on this machine or by system wide service Figure 2 8 Machine Data Source tab 3 Ifthe database exists i e PSQ 96 select it in the list and click on OK Otherwise click on the New button 4 Click on System Data Source Click on Next 5 Select the database driver in most cases the Microso
9. PYROSEQUENCING 9 PYROSEQUENCING Index System requirements Hardware suis aient lin XR E Pace 9 EAN sarte Le ne 10 Operating system 10 T Trademarks and Patents 4 Troubleshooting Guide ss ies mee LU eR Penn ein 80 Obtaining system information 77
10. Remove Instrument henge aucress Change heme Figure 2 3 System Administration Instruments Chapter 2 System Installation and Administration 2 15 To add an instrument 1 Click on the Find Instruments button message is broadcast on the network and responding instruments will be listed in the Instruments found field 2 Select the desired instrument from the list in the Instruments found field Click on the Add Instrument button 4 Inthe dialog that opens assign a name to the instrument and then click OK 5 The instrument will be listed in the Instruments used field 6 Click on the Close button to exit Note If you have problems finding the desired instrument in the Instruments found list consider the following reasons Instruments can only be accessed if they are switched on and if the PSQ 96 SNP Software applications on the operator s computers are closed Conflicting IP addresses will prevent instruments to be identified If the instrument is connected to a LAN check if the instrument is in use from another operator s computer To change the name of an instrument listed in the Instruments used field 1 Select the desired instrument from the list in the Instruments used field 2 Click on the Change Name button 3 Enter a new name for the instrument in the dialog that will open then click OK 4 Click on the Close button to exit To remove an instrument 1 Select the name of th
11. spa E T 86 A T E i 2 FEn e AA C T 6 G6 A T C s a IE LX cC T 6 AA T C r Quality 4 Passed _ Check Failed GIT Figure 5 13 Results from individual well The top graph in the dialog shows the pyrogram from the run The three histograms show the three possible genotypes for example G G G T and T T respectively The radio button for the chosen genotype is Chapter 5 Evaluating results 5 71 marked 1 and the genotype assessment on a background corresponding to the assigned quality of the result is displayed at the bottom right of the dialog 2 In addition the Quality field button is depressed 3 Tips To zoom in the pyrogram left click and drag with the mouse pointer in the pyrogram window to select the area of interest Right click to reset the zoom To get an expanded view of the pyrogram press the lt Alt gt key while double clicking on the specific well It is also possible to save any of the four graphs in the Edit SNP window or in the expanded view of the pyrogram as a jpg file by pressing the lt Alt gt key while pointing at the graph to be saved and right click standard Save dialog window will open Name the file and save in a desired directory 5 3 8 Editing results There may be reasons to change the quality classification or the genotype assignment of an individual well For instance you may wish to change from Check to Failed if when l
12. Once the SNP Analysis button has been clicked on the following screen will open Instrument Test 1 Process Identification Process Parameters Process Output r General r Sample Notes Ween z neng po zl Close ap M Pe ores as SE Noles af Hun a f as Wells ss ad e EE SEDET NIS anf AFFFFFFFFFFFEF BRR RRR RR PR CRFFFFFFFFFFF a lt lt lt AA A aa DFFFFFFFFFFFF 84 ER PRP PR PRP RP as EFFFFFFFFFFFF A GRR RRR PRP Pe ei HFFFFFFFFFFFF as DEI d enf el c D m Full plate Define wells E Sotby Row FEN Figure 4 8 PSQ 96 Instrument Control Process Identification tab The instrument name is shown at the top left hand corner of the entry field area If there are multiple PSQ 96 Instruments connected each instrument will appear in a separate window The New Run button is normally grayed and is only activated after the completion of a run and when the status bar text states Idle Before starting a run information regarding the run must be entered as follows Process Identification information The tab is divided into two sections The left hand section regards General and Wells information the right hand side is used to enter Sample Notes optional CG PYROSEQUENCING 4 46 9 PYROSEQUENCING Chapter 4 Running PSO 96 Instrument Dat
13. There may be occasions when it is necessary to enter a new SNP using PSQ 96 SNP Entry via Modules SNP Entry while the Process Parameters tab is still open If this is the case the newly entered SNP will not appear in the list until the Update SNP button has been clicked on 5 The SNP Dispensation field will be automatically filled in with the dispensation order specifically entered in SNP Entry for the selected SNP 6 Inthe well indication field the SNPs used are entered into the desired wells as follows Click in a well to enter the SNP shown in the SNP Name field To enter the same SNP in multiple wells use the click and drag technique described in point 6 of Process Identification information on page 4 46 To enter different SNPs into other wells select another SNP from the SNP Name field and continue clicking in the wells to enter the SNPs 7 An SNP Panel microtiter plate layout can be saved for future use by naming it in the SNP panel field and clicking on the Store SNP Panel button Stored SNP Panels can be recalled by clicking on the down arrow to the right of the Stored SNP Panel field and selecting the SNP Panel from the list The SNP panel will contain both SNP data and sample notes for the wells included 8 Once all the information has been entered in the Process Identification and Process Parameters tabs you are ready to start a run Tip If you want to check the dispensation order for a certain SNP during a
14. eres 22 3 PSQ 96 SNP Entry EE 27 3 1 Opening SNP Entry nier 27 3 2 SNP Entry screen rrmssvrnnsvvnnnnnvnnnnnvnnnnnvnnnnnnnnnnnnnnnnnnnnnner 29 3 3 Removing SNP data is 34 3 4 Guidelines for entering data in the Sequence to analyze field esses 34 3 5 How a dispensation order is generated 35 3 6 Guidelines for editing a dispensation order 36 3 7 Warning messages eene 38 Table of Contents 1 Table of Contents 4 Running PSQ 96 Instrument 39 4 1 Assumptions eebe ee 39 4 2 Starting the system es 39 4 3 PSQ 96 Instrument Control Software start screen description ses 41 4 4 Selecting an application rrsanrnnnnnvnnnnnvnnnnnvnnnnnvnnnnnnnnnnn 44 4 5 Performing an SNP analysis 45 4 6 After a TEE 55 5 Evaluating results ssss 59 5 1 Opening the evaluation module 59 5 2 Menu alternatives and buttons 60 5 3 Analysis of run data eene 64 5 4 Algorithm for analysis eere 74 6 Troubleshooting eeeeeeeeeeeee TT 6 1 Obtaining system information 77 6 2 Troubleshooting guide sees 80 Appendix i
15. 1 1 Hardware ZIP drive or similar for backing up the result file and database Required if the computer used to analyze the data is stand alone and is not connected to the LAN Hub for connecting multiple PSQ 96 Instruments to an operators computer in a stand alone configuration Use an Ethernet hub for four RJ 45 10Base T connectors 10 Mbps twisted pair suitable four port hub is manufactured by 3Com model OfficeConnect Hub TP4 art no 3C16704 2 22 Installation stand alone When purchased the PSQ 96 Instrument and operator s computer are preconfigured as a stand alone system No further software installation is required in the operator s computer or in PSQ 96 Instrument 2 23 Installing PSQ 96 SNP Software complete installation This refers to a complete installation of the software in a new or different operator s computer not the operator s computer supplied with the PSQ 96 System but another computer fulfilling the hardware requirements see section 2 1 1 Hardware Proceed as follows 1 Connect the PSQ 96 Instrument and operator s computer in a stand alone configuration see PSQ 96 Instrument Reference Manual 2 Insert the PSQ 96 SNP Software CD ROM into the CD ROM drive in the computer 3 Follow the installation wizard that will automatically start if the wizard does not start open Run under the Windows Start button specify the path to your CD drive and type autorun exe e g D Naut
16. 96 Plate All wells used If samples have been added into all wells of the PSQ 96 Plate click in the Full plate radio button at the bottom of the field to select all the wells Fach well in the graphical representation of the PSQ 96 Plate will be checked with a tick and the whole plate will appear grayed Only some wells used You can define specific wells filled in the PSQ 96 Plate by first clicking in the Define wells radio button at the bottom of the field Then position the mouse pointer in a well in the graphical representation of the plate to mark the well A tick character will appear Continue until all the wells that you have filled with samples are marked Chapter 4 Running PSQ 96 Instrument 4 47 Alternatively if the wells used are in a square or rectangular pattern click in the top left hand well or the bottom right hand well and hold the left mouse button down Drag the mouse pointer to include all the wells filled with samples and then release the mouse button You can use the same technique to rapidly deselect previously selected wells or to create an uneven pattern Click in the well to be deselected Hold the left mouse button down and drag the mouse pointer to include all the wells to be deselected Release the mouse button Clear checked wells If you make a mistake when defining the wells to be used either click in the well again to remove the tick character or click on the Clear button to remove all the tick
17. been saved Sample Details History Log 7 14 00 10 59 16 AM Analyzed v 1 1 3 Administrator T T Passed Window 7 Stringency High SNP Entry Warnings Off 7 14 00 10 58 40 AM Analyzed v 1 1 3 Administrator T T Passed Window 7 Stringency Medium SNP Entry Warnings On 7 5 00 11 38 56 AM Analyzed v 1 1 3 Maria T T Passed Window 0 Stringency Low SNP Entry Warnings On Figure 5 15 History log Tip You can easily display and navigate in the Edit SNP window using the keyboard Select a well using the lt Arrow gt keys on the keyboard Press the lt Space bar gt to open the window Move between fields using the lt Tab gt key Edit genotype or quality assignment using lt Arrow gt keys pa nec pi d Highlight OK Apply or Cancel buttons using the Tab key and make your choice by pressing Enter 6 Exit the window by pressing the Esc key Chapter 5 Evaluating results 5 73 5 3 9 Print Select print parameters in the dialog that appears when the Print button is clicked on Print c x iui _ _ Ft C Report 3 r Sections to print Landscape fi Diagrams per column 1 6 VIE iun date P i Nd 1 2 Colums FF Results Rescale all with Qrder by IV Quality messages Ymax o Row C Column History log Print All Wells Print Selected Wells Cancel Pint Selected Wels Cancel Figure 5 16 Print screens Graph Produces a p
18. by clicking in the appropriate radio button Auto time interval can be selected or a Time Range can be stated MaxY and MinY values can also be entered to limit the display 6 1 1 General The General tab contains a number of information fields Software versions This field is used to identify the version of the components of PSQ 96 SNP Software You will need this information when contacting Pyrosequencing AB Log Files Shows the locations where the various log files are stored Events Shows an event log of the events generated since the System Information function was opened 6 1 2 Pressure Pressure Shows the current pressure in the system Pump Status Shows the status of the pump on off and whether or not the pump is working The pump can be tested by clicking on the Turn pump on toggle button The pressure will increase to 700 mBar whereafter the pump will be automatically switched off The pump can also be switched off manually by clicking on the button again Valve Status Shows the status of the valve selected in the Valve field The valve can be tested by clicking on the Close Valve button Valve Select a valve to test by clicking on the appropriate button The valve will open for 10 milliseconds and a clicking sound will be heard in PSQ 96 Instrument 6 1 3 Lamps Speaker Lid Offers a means of checking the Busy Lamp Status Message Lamp Chapter 6 Troubleshooting 6 79 Status and Speaker Status and indic
19. characters from the plate representation If you have selected Full plate the Clear button will appear grayed 7 If applicable enter sample information for the wells used in the form of free text in the fields on the right hand side of the screen You can only enter information in those fields that correspond to the wells ticked on the plate representation A maximum of 40 characters of text can be entered for each well The two radio buttons at the bottom of the field Sort by Row and Column can be selected to sort the well fields by row and column respectively This is useful if the same notes are to be filled in for multiple wells To fill in the same notes for multiple wells sort the well list as desired by row or column and enter the notes for the first well Then mark the well note that you have written and holding down the left mouse button drag the pointer to cover all the wells that should have the same text Release the left mouse button to fill in the note in all wells selected To delete multiple well notes delete a well containing a note click in this empty well note field and holding down the left mouse button drag the pointer to cover all the well notes that you want to delete CG PYROSEQUENCING 4 48 t PYROSEQUENCING Chapter 4 Running PSO 96 Instrument Process Parameter information Click on the Process Parameters tab Instrument Test 1 Process Identification Process Parameters Process Outpu
20. dialog will open when this option is selected see section 4 5 5 Exporting data Exit and Log Off Closes down PSQ 96 Instrument Control returns to Windows NT desktop Modules SNP Entry Used to enter SNPs into the database and to automatically generate a dispensation order See chapter 3 Evaluation Used to evaluate and analyze data see Chapter 5 Window The undermenus are standard Windows functions for arranging multiple windows Also used to select different instrument windows in multiple instrument set ups Help Provides help information for PSQ 96 Instrument Control 4 3 2 Status Bar The Status Bar appears as follows TEST Figure 4 6 Status Bar If there are multiple PSQ 96 Instruments connected to the same operator s computer there will be one status bar for each PSQ 96 Instrument Chapter 4 Running PSQ 96 Instrument 4 43 Instrument name At the left hand side of the status bar the name of the instrument is displayed in this example TEST Application button To the right of the instrument name is the Application button When clicked on the Select Application dialog will open Click on the application desired and the appropriate screen will open Run button Starts a run after the system has been loaded and programmed Pause button Pauses a run that is underway The run is not paused until the ongoing dispensation cycle has been completed To restart the run click on the Run button
21. or a group of individuals The User name is used for identification when logging in as well as when saving information in order to verify the origin of information and to be able to trace actions In PSQ 96 Instrument Control select Instrument System Administration Note This dialog can only be accessed if there is no instrument window currently open The System Administration dialog will open The Users tab is open as default Chapter 2 System Installation and Administration 2 17 System Administration x Instruments Preferences User name Password Add User Close Figure 2 4 System Administration Users dialog The list of current users is shown in the Users field To add a new user proceed as follows 1 Enter the user s name in the User name field 2 Enter a password if desired in the Password field 3 Click on the Add User button 4 Click on the Close button to exit To remove an existing user proceed as follows 1 Click on the name of the user to be removed from the list in the Users field The name will appear in the User name field ps Click on the Remove User button 3 Awarning dialog will appear asking you to confirm removal of the user 4 Click on the Close button to exit Note Ifa user should forget his her password the user will have to be removed and re registered CG PYROSEQUENCING 2 18 9 PYROSEQUENCING Chapter 2 System Installation and Admin
22. removing the SNP Click on Exit to close 3 4 Guidelines for entering data in the Sequence to analyze field 9 PYROSEQUENCING In order to generate optimal conditions for evaluating the data certain recommendations for the entry of data in the Sequence to analyze field should be considered 1 The polymorphic position should be located within the first five bases 3 of the sequencing primer Enter enough bases to specify at least five non polymorphic peaks adjacent to the polymorphic position In order to save an SNP at least one non polymorphic peak is required This will generate a minimum of three reference dispensations one positive and two negative controls surrounding the SNP position Homopolymeric stretches of more than three or more than five C G or T should be avoided If possible do not enter the polymorphism at the terminal position of the entered sequence Two or more polymorphic positions cannot be entered in the sequence to be analyzed At present the software does not support automated genotyping of more than one polymorphism However for this purpose a suitable dispensation order can be entered and run data evaluated manually for the additional polymorphisms Avoid homopolymeric stretches on both sides of the polymorphic position In such cases consider a sequencing primer design overlapping one of the homopolymeric stretches Never end the sequence input in the middle of a homopolymeric stret
23. run double click on the desired well in the well indication field The SNP ID and dispensation order will be shown in the SNP name and SNP dispensation fields respectively 4 5 2 Setting up PSO 96 Instrument The section assumes that you have prepared your samples and pipetted them into a PSQ 96 Plate Loading sample and cartridge To load a PSQ 96 Plate into PSQ 96 Instrument proceed as follows CG PYROSEQUENCING 4 50 Chapter 4 Running PSQ 96 Instrument 1 Open the lid of PSQ 96 Instrument Figure 4 10 PSQ 96 Instrument with lid open 2 Lift the heating block lid and the PSQ 96 Plate holding frame 3 Position the PSQ 96 Plate containing your samples on the heating block The plate is keyed so that it can only be placed on the heating block in one position i e well A1 is at the farthest left hand corner from you Figure 4 11 PSQ 96 Instrument with heating block lid and plate holding frame up Close the PSQ 96 Plate holding frame Close the PSQ 96 Plate heating block lid Release the bayonet catch on the cartridge cover by pressing down and turning the black knob counter clockwise 7 Open the cartridge cover lid 9 PYROSEQUENCING Chapter 4 Running PSQ 96 Instrument 4 51 Figure 4 12 PSQ 96 Instrument with reagent cartridge holder lid open 8 Insert the filled reagent cartridge from PSQ 96 SNP Reagent Kit into position The reagent cartridge will only fit if the label and slot is facing the u
24. 1 BE4PN SNP SNP name BE4PN SNP annotation Instrument test oligo Collection PSQ 96 SNP Sequence to analyze CTAA AGG A TGC Dispensation order manually defined Dispensation order ACGT ACGA CGTA CGTA CGT Appendix A 5 1 3 2 BE4PNG SNP SNP name BE4PNG SNP annotation Application test oligo Collection PSQ 96 SNP Sequence to analyze CT GAAA GGTG Click on Check to retrieve Dispensation order TCTG CAGTG 1 3 3 Wash SNP SNP name Wash SNP annotation Cartridge wash Collection PSQ 96 SNP Sequencing primer Sequence to analyze A CGTACGT Dispensation order manually defined Dispensation order ACGTACGT 9 PYROSEQUENCING Appendix 9 PYROSEQUENCING A 6 Index 1 Index A Adding Removing an instrument 14 Adding removing user 16 Administration Adding Removing an instrument 14 Adding removing user 16 B ckup ofdata ce EDIIE uw ARIA Task re 21 Remvoing SNP panels and methods 18 Setting preferences 19 B Backup of data setsi ai eu DR RE ES 21 C Cartridges cleaning 55 Cleaning cartridges 55 Closing down PSQ 96 SNP Software 56 D Data backups co ceu rper per ue e tS 21 Defining a new SNP method A 1 Dispensation order Generation method
25. 6 Instrument Will clsoe the PSQ 96 Instrument Control software on the operator s computer as well as all applications on the instrument embedded computer Close the instrument control window Instrument reboot Will close the PSQ 96 Instrument Control software on the operator s computer and reboot the PSQ 96 Instrument embedded computer Confirm your choice by clicking OK You will return to the Windows NT desktop 4 6 4 Closing down PSO 96 Instrument Ideally PSQ 96 Instrument should never be turned off but if the instrument is not to be used for a longer period of time it may be wise to do so Use the Close button to exit the PSQ 96 Instrument Control software Choose alternative three in the Close Option window Close the instrument control window Instrument shutdown Wait until the shutdown is ready before turning off the power switch on the front of the PSQ Instrument If desired disconnect the mains power cable from the mains power socket Chapter 4 Running PSQ 96 Instrument 4 57 4 6 5 Disposal of PSQ 96 Plates and reagent cartridges PSQ 96 Plates and reagent cartridges should be disposed of according to local regulations for plastic ware containing potentially hazardous materials Replace the plastic needle cover on the reagent cartridge to make sure that nobody gets hurt by the needles 9 PYROSEQUENCING 4 58 Chapter 4 Running PSQ 96 Instrument 9 PYROSEQUENCING Chapter 5 Evaluating results 5 59 5 Eva
26. 96 Instrument Control Software Start the computer containing PSQ 96 SNP Software and log on as normal When you reach the Windows NT desktop double click on the PSQ 96 Instrument Control icon to open the software 9 PSO 96 Instrument Control Figure 3 1 PSQ 96 Instrument Control icon on Windows desktop The PSQ 96 Instrument Control start screen then opens 9 PYROSEQUENCING 3 28 9 PYROSEQUENCING Chapter 3 PSQ 96 SNP Entry Figure 3 2 PSQ 96 Instrument Control start screen Select Modules SNP Entry The PSQ 96 SNP Entry screen appears 3 1 2 Opening via PSO 96 SNP Entry icon Start the computer containing the PSQ 96 SNP Software and log on as normal When you reach the Windows NT desktop double click on the PSQ 96 SNP Entry icon to open the software t PSQ 36 SNP Entry The PSQ 96 SNP Entry screen appears 3 1 3 Opening via Start menu From the Start menu choose Programs PSQ 96 Software PSQ 96 SNP Entry to open the software Chapter 3 PSQ 96 SNP Entry 3 2 SNP Entry screen Description of SNP Entry screen PSQ 96 SNP Entry Administration Help SNP ist BEAPN 1 Wash T SNPID BEAPNG T SNP name BE4PNG TIT SNP annotation Collection PSQ 96 SNP X Sequencing primer Sequence to analyze CT GAAAGGTG TIG Dispensation order TCTGCAGTG PCR pimer 1 PCR primer 2 Note G G T C T G c A G T G 3 29 BEES c T G c A G T G T c T G c A G T G
27. AUTION or ATTENTION is used to call attention to an instruction or condition that shall be followed to avoid damage to the product or other equipment Be sure not to proceed until the instructions are clearly understood and all stated conditions are met The Note symbol is used to indicate information that is important for trouble free or optimal use of the product 9 PYROSEQUENCING 9 PYROSEQUENCING Important User Information The following safety warning labels can be found on PSQ 96 Instrument Please read the warnings before using the instrument CAUTION ATTENTION Pinch and impact hazards N robot may move at any time Risques de pincement et d impact le robot peut bouger tout moment Figure IUI 3 Pinch impact warning WARNING For continued protection against risk of fire replace only with fuse of the specified type and current rating Always disconnect mains before service Figure IUI 4 Fuse replacement warning English AVERTISSEMENT Puor conserver la protection contre les risques d incendie effectuer le remplacement uniquement avec un fusible du type et de l intensit nominale sp cifi s Toujours d connecter le r seau avant toute mesure d entretien Figure IUI 5 Fuse replacement warning French MAINS R SEAU Connect to grounded outlet only Brancher uniquement sur une prise de terre Figure IUI 6 Grounding warning Voltage Auto ranging Frequency Powe
28. Bemave Es Figure 3 3 SNP Entry screen The left hand field shows a list of SNPs that have already been entered into the database The right hand half graphically displays theoretical sequence outputs for the three genotypes Details about the SNP selected are shown in the center of the screen Note Some of the buttons at the bottom of the screen will appear grayed until their functions can be used For instance Save cannot be clicked on until Check has been clicked on and a dispensation order has been presented 3 2 1 Menu bar Users can be added removed under Administration Administration 9 PYROSEQUENCING 3 30 t PYROSEQUENCING Chapter 3 PSQ 96 SNP Entry Add Remove User Bisi x Users Administrator User name Password D Add User Figure 3 4 Add Remove User dialog The list of current users is shown in the Users field A User can be a person or a group of individuals The User name is used for identification when logging in as well as when saving information in order to verify the origin of information and to be able to trace actions To add a new user proceed as follows 1 Enter the user s name in the User name field 2 Enter a password if desired in the Password field 3 Click on the Add User button 4 Click on the Close button to exit To remove an existing user proceed as follows 1 Click on the name of the user to be removed from the list in the Users f
29. PSQ 96 SNP Software User Manual Version 1 1 E PYROSEQUENCING Code number 60 0016 Article no 50 0016 AB Copyright 2000 Pyrosequencing AB All rights reserved No part 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 Pyrosequencing AB Vallongatan 1 SE 752 28 Uppsala Sweden Tel Int 46 18 56 59 00 Fax Int 46 18 59 19 22 e mail info pyrosequencing com Website www pyrosequencing com Table of Contents Contents lUI Important User Information 1 IUI 1 Important note sssvennnnnnnnnvnnnnnnnnvnnnnnnnnnvennnnnnnvnnnnnnnnnnr 1 IUI 2 Safety symbols ns 1 IUI 3 Declaration of Conformity eeeesss 3 IUI 4 External equipment for connection to PSQ 96 Instrument mes 3 IUI 5 Warranty and Liability eee 4 IUI 6 Trademarks and Patents 4 1 Introduction 3 rise ent ecu cen EEN 7 1 1 Background eene 7 1 2 Product family iere 7 UE NEO e LE 8 2 System Installation and Administration 9 2 1 System requirements ses 9 2 2 Installation and set up nnmnnn 11 2 3 Backup of data nine 21 2 4 Installation testing
30. a are entered as follows Tip move between data entry fields either by clicking in the field with the mouse pointer or by pressing the lt Tab gt key on the keyboard 1 The User name field is automatically filled in with the user name that you logged on with The user name cannot be changed except by logging off and logging on again with a new user name 2 Enter Run ID mandatory field for data to be stored The data will be stored in the default directory specified under Instrument System Administration Preferences 3 Enter the Kit ID mandatory field for the PSQ 96 SNP Reagent Kit that you are using for the analysis 4 Enter the Plate ID mandatory field for the PSQ 96 Plate that you have just put into PSQ 96 Instrument The Fetch button is used to retrieve information stored about the plate into the Sample Notes fields The information to be retrieved first has to be entered into the INWELLINFO table in the current database using Microsoft Access It is advisable to make a back up copy of the database before making any changes In the INWELLINFO table specify the PLATEID has to be the same as entered in the Plate ID field WELLNAME A1 H12 and WELLNOTE max 40 characters Note Unless you are very familiar with Microsoft Access do not attempt to make changes in the database 5 Enter any further information free text max 90 characters regarding the run under Notes 6 Define the wells to be used in the PSQ
31. age will be shown if the SNP is entered after base position 5 6 When the user has entered the SNP at a distance of five or more bases from the primer the additions following the SNP dispensation will be a negative control plus two non polymorphic bases regardless of whether there are more bases entered as sequence to analyze An entered sequence which ends with the SNP position will generate a warning 7 Ifthe user tries to enter two SNP positions which is not possible a warning will be generated 3 6 Guidelines for editing a dispensation order 9 PYROSEQUENCING PSQ 96 SNP Entry automatically determines the optimum dispensation order for each individual SNP to be analyzed When entering a new SNP PSQ 96 SNP Entry suggests a dispensation order based on a number of simple rules see section 3 5 How a dispensation order is generateds However under certain conditions it may be desirable or necessary to use a dispensation order that deviates from that suggested The descriptions below state how under certain conditions one can achieve more informative data by manually changing the suggested dispensation order Chapter 3 PSQ 96 SNP Entry 3 37 3 6 1 Negative controls Two negative control dispensations are entered as default These can be removed changed or extended as desired If the sequence will generate a four fold peak or higher it can be useful to add this base once more to ensure that all templates have been full
32. aks that are too wide may result in an uncertain analysis High pre sequencing signal If a very large peak appears at substrate dispensation the well is marked as Check since nucleotide contamination in the sample may have resulted in uncontrolled incorporation of nucleotides Contamination by nucleotides and or pyrophosphate might also result in consumption of the substrates SNP Entry warning s When the SNP was entered in PSQ 96 SNP Entry at least one of the following warnings appeared SNP entered at gt 5 bases from primer If the SNP was entered after the fifth base Pleas nter some bases after the SNP position for control Addition of some bases after the SNP will improve data evaluation Entered sequence contains a homopolymer of value If there are more than five G C T or three A in a row You have homopolymeric stretches on both sides of the SNP If possible redesigning your sequencing primer may improve genotype assessment Well Result By clicking on the Well Result tab the well results are displayed in a different format 9 PYROSEQUENCING 5 70 9 PYROSEQUENCING Chapter 5 Evaluating results Well Overview Figure 5 12 Results displayed under Well results tab 5 3 7 Displaying individual well results To view the pyrogram and theoretical outcomes for an individual well double click on the well 3 2 G G
33. ates the Lid Status 6 1 4 Temperatures Displays the Block Chamber and Environment temperatures and a means of testing the Block and Chamber temperatures When a Test button is clicked on the temperature will change by approximately 2 C message window will show the result of the test 6 1 5 X Y Motors Mixer X Y Settings It is advisable to move the X Y table to the Home position before starting this test Home Click on the Home button The X Y table should move to its home position The status for the motors will be shown in the Status field End position Click on the End Position button The X Y table should move to the end position The status for the motors will be shown in the Status field Parking Position Click on the Parking Position button The X Y table should move to its parking position The status for the motors will be shown in the Status field Cycles This function is used to test the movement of the X Y table without dispensations being carried out To start the function click first on the Home button and then enter the number of cycles for the test in the Total number of cycles field Click on the Apply button to start The number of cycles performed is shown in the Cycles done field Mixer To test the mixer click on the Test button The mixer will start and come up to speed for 10 seconds The speed shown in the Mixer speed Hz field should be 30 Hz When the mixer has come up to speed a mes
34. ation 45 Selecting an application 44 IEN E EE ERE nux 41 Starting NEEN 51 Status bar gassens net ren E RR RUE rc e ee 42 PSQ 96 SNP Entry Guidelines for entering data 34 IUPAGstandard i362 ew ex eR eek eR aS ex 32 Opening uses P OE REI RU Ae 27 28 Removing SNP data 34 Pyrosequencing AB contacting the company 5 R Removing SNE data coke su E gi hens eins Payee VR 34 Removing SNP panels and methods 18 Running PSQ 96 Instrument Indicator lights 40 Starting PSQ 96 Instrument Control 40 Starting the system 39 S Safety symbols 3 5 sanger ee EE HE ne 1 Setting up PSQ 96 Instrument Control Process Identification information 45 Process Parameter information 48 SNP Entry Warning m ssages 5 28 4 A IA hr ras RS 38 SNP entry ass bee eR Xe RES SUPR 31 33 SNP Entry screen 29 SNP Entry screen menu bar 29 Stand alone installation 11 Standard methods A 4 Standard SNPS 224 raste vg emo V UR ER A 4 Starting run icc Gre rikke eee aus ekke Fre TERR e eee s 51 Starting evaluation module 59 9
35. ature should normally be set to about 10 C lower than the Block temperature Nucleotide pressure Permitted range 300 1000 mBar The Nucleotide pressure is the pressure of the air pulse used for dispensing Too low a pressure can cause faulty dispensations too high a pressure can cause splashing from the well The lower the pressure the longer the pressure pulse that is necessary to dispense the same volume Nucleotide pulse time Permitted range 4 600 ms The nucleotide pulse time along with the pressure inner diameter of the tubing in the instrument and the viscosity of the fluid determines the volume dispensed into each well Appendix Cycle time Permitted range 60 600 sec The Cycle time defines the shortest time between two dispensations into the same well For a full plate 96 wells the shortest Cycle time is about 65 seconds Mixer frequency Permitted range 0 off or 20 50 Hz This value controls the oscillation frequency of the PSQ 96 Plate The frequency required depends on the shape of the wells and the viscosity of the fluid Too high a frequency with too large a sample volume may result in fluid splashing out of the well Reagent pulse time 1 2 two fields Permitted range 0 600 ms Together with the pressure inner diameter of the tubing in the instrument and the viscosity of the fluid the duration of the Reagent pulse determines the volume that is dispensed into each well Reagent pressure Permitte
36. ch Chapter 3 PSQ 96 SNP Entry 3 35 3 5 How a dispensation order is generated Based on the entered sequence in the Sequence to analyze field PSQ 96 SNP Entry suggests a nucleotide dispensation order based on the rules listed below 3 5 1 Primary rules The primary rules are the basis for a suggested dispensation order when the user has entered sequence information in accordance with the given recommendations The polymorphic position SNP is within the first five bases 3 of the sequencing primer and enough sequence information is available to generate at least five non polymorphic reference peaks The dispensation order follows the sequence considering the following 1 Inaddition to the dispensations for the polymorphic position PSQ 96 SNP Software will suggest five additional dispensations for confirmation of sequence and sample quality plus two dispensations for negative control This yields a final number of at least nine nucleotide dispensations for every sample 2 Ifasequencing primer has been entered the last base of the primer will be suggested as the first in the dispensation order as a negative control If this base is the same as the base in the first position in the sequence to be analyzed then C is suggested according to the following priority C gt T gt G In cases where the polymorphic position is located directly 3 of the sequencing primer none of the polymorphic bases are suggested as a negative control
37. change from being inactive gray to being active black 4 5 5 Exporting data The Export Data function is used to copy a run file to a new location To export data select Instrument Export Data The dialog will open 9 PYROSEQUENCING 4 54 9 PYROSEQUENCING Chapter 4 Running PSO 96 Instrument Export Data x r Search Profile Run ID o Instrument name E User Lo Method F x Kit ID PaelD Find RunlD User ku Plate ID Instrument name Method Figure 4 15 Export Data dialog It is necessary to specify search criteria to identify run s to be exported To select a run use one or several of the following search criteria If no run parameters are entered and fields only contain asterisks all runs will be listed when the Find button is clicked on The asterisk can also be used as a wild card in combination with any other character 1 Enter the Run ID Enter the identification for the run 2 Enter the User name Enter the name of the user 3 Enter the Kit ID Enter the identification for the kit 4 Enter the Plate ID Enter the identification for the plate 5 Select the Instrument name Click on the down arrow to the right of the field and select the instrument name from the list when multiple instruments are connected 6 Select the Method Click on the down arrow to the right of the field and select the method from the list 7 Click on the Find button C
38. characters otherwise leave the field empty Position the cursor in the PCR primer 2 field If desired enter the PCR primer 2 sequence max 40 characters otherwise leave the field empty Position the cursor in the Note field If desired enter a free text note about the SNP max 80 characters otherwise leave the field empty Click on the Check button A nucleotide dispensation order is then suggested by PSQ 96 SNP Entry in the Dispensation order field see below Simultaneously the related theoretical graphic output is shown If desired the dispensation order can be manually modified Change the order of the nucleotides to be dispensed as desired max 60 characters see section 3 6 Guidelines for editing a dispensation order for assistance If data have been incorrectly entered into any of the fields a warning dialog will open clarifying the problem Correct the fault and click on the Check button again Chapter 3 PSQ 96 SNP Entry 3 33 11 If the Dispensation order field already contains data when clicking on the Check button a dialog opens asking whether or not you want to change the dispensation order If you answer Yes the software will suggest a new dispensation order 12 If you do not want to save the SNP data entered click on the New button to clear the entry 13 Click on the Save button to store the SNP information The SNP ID will then appear in the SNP list on the left hand side of the screen If enter
39. cing AB PSQ 96 is a trademark owned by Pyrosequencing AB The methodology of Pyrosequencing is covered by patents and patent applications owned by Pyrosequencing AB In view of the risk of trademark degeneration authors intending to use the trademarked designations are respectfully requested to acknowledge the trademark status of the products at least once in each article Important User Information L IUI 6 1 Other trademarks Pyrosequencing AB acknowledges the following trademarks that have been mentioned in the text Microsoft Excel Microsoft Access and Windows NT are either registered trademarks or trademarks of Microsoft Corporation in the United States and or other countries 3Com is a trademark of 3Com Corporation Intel and Pentium are registered trademarks of Intel Corporation Copyright 2000 Pyrosequencing AB All rights reserved No part 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 Pyrosequencing AB Vallongatan 1 SE 752 28 Uppsala Sweden Tel Int 46 18 56 59 00 Fax Int 46 18 59 19 22 e mail info pyrosequencing com Website www pyrosequencing com CG PYROSEQUENCING L Important User Information 9 PYROSEQUENCING Chapter 1 Introduction 1 7 1 Introduction 1 1 Background The methodology used in Pyrosequencing was developed by researchers at th
40. d range 300 1000 mBar The Reagent pressure is the pressure of the air pulse used for dispensing Too low a pressure can cause faulty dispensations too high a pressure can cause foaming in the wells as well as splashing from the wells The lower the pressure the longer the pressure pulse that is necessary to dispense the same volume Priming time Permitted range 0 600 ms To prime the needles prior to dispensation normally 100 ms 1 13 Saving a method Once all the required parameters for a method have been entered enter a name for the method in the Method field enter any notes regarding the method in the Method Note field max 60 characters and click on the Save Method button 1 1 4 Exiting from Method Advanced Click on the Hide Advanced button to hide the method definition fields 9 PYROSEQUENCING Appendix 1 2 Standard methods 1 2 1 SNP Method Block temp 28 C Chamber temp 17 C Nucleotide pressure 650 mBar Nucleotide pulse time 9 ms Cycle time 65 Mixer frequency 35 Hz Reagent pulse 1 130 ms Reagent pulse 2 116 ms Reagent pressure 400 mBar Priming time 100 ms 1 22 Wash Method Block temp 28 C Peltier element temp 20 C Nucleotide pressure 700 mBar Nucleotide pulse time 100 ms Cycle time 65 Mixer frequency 0 Hz Reagent pulse 1 100 ms Reagent pulse 2 100 ms Reagent pressure 700 mBar Priming time 100 ms Note Choose all wells in the PSQ 96 Plate 1 3 Standard SNPs 9 PYROSEQUENCING 1 3
41. e light on PSQ 96 Instrument will go out CG PYROSEQUENCING 2 26 Chapter 2 System Installation and Administration 11 Click on the Stop A window will be opened asking the button user to confirm the termination of the run When confirmed the run will be stopped immediately and data will be saved Idle will then appear on the status bar 9 PYROSEQUENCING Chapter 3 PSQ 96 SNP Entry 3 27 3 PSQ 96 SNP Entry PSQ 96 SNP Entry is used to enter edit and organize sequences to be analyzed The software uses this sequence information to determine the nucleotide dispensation order and graphically displays the theoretical sequencing outcomes The bar graphs representing the results for different genotypes are later used in the PSQ 96 Evaluation module when analyzing data obtained using the PSQ 96 System powerful feature of the software is that any change in the dispensation order is accompanied by a real time update of the theoretical graphic display making it flexible and easy to customize the dispensation order used for analysis of specific SNPs Note There is no other way to enter SNP data Note In this manual SNP sometimes denotes the collective information entered in the SNP Entry dialog for a certain single nucleotide polymorphism to be analyzed rather than the specific polymorphic position 3 1 Opening SNP Entry There are three ways of opening this module 3 1 1 Opening via PSQ
42. e Royal Institute of Technology in Stockholm Sweden in 1996 Since then the methodology has been continuously refined by Pyrosequencing AB in Uppsala Sweden Pyrosequencing AB was founded in 1997 1 2 Product family Pyrosequencing AB has developed a family of products designed for rapid and accurate Pyrosequencing The PSQ 96 product family consists of e PSQ 96 System instrument operator s computer monitor and printer e PSQ 96 Instrument e PSQ 96 SNP Software PSQ 96 SNP Reagent Kit e PSQ 96 Plate special microtiter plate designed for use with PSQ 96 Instrument e PSQ 96 Sample Prep Workstation including PSQ 96 Sample Prep Tool PSQ 96 Sample Prep Thermoplate PSQ 96 Sample Prep Rack e PSQ 96 Sample Prep Tool Cover To perform a Pyrosequencing analysis PSQ 96 Instrument is required and must be connected to an external operator s computer containing PSQ 96 SNP Software In the assay PSQ 96 SNP Reagent Kit and a PSQ 96 Plate must be used PSQ 96 Sample Prep Workstation and its constituent parts is an accessory that simplifies preparation of the samples 9 PYROSEQUENCING Chapter 1 Introduction Figure 1 1 PSQ 96 System New products are being continually developed Please refer to Pyrosequencing s website for further details www pyrosequencing com 1 3 Assumptions t PYROSEQUENCING 1 3 1 Pyrosequencing analysis Use of PSQ 96 Instrument requires special preparation of sample
43. e instrument to be removed from the list in the Instruments used field 2 Click on the the Remove Instrument button 3 Confirm the removal in the window that will appear 4 Click on the Close button to exit 2 2 7 Changing IP addresses 1 In PSQ 96 Instrument Control select Instrument System Administration and select the Instruments tab CG PYROSEQUENCING 2 16 9 PYROSEQUENCING Chapter 2 System Installation and Administration 2 Select the instrument in the Instruments used field If the instrument is not listed please refer to 2 2 6 Adding removing an instrument Click on the Change IP address button Enter a new IP address for the instrument in the dialog that will open Then click OK To exit click on the Close button 6 Check the contact between the instrument and the operator s computer Note Ifthe PSQ 96 System is connected to a LAN contact the network administrator regarding changes of IP addresses If any of the IP addresses are incorrectly specified it will be impossible to use the corresponding PSQ 96 Instrument until the error is rectified Incorrect IP addresses might also cause communication conflicts If multiple PSQ 96 Instruments are connected to one operator s computer the IP addresses of the different instruments will need to be changed see 2 2 5 Setting up multiple PSQ 96 Instruments to one operator s computer 2 2 8 Adding removing a user User can be a person
44. ed data need to be corrected or confirmed a dialog will open 14 If you want to enter additional SNPs click on the New button 15 Click on Exit to close PSQ 96 SNP Entry 3 2 3 Editing SNP information 1 Select the SNP to be edited by highlighting it in the SNP list 2 Edit the data as desired 3 In order to save the edited SNP you first need to click on the Check button If you have made alterations to the dispensation order select No when asked if you want to create a new dispensation order 4 Click on Save to store the edited information The SNP will get a new unique SNP ID 3 2 4 Creating a new Collection 1 Enter SNP information for a new SNP or choose an SNP from the SNP list 2 Enter a name for the new collection in the Collection field 3 In order to save the SNP in the new Collection you first need to click on the Check button If you have made alterations to the dispensation order select No when asked if you want to create a new dispensation order 4 Click on Save to store the information CG PYROSEQUENCING 3 34 Chapter 3 PSQ 96 SNP Entry 3 3 Removing SNP data To remove a previously stored SNP proceed as follows 1 Scroll through the SNP list on the left hand side of the screen and highlight the SNP to be removed Click on the Remove button A dialog will open asking you to confirm removal of the SNP from the database Click on Yes to remove or No to return to the screen without
45. ed in this field determines how many dispensations apart from the SNP position that are to be used in the quality measurement The default value is 7 The window of the quality measurement is positioned as evenly as possible around the SNP position e g in the case of 7 this means 4 before the SNP and 3 after The largest number always comes before the SNP If the SNP is positioned first in the sequence to analyze the following seven positions will be used as controls if available 3 Consider SNP Entry warnings If this box is checked warnings generated and overruled when entering data about the SNP in SNP Entry will affect the quality assignment and the user will be prompted to check the results 4 Show results from failed analyses If this box is checked genotyping results from failed analyses will also be displayed Otherwise failed genotype results will not be displayed Chapter 5 Evaluating results 5 67 Set as Default The criteria selected in the whole dialog can be set as default criteria for future runs by clicking on this button You will be asked to confirm the choice Revert to Default Resets any changed values in the dialog to the last chosen default values OK Saves the chosen criteria parameters for the current analysis and returns to the Analyze SNP dialog Cancel Aborts any changes and returns to the Analyze SNP dialog Note When a new run is selected for analysis the Criteria are reverted to the defaul
46. emp Block temp Environmental temp Pressure Mixer speed Click on the arrow at the right of the combination box and select the parameter of choice from the list Auto time interval if this box is checked the graphical display follows the actual time If it is not checked the desired Time range should be entered see below Time range either enter the time range desired in seconds or click on the arrow at the right of the combination box and select the time from the list MaxY MinY used to set a specific range for the Y axis If you enter a value and wish to return to the default auto press the button Set Auto Scale or enter the word auto in field Well Overview area Displays a graphical representation of the wells indicating what is happening in each well Click on a well to display real time data information Process Parameters tab This field area shows the status of the process parameters The current value is shown in the box below each parameter If the block is red the parameter is outside the acceptance level for the preset value If it is white with black text it has reached the preset value The Time box shows the elapsed time since the Run button was pressed Events tab Shows run specific information for each well When the run has finished a dialog will open stating that run information is being saved and the word Idle will appear in the status bar at the top of the screen The New Run button will
47. es comes completely out of phase This phenomenon may also occur as a result of a cyclical dispensation order The advantage is that a large number of peaks are obtained containing information about the polymorphic position The disadvantage is that the sequence following the polymorphic position cannot be confirmed e g AGTGAGCAGCAT for the analyzed AGTG CAGGGGCAT Note PSQ 96 Evaluation software algorithm is optimized to work with the dispensation orders suggested by the SNP Entry software Therefore out of phase analysis should always be used with care and be accompanied by a higher degree of visual inspection of the data displayed CG PYROSEQUENCING 3 38 Chapter 3 PSQ 96 SNP Entry 3 7 Warning messages 9 PYROSEQUENCING When entering data in the Sequence to analyze field that are deviating from the recommendations given in section 3 4 one or several warning messages will be displayed informing the user about this The user can choose whether or not to follow the recommendations If the run has been performed with an SNP that generates any of the messages below the following warning message will be displayed for relevant wells after analysis of the run data in PSQ 96 Evaluation SNP Entry warning s This will also result in an uncertain quality assignment yellow for the relevant wells if the criterion Consider SNP Entry warnings is selected see section 5 3 4 Criteria and see section 5 4 Algorithm for a
48. ft Access t PYROSEQUENCING Chapter 2 System Installation and Administration 2 21 Driver mdb is the selection of choice Click on Next 6 Click on Finish Enter a Data Source Name of your choice and click on Select 8 Select the path to the database according to the instructions above for the Result directory 9 Select the database in the left hand field The database name will now appear in the Database Name field Click on OK 10 Thereafter click on the Options button and check that the Page Timeout is set to 5000 Note This Page Timeout value is only valid for the Microsoft Access ODBC driver MDAC 2 1 or later shipped with ver 1 1 of the PSQ 96 SNP Software For earlier versions of the ODBC driver MDAC 2 0 this value should be set to 5 since these versions of the driver interpret the Page Timeout in seconds rather than in milliseconds 11 Click on the OK button to exit Note Under Windows NT the location of result and database directories can only be changed if you have Administrator s rights 2 3 Backup of data Good data management requires that data backups are made on a regular basis In the case of data produced in a stand alone system the result files and database should be backed up at regular intervals If the system is connected to a LAN ask your LAN administrator for assistance regarding backups A ZIP drive or similar can be used to make backups in a stand alone system The direct
49. hand side of the status bar is a process progress bar This indicates the time that the process has been running as a graphical scale that shows with advancing bars the progression of the process The time displayed to the left of the process progress bar indicates the time remaining for the run 4 4 Selecting an application t PYROSEQUENCING From the start screen click on the Application button The dialog will open Select Application x User Administrator h Password System Information Figure 4 7 Select application dialog To be able to select an application you must first enter your user name and password if applicable in the User and Password fields The user name can be selected from the drop down list Select the application desired by clicking on the appropriate button The SNP Analysis application is described below The System Information application is described under Troubleshooting see section 6 1 Obtaining system information Chapter 4 Running PSQ 96 Instrument 4 45 4 5 Performing an SNP analysis The first stage in an SNP analysis is to set up the PSQ 96 Instrument Control software with sample and SNP information Then the reagent cartridge and the sample plate are loaded in the instrument and the run started Note Make sure that the PSQ 96 Instrument has been switched on for at least 90 minutes before the start of the run 4 5 1 Setting up PSO 96 Instrument Control
50. hapter 4 Running PSQ 96 Instrument 4 55 4 6 After a run 8 A list of runs stored in the defined database which match the specifications will then appear in the lower half of the dialog Click on a Run ID from this list and click on the Export button to export the run file A standard Windows Save As dialog will open Save the run in the desired directory Note Only one run can be exported at a time 9 Click on Close to exit 4 6 1 Removing PSQ 96 Plate and cartridge After a run is finished it is necessary to remove the PSQ 96 Plate and reagent cartridge from the instrument If another run is to be performed reload the instrument and continue as in section 4 5 Performing an SNP analysis Make sure that there are no spillages in the instrument Refer to the chapter on Maintenance in the PSQ 96 Instrument Reference Manual 4 6 2 Cleaning cartridges If the cartridge is to be reused it has to be cleaned directly after use We recommend that the cartridge is used no more than 5 6 times 1 Discard remaining solutions 2 Rinse the cartridge twice with water 3 Fill the compartments almost to the top with high purity water and check for obstruction by pressing on top of the compartment with a finger Ensure that the needles are clear a droplet should be visible at the tip of the needle If there is a blockage wet the needle tip with a moist lint free tissue and test again 4 Discard the water 5 Half fill with h
51. id erae ind ii ava suus Sun qe are eranl A 1 1 1 Defining Methods erre A 1 1 2 Standard methods esses A 4 1 3 Standard SNPS esee A 4 Table of Contents 2 Important User Information l 1 IUI Important User Information IUI 1 Important note The PSQ 96 System and all associated products from Pyrosequencing AB are for research use only and are not for use in clinical procedures This manual is an integral part of PSQ 96 Instrument The instructions contained in the manual regarding system operation and test set up are to be strictly observed Pyrosequencing AB and its representatives are not responsible for damage to persons animals property and equipment by non observance of the safety rules and precautions in the manual Pyrosequencing AB reserves the right to make changes in the information contained herein without prior notice IUI 2 Safety symbols A WARNING AVERTISSEMENT Figure IUI 1 Warning symbol and text The warning triangle together with the text WARNING or AVERTISSEMENT is used to call attention to the necessity to follow an instruction in detail in order to avoid personal injury Be sure not to proceed until the instructions are clearly understood and all stated conditions are met N CAUTION ATTENTION Figure IUI 2 Caution symbol and text The warning triangle together with the text C
52. ield The name will appear in the User name field Click on the Remove User button 3 A warning dialog will appear asking you to confirm removal of the user 4 Click on the Close button to exit Note Ifa user should forget their password the user will have to be removed and re registered Chapter 3 PSQ 96 SNP Entry 3 31 3 22 Entering SNP Information To enter a new SNP proceed as follows 1 Click on the New button Alternatively if the SNP information that you wish to enter is very similar to one already entered highlight the SNP in the SNP list The data on this SNP will appear in all the fields and can then be modified Position the cursor in the SNP name field Enter a name for the SNP max 10 characters Filling in data in this field is mandatory The SNP will automatically be assigned an SNP ID when the Check button is clicked on see below The SNP ID will appear in the SNP list to the left and consists of the specified SNP name together with an ID number which makes each SNP ID unique The SNP ID cannot be changed Note SNPs with identical names will be assigned different ID numbers Position the cursor in the SNP annotation field Enter a description of max 40 characters Filling in data in this field is optional Choose a Collection for the SNP or create a new Collection by entering a new Collection name if desired max 20 characters SNPs can be grouped together in collections making them eas
53. ier to locate and limiting the number of SNPs to review e g when setting up a run Each SNP must belong to a collection Position the cursor in the Sequencing primer field Enter the full primer sequence max 40 characters Filling in data in this field is optional Position the cursor in the Sequence to analyze field Enter the full SNP sequence i e the bases that will be incorporated by extension of the 3 end of the sequencing primer max 32 characters Filling in data in this field is mandatory Polymorphic positions are marked with a slash e g A C or are filled in using the standard IUPAC nomenclature as shown below For more specific information see section 3 4 Guidelines for entering data in the Sequence to analyze field Note Ifrunning an SNP with an unknown sequence to analyze use a cyclic dispensation order e g A C G T A C The software does not support automatic genotyping for unknown sequences which is why genotype assignment has to be performed manually by visual inspection in this case CG PYROSEQUENCING 3 32 9 PYROSEQUENCING Chapter 3 PSO 96 SNP Entry Code to be ner entered nucleotide s G G A T T C C R G A M A C S G C Y T C K G T Ww A T Figure 3 5 IUPAC standard for single letter codes for nucleotides Te 10 Position the cursor in the PCR primer 1 field If desired enter the PCR primer 1 sequence max 40
54. igh purity water the compartments fitted with needles 6 Place the cartridge in the PSQ 96 Instrument together with a used PSQ 96 plate 7 Run a full plate with the pre defined SNP called Wash using the Wash method see Appendix 1 3 3 Wash SNP and 1 2 2 Wash Method 9 PYROSEQUENCING 4 56 9 PYROSEQUENCING Chapter 4 Running PSO 96 Instrument 8 Discard remaining water and leave to dry If the cartridge is to be left in PSQ 96 Instrument overnight it should be cleaned as described in the chapter on Maintenance in the PSQ 96 Instrument Reference Manual 4 6 3 Closing down PSO 96 Instrument Control Select Instrument Exit and Log Off warning dialog will open asking you to confirm log off You will return to the Windows NT desktop Alternatively use the Close button The Close Option window will open displaying four different alternatives Close the instrument control window This alternative is selected as default and should be used when exiting the PSQ 96 Instrument Control software application e Close the instrument control window Exit the instrument control application Is used only for instrument service related actions Will close the PSQ 96 Instrument Control software on the operator s computer as well as the instrument applications on the PSQ 96 Instrument embedded computer Close the instrument control window Instrument shutdown Use this alternative when you want to shut down the PSW 9
55. ine noise level Light leaking in to the PSQ 96 Plate Contaminated nucleotides Background sequence of contami nating sequence from PCR Contact your local Pyrose quencing engineer if problem persists High back Light leaking in to the PSQ 96 ground Instrument No contact Loose network cables between the oper ator s computer and PSQ 96 Instrument even though the net work cables are correctly plugged in and PSQ 96 Instrument is switched on Power light is illuminated No match between IP numbers for Instrument and Operator PC IP number conflicts with other equipment on the network The performance of the PSQ 96 SNP Software appears to be slow Wrong Page Timeout setting for the ODBC data source Microsoft Access ODBC driver MDAC 2 1 Change the Page Timeout set ting to 5000 according to sec tion 2 2 10 Setting preferences Note Only valid for Microsoft Access ODBC driver MDAC 2 1 installed by PSQ 96 SNP Software ver 1 1 All lights on PSQ 96 Instrument remain illumi nated 5 minutes after it has been switched on Instrument software problem Contact your local Pyrose quencing service engineer 9 PYROSEQUENCING Chapter 6 Troubleshooting 6 83 Heating block temperature con trol is not work ing Heating block incorrectly inserted Fluid between the heating block contact pins and the instrument
56. is way data can be backed up frequently and will be accessible for analysis on other computers on the LAN running PSQ 96 SNP Software The LAN administrator should be contacted regarding the network set up and back up If desired copy the PSQ 96 database from the operator s computer C Program Files Pyrosequencing PSQ 96 Software Db PSQ96db mdb to the desired directory on a network server In order to store the result files dat on the LAN create a results directory on the desired network server Set the the preferences for both the results directory and the database according to 2 2 10 Setting preferences If a separate computer is used to analyze data this may need to be connected to the LAN in order to retrieve results The LAN administrator should be contacted regarding the set up of this network connection Note In order for the operator s computer to communicate with the instrument the following settings are necessary In the Properties of Network Neighborhood under the Bindings tab choose to show bindings for all protocols Expand the TCP IP Protocol and make sure that the network card that communicates with the instrument is at the top of the list This is accomplished using the Move Up button Chapter 2 System Installation and Administration 2 11 2 2 Installation and set up 2 2 1 Items required but not provided Computer for Office installation of the software not provided by Pyrosequencing AB see section 2
57. istration 2 29 Removing SNP Panels and Methods In PSQ 96 Instrument Control select Instrument System Administration Note This dialog can only be accessed if there is no instrument window currently open Click on the Database tab System Administration x Users Instruments D e Preferences SNP panels S Remove SNP panel X Remove SNP method SNP methods Figure 2 5 System Administration Database dialog To remove an SNP panel proceed as follows 1 Q Select the SNP panel to be removed from the SNP Panel combination box by clicking on the arrow at the right hand end of the box scrolling through the list and clicking on the SNP panel of choice Click on the Remove SNP Panel button warning dialog will appear asking you to confirm removal of the SNP panel To remove an SNP Method proceed as follows 1 Select the SNP Method to be removed from the SNP Method combination box by clicking on the arrow at the right hand end of the box scrolling through the list and clicking on the SNP Method of choice Click on the Remove SNP Method button Chapter 2 System Installation and Administration 2 19 3 A warning dialog will appear asking you to confirm removal of the method 4 Click on the Close button to exit 2 2 10 Setting preferences In PSQ 96 Instrument Control select Instrument System Administration Note This dialog can only be accessed if there is no instrument windo
58. lated 9 The average value of all peak widths within the quality control window is calculated The quality assessment is based on the quality calculations stated 9 PYROSEQUENCING Chapter 5 Evaluating results 5 75 above A well can be displayed yellow check if The assessment of the genotype is somewhat uncertain depending on the chosen stringency criteria low medium or high The agreement with the theoretical sequence is somewhat poor The presequencing signal at substrate addition is unusually high The signal to noise ratio is somewhat low The peaks are unusually wide The Sequence to analyze has not been entered according to recommendations can be deselected in the Criteria dialog under SNP warnings A well can be displayed red failed if The assessment of the genotype is very uncertain The agreement with the theoretical sequence is very poor The signal to noise ratio is very low 9 PYROSEQUENCING 5 76 Chapter 5 Evaluating results 9 PYROSEQUENCING Chapter 6 Troubleshooting 6 77 6 Troubleshooting 6 1 Obtaining system information Before proceeding with troubleshooting and before contacting Pyrosequencing AB you can perform a series of checks to assess if the system is working properly The System Information function enables you to test individual system functions and pinpoint possible sources of error From the PSQ 96 Instrument Control main screen click on the Applica
59. ll appear asking you to confirm removal of the user 4 Click on the Close button to exit 5 2 2 Help Provides help for the Evaluation module 9 PYROSEQUENCING 5 62 t PYROSEQUENCING Chapter 5 Evaluating results 5 2 3 Set category button The Set Category function is used to organize the run files Each run file may belong to multiple categories In order to set a category for a certain run file mark the Run ID in the Run Information window under the Selection tab and click on the Set Category button Alternatively open the run file see 5 3 1 and with the Analyze SNP tab selected click on the Set Category button Set Category lolx Categories New category Add New Category v Figure 5 6 Set categories The existing categories are shown on the left hand side Click in the box for the category under which to store the results or enter a new category in the New category field and then click on the Add new category button 5 2 4 Export button The Export function is used to copy a run file to a new location or to generate and export a report including information associated with the run and or analysis results In order to export a certain run file mark the Run ID in the Run Information window under the Selection tab and click on the Export button Alternatively open the run file see section 5 3 1 Selecting a run and with the Analyze SNP tab selected click on the Export button In the Expo
60. luating results The PSQ 96 Evaluation module automatically evaluates data obtained from a run and presents genotypes as well as quality assignments for all samples analyzed Criteria for the analysis can be set to different stringency levels by the user 5 1 Opening the evaluation module There are three ways of opening this module 5 1 1 Opening via PSQ 96 Instrument Control Software Start the computer containing PSQ 96 SNP Software and log on as normal When you reach the Windows NT desktop double click on the PSQ 96 Instrument Control Software icon to open the software b PSG 36 Instrument Control Figure 5 1 PSQ 96 Instrument Control icon on Windows desktop The PSQ 96 Instrument Control Software start screen then opens Figure 5 2 PSQ 96 Instrument Control software start screen Select Modules Evaluation The PSQ 96 Evaluation screen appears 9 PYROSEQUENCING 5 60 Chapter 5 Evaluating results 5 1 2 Opening via PSQ 96 Evaluation Start the computer containing PSQ 96 SNP Software and log on as normal When you reach the Windows NT desktop double click on the PSQ 96 Evaluation icon to open the software b PSQ 96 Evaluation Figure 5 3 PSQ 96 Evaluation icon on Window desktop 5 1 3 Opening via Start menu From the Start menu choose Programs PSQ 96 Software PSQ 96 Evaluation to open the software 5 2 Menu alternatives and buttons 9 PYROSEQUENCING PSQ 96 Evaluation BEES Ad
61. ministration Help Selection Search Profile Rnb 5 JD Wewennme 4 Ue Method y RBE Plate D Category r Run Information Plate ID Instrument name Method Delete Run SetCategowy Export Import Ext Figure 5 4 PSQ 96 Evaluation start screen The menu bar contains two choices Administration and Help 5 2 1 Administration The menu selection Administration Administration opens the dialog Chapter 5 Evaluating results 5 61 Add Remove User Bisi E Users User name Password Administrator Add User Figure 5 5 Users The Users tab shows a list of the registered users A User can be a person or a group of individuals The User name is used for identification when logging in as well as when saving information in order to verify the origin of information and to be able to trace actions To add a new User proceed as follows 1 Enter the user s name in the User name field 2 Enter a password if desired in the Password field 3 Click on the Add User button The new user name will appear in the list of registered users 4 Click on the Close button to exit To remove an existing user proceed as follows 1 Click on the name of the user to be removed from the list in the Users field The name will appear in the User name field Click on the Remove User button b 3 Awarning dialog wi
62. n and follow the instructions that appear on screen Please refer to PSQ 96 SNP Software Office User Manual for further details 2 25 Setting up multiple PSO 96 Instruments to one operator s computer Up to four PSQ 96 Instruments can be connected to one operator s computer To set up the system in this configuration each instrument must have a unique IP address irrespective of whether or not the system is set up in a stand alone or LAN configuration IP addresses should be changed only if you plan to have more than one instrument connected to an operator s computer or to a LAN To set up multiple PSQ 96 Instruments to a single operator s computer either in a stand alone configuration or to a LAN Pyrosequencing AB recommends the set up described in the PSQ 96 Instrument Reference manual LAN installation with several instruments and office computers If you plan to connect the PSQ 96 system to a LAN contact your LAN administrator regarding installation and for set up of the second Ethernet network adapter The host IP address 192 168 255 200 operator s PC does not need to be changed if a separate Ethernet network adapter is used referred to as the second Ethernet network adapter for connection of the operator s computer to the LAN For example Instrument 1 IP address 192 168 255 201 Instrument 2 IP address 192 168 255 202 Instrument 3 IP address 192 168 255 203 Instrument 4 IP address 192 168 255 204 Si
63. nalysis The messages prompting the above message in PSQ 96 SNP Entry are SNP entered gt 5 bases from primer if the SNP was entered after the fifth base Pleas nter some bases after the SNP position for control Entered sequence contains a homopolymer of lt value gt if there are more than five G C or T or three A in a row You have homopolymeric stretches on both sides of the SNP If possible redesigning your sequencing primer may improve genotype assessment Chapter 4 Running PSQ 96 Instrument 4 39 4 Running PSQ 96 Instrument This chapter describes how to set up and run an SNP analysis on the PSQ 96 Instrument 4 1 Assumptions The descriptions in this chapter assume that the system has been correctly installed and set up and that an SNP method has been defined and stored The descriptions also assume that a single stranded DNA sample has been prepared and annealed to a sequencing primer according to recommendations To perform a run you will also need a PSQ 96 SNP Reagent Kit and PSQ 96 Plate 4 2 Starting the system Starting the system involves starting both the PSQ 96 Instrument and the operator s computer containing the PSQ 96 SNP Software 4 2 1 Starting PSO 96 Instrument To start PSQ 96 Instrument ensure that the mains power cable is correctly inserted into PSQ 96 Instrument and connected to a safety grounded mains power socket Turn the power switch to On
64. nce all PSQ 96 Instruments are delivered with the IP address 192 168 255 201 as the default configuration only one instrument should be connected at a time to prevent address conflicts CG PYROSEQUENCING 9 PYROSEQUENCING Chapter 2 System Installation and Administration Follow the procedure below 1 Connect one PSQ 96 Instrument to a suitable hub using a twisted pair network cable Connect the hub to the operator s computer using a cross connected twisted pair network cable Add the PSQ 96 Instrument and assign an available IP address for the instrument according to 2 2 6 Adding removing an instrument and 2 2 7 Changing IP addresses In the example above it is advisable to assign the IP addresses in reversed order starting with instrument 4 then instrument 3 and so on Check that the instrument functions Connect the next PSQ 96 Instrument to the hub using a twisted pair network cable and proceed as in the paragraph above For the last PSQ 96 Instrument to be connected the IP address does not need to be changed 2 2 6 Adding removing an instrument In PSQ 96 Instrument Control select Instrument System Administration Note This dialog can only be accessed if there is no instrument window currently open Click on the Instruments tab The dialog will open System Administration x Instruments Database Preferences Instruments Instrument found Instrument used
65. nts 2 1 1 Hardware Operator s computer The operator s computer for the PSQ 96 Instrument is supplied with the instrument The rest of this section is only relevant if the software modules PSQ 96 SNP Entry and PSQ 96 Evaluation are going to be installed Office installation on additional computers not supplied by Pyrosequencing AB Office Computer The Office Computer used to analyze sequence data should be a PC type with the following minimum specifications Processor Min Intel Pentium II 233MHz RAM Min 64 MB recommended 128MB Free hard disk space Min 200 MB CD ROM Min 4x Graphics card Super VGA CG PYROSEQUENCING 2 10 9 PYROSEQUENCING Chapter 2 System Installation and Administration Monitor Color Super VGA 1024 x 768 pixels 256 colors Pointer device Mouse or similar Printer All printers supported by Windows NT 4 0 are suitable Pyrosequencing AB recommends the use of a laser printer 2 1 2 Operating system The computer used to analyze sequence data must be running Microsoft Windows NT 4 0 Workstation service pack 5 English version only 2 1 3 Local area network The PSQ 96 Instrument is usually connected to the operator s computer which in turn is connected to a local area network LAN using a standard Ethernet cable If the operator s computer is connected to a LAN it is usually favorable to put the PSQ 96 database PSQ96db mdb and results directory on a network server In th
66. od Advanced fields The method shown will be one of the default methods supplied with PSQ 96 SNP Software To define a new method click on the Define Method button All fields will be emptied including the Method field 9 PYROSEQUENCING Appendix 9 PYROSEQUENCING A 2 There are two ways of defining a new method 1 Fillin all the empty fields with suitable parameter values as suggested in the next section Enter a Method name and click on the Save Method button 2 Select an existing method by pressing the arrow in the Method field and clicking on the desired method Change the parameter values as desired Enter a new method name and click on Save Method Click on the Cancel Method button to exit from the dialog without saving the changes made 1 12 Suggested parameter values The following guidelines should be used when entering new parameter values Block temp Permitted range 20 50 C The Block temperature is the temperature maintained by the heating block during the process Following equilibration the samples will maintain the same temperature The block can only be heated not cooled Chamber temp Permitted range 10 30 C The Chamber temperature is the temperature at the bottom of the process chamber The Chamber temperature maintains the level of the block temperature no matter the amount of internal heating due to the instrument The chamber can only be cooled not heated Note The Chamber temper
67. ooking at the pyrogram you feel that the sequence data are too poor for reliable genotyping To change the quality classification click on the appropriate classification button in the Quality field The background color of the displayed genotype shown at the bottom right of the dialog will change to reflect the new quality classification The genotype assignment can similarly be changed by clicking in the desired radio button Click on Apply to confirm the changes or click on Cancel to exit the dialog without making any changes Click on OK to accept the genotype and quality assignments and exit from the dialog The Well Overview field will indicate that the genotype and or quality for this particular well has been changed edited by displaying a black ring around the well 9 PYROSEQUENCING 5 72 9 PYROSEQUENCING Chapter 5 Evaluating results Well Overview m Well Overview Well Result 10 11 12 Legend rommooor Figure 5 14 Well result edited Information about the analysis history and changes made by any user can be viewed under the History Log tab To access this information select the desired well in the Well Overview and click on the History Log tab Information shown include the changes made who made them and when they were made Note When editing data the identity of the user making the changes will not be visible in the History Log until the results changes have
68. ories that need to be backed up are unless other directories have been defined c Program Files Pyrosequencing PSQ 96 SNP Software Db c Program Files WPyrosequencing PSQ 96 SNP Software Result No backup software is included in PSQ 96 SNP Software CG PYROSEQUENCING 2 22 Chapter 2 System Installation and Administration 2 4 Installation testing 9 PYROSEQUENCING 2 4 1 Stand alone system A stand alone system should be tested after installation as described in 2 4 3 Test method 2 4 2 LAN system A LAN system should be tested after installation as described in 2 4 3 Test method Chapter 2 System Installation and Administration 2 4 3 Test method 2 23 This test will check the main functions of the hardware and software If the test functions correctly PSQ 96 Instrument can be used for running samples Step Activity Result 1 Start the The NT log on dialog will appear operator s When logging on for the first time computer you will be logged on as Administrator No password is required 2 Log on to NT Windows NT Desktop will appear 3 Start PSQ 96 All lights on the front of PSQ 96 Instrument Instrument will illuminate The software in PSQ 96 Instrument can take 1 3 minutes to load The yellow light on the PSQ Instrument will then go out and the red light will start to flash The instrument is now ready to receive instructions from the operator s computer
69. orun exe 4 Click on Install PSQ 96 SNP Software CG PYROSEQUENCING 2 12 Chapter 2 System Installation and Administration lt 2 PSQ 96 SNP Software x 4 Install b PSQ 96 SNP Software PYROSEQUENCING Install internet K Explorer 5 A Install Adobe Acrobat Reader Figure 2 1 Software selection dialog 5 Select Complete installation and follow the instructions that appear on screen InstallShield Wizard Lx Setup Type Choose the setup type that best suits pour needs Click the type of Setup you prefer m Description This installs the software for creating SNP s and evaluating results Instale lt Back Next gt Cancel Figure 2 2 Software installation dialog 2 24 Installing PSO 96 SNP Software Office version An Office installation means that the software modules PSQ 96 SNP Entry and PSQ 96 Evaluation are installed on a separate computer Proceed as follows 1 Ensure that a suitable computer is available as specified in 2 1 1 Hardware G PYROSEQUENCING Chapter 2 System Installation and Administration 2 13 2 Insert the PSQ 96 SNP Software CD ROM into the CD ROM drive in the computer 3 Follow the installation wizard that will automatically start if the wizard does not start open Run under the Windows Start button specify the path to your CD drive and type autorun exe e g D autorun exe 4 Select Office installatio
70. ple preparation 9 PYROSEQUENCING Chapter 6 Troubleshooting 6 81 Faulty or poor sequence Contaminated samples Too much template Non homogeneous sample Reagents incorrectly diluted or incorrectly stored Sample not prepared according to recommendations Sample contains too much mag netic beads Use a smaller amount of template Wide peaks Too much template Reagents incorrectly diluted or incorrectly stored Contaminated sample Too much magnetic beads Incorrect sample preparation Unspecific peaks Sequence signals from the self annealed sequencing primer and or biotinylated PCR primer and or DNA template Unspecific annealing of the sequencing primer Run DNA template sequenc ing primer and biotinylated PCR primer on their own sep arate wells If significant peaks appear consider rede signing PCR and or sequenc ing primer s Redesign sequencing primer Low signals Too little template Reagents incorrectly diluted or incorrectly stored Too much salt in the sample Drifting baseline CCD camera temperature is not sufficiently stable Large variations in the ambient temperature Kinase contaminated sample No action necessary since the algorithm in the analysis software will compensate for the drift 9 PYROSEQUENCING 6 82 Chapter 6 Troubleshooting High basel
71. provided with PSQ 96 SNP Software An exception is when new PSQ 96 SNP Reagent Kits are released The new methods will then be provided with the new PSQ 96 SNP Reagent Kits The parameters are optimized to give the best possible quality for sequencing Since the analysis software is based on these parameters the quality of the result analysis is also optimized If you still wish to change a standard method proceed as stated below 1 1 1 Defining a new SNP method When in the PSQ 96 SNP application click on the Process Parameters tab Click on the Method Advanced button A field area will open in the lower part of the screen Instrument Test 1 Process Identification Process Parameters Process Dutput Method metode XP Dispensation SNP name y Colection v UpdateSNP SNP panel v Store SNP Panel m RN Z _Update SNP El SNP dispensation e 8 3 10 1 12 I EE D 01 L L LLL Lr cl HL T Method name Block temp 20 50 Celsius CT Reagent 1 pulse time 0 600ms Chamber temp 10 30 Celsius EE Reagent 2 pulse time 0 600ms Nucleotide pressure 300 1000 mBar Nucleotide pulse time 4 600 ms mm at Cycle time 60 600 sec Po Reagent pressure 300 1000mBa Mixer frequency 0 20 50 Hz I Priming time 0 600 ms Define Method Figure 1 1 SNP Meth
72. r max Leakage current 100 120 V Ko PN cm CE 220 240 V Figure IUI 7 Voltage table Important User Information 1 3 IUI 3 Declaration of Conformity The PSQ 96 meets the requirements of the following standards e EN 55022 1994 Class B Emission e EN 50 082 1 1992 Generic Immunity standard EC 801 2 ed 1 1984 Immunity against ESD EC 801 3 ed 1 1984 Immunity against Electromagnetic Fields e EC 801 4 ed 1 1988 Immunity against Fast Transients e ENV 50204 ed 1 1995 Immunity against Electromagnetic Fields e EN 61010 1 ed 1 1993 including A1 and A2 Safety The PSQ 96 meets the requirements of the following directives Low Voltage Directive 73 23 EEC EMC Directive 89 336 EEC including amendments in the CE marking Directive 93 68 EEC IP code 21 Enclosure Safety The PSQ 96 fulfils the following requirements for U S A and Canada Meets the requirements in UL 3101 1 and CAN CSA C22 2 no 1010 1 92 The PSQ 96 is certified by SEMKO Sweden as regards product safety S marking IUI 4 External equipment for connection to PSO 96 Instrument External equipment intended for connection to signal input signal output or other connectors shall comply with relevant IEC standards e g IEC 60950 for IT equipment 9 PYROSEQUENCING l 4 Important User Information IUI 5 Warranty and Liability Pyrosequencing AB warrants that the product supplied has been tho
73. rintout of the pyrograms in graphical form including annotations Report Select which sections to include in the report under Sections to print Run details includes the information found under the Run Infor mation and Method Details tabs as well as the Event Log Results shows wellspecific information such as the analyzed sequence SNP ID genotype quality and sample notes Quality messages a list of messages stating the reason why the analyses in certain wells did not pass the quality requirements History log the information found under the History Log tab Landscape or Portrait Select an orientation for the printout Ymax value Means that a fixed value for the Y axis is entered This can be used to simplify comparisons between curves If no change is made in the dialog the Y axis value is scaled automatically Diagrams per column Can be set between 1 and 6 with 1 or 2 columns per page page can contain a maximum of 12 pyrograms i e two columns each containing 6 curves Row or Column The order of the diagrams on the printout can be set by Row or Column i e across the plate or down the plate CG PYROSEQUENCING 5 74 Chapter 5 Evaluating results Print All Wells To print all wells click on the Print All Wells button Print Selected Wells Clicking on the Print Selected Wells button presumes that wells have been marked in the Well Overview dialog 5 3 10 Saving results To permanently store re
74. roughly tested to ensure that it meets its published specifications The warranty is only valid if the product has been installed and used according to the instructions provided by Pyrosequencing AB Pyrosequencing AB makes no warranties expressed or implied including without limitation the implied warranties of merchantability and fitness for a particular purpose regarding the product Pyrosequencing AB does not warrant guarantee or make any representations regarding the use or the results of the use of the product in terms of its correctness accuracy reliability currentness or otherwise The entire risk as to the results and performance of the product is assumed by the user Since the exclusion of implied warranties is not permitted by some jurisdictions the above exclusion may not necessarily apply Pyrosequencing AB shall in no event be liable for any direct indirect special or consequential damages including without limitation damages for loss of business income business profits business interruption loss of business information and the like arising out of the use or inability to use the product Since the exclusion of implied warranties is not permitted by some jurisdictions the above exclusion may not necessarily apply IUL6 Trademarks and Patents 9 PYROSEQUENCING All rights to the trademark Pyrosequencing are owned by Pyrosequencing AB The firefly symbol in Pyrosequencing s logo is a trademark owned by Pyrosequen
75. rt window choose Raw data and click on the Export button Chapter 5 Evaluating results 5 63 Sections to export f Bur deta Jv Result f ouai messages f Histor loa Export Cancel Figure 5 7 Export data A dialog will then open similar to the standard Windows Save As dialog where you state the directory and name under which the file should be stored Click on Save In order to generate and export a report the run file has to be opened and the Analyze SNP tab selected Click on the Export button and select Report as Choose between Plain text or Tab delimited format in the list box and select which sections to include in the report under Sections to export Run details includes the information found under the Run Information and Method Details tabs as well as the Event Log Results shows well specific information such as the analyzed sequence SNP ID genotype quality and sample notes Quality messages a list of messages stating the reason why the analyses in certain wells did not pass the quality requirements History log the information found under the History Log tab Click on the Export button and in the dialog window state the directory and name under which the file should be stored Click on Save 5 25 Import button Use Import to import a copy of a run file raw data dat to your present results directory and database Click on the Import button and locate the director
76. runs Therefore it may be useful to limit the number of runs in the list Proceed as follows Enter any information to search for desired run files in the appropriate fields under Search Profile and click on the Find button Under each category runs matching the specified search criteria will be shown in the list 2 Double click on the run ID in the Run Information list to display the data for the run it may take several seconds for the results to appear 3 An Analyze SNP tab will appear alongside the Selection tab showing data from the run Chapter 5 Evaluating results 5 65 5 3 2 Analyze SNP tab The Analyze SNP tab shows the results in the following format PSQ 96 Evaluation MIE Administration Help Selection if Analyze SNP Run Information Method Details Well Overview if Well Result Well Overview ronmoner 00000000 Sample Details if History Loa Set Category Export Import Exit Figure 5 8 Analyze SNP tab The Run Information and Method Details tabs and the Event Log field contain information about the run The information cannot be edited In the Well Overview field the wells in the micro titer plate that have been used in the run are colored black The color sequence shown under Legend is not relevant until the run has been analyzed The Sample Details field shows information about the sample for the well highlighted boxed in the Well Ove
77. rview field The History Log field and the Well Result tab do not show any information until the run has been analyzed 5 3 3 Well navigation To move between wells on the plate display point and click on a well must be a filled well to display the sample details Having selected a well you can also move between the wells by using the arrow keys on the keyboard To select a number of wells in the plate display click on a well while holding down the lt Ctrl gt key or lt Shift gt key The lt Shift gt key is used to select a continuous range of wells while the lt Ctrl gt is used to select individual wells 9 PYROSEQUENCING 5 66 9 PYROSEQUENCING Chapter 5 Evaluating results 5 3 4 Criteria If desired the stringency for the analysis and or criteria for quality assignment can be altered by the user Click on the Criteria button to set the quality criteria for the run Set Criteria i OF EG Quality control window 7 Consider SNP Entry E warnings Show results from failed r analyses Set as Default Revert to OK Cancel Default Figure 5 9 Set criteria Criteria for the analysis should be considered as follows 1 SNP Analysis Stringency Level Can be set to use Low Medium or High stringency for the analysis At High stringency the discrimination between the genotype scored and any of the other two genotypes must be very clear 2 Quality control window The number enter
78. s to be analyzed Please refer to separate printed information or Pyrosequencing s website www pyrosequencing com for details on preparing samples for SNP analysis When interpreting the results a basic knowledge of the methodology of Pyrosequencing is recommended 1 3 2 Computer knowledge PSQ 96 SNP Software is simple to use since it is based on standard Windows M conventions However experience of the Windows NT operating system will ensure that the software can be learned faster Chapter 2 System Installation and Administration 2 9 2 System Installation and Administration Note The term operator s computer refers to the computer containing the PSQ 96 SNP Software that is used to control the PSQ 96 Instrument Note Making changes as described in this chapter may adversely affect the functioning of the PSQ 96 Instrument and the local area network LAN where applicable if carried out incorrectly If you are not familiar with networks do not make changes in IP addresses and system set up parameters in the operator s computer Unless you fully understand the consequences of the changes you are considering do not make them The chapter describes the requirements for equipment to be used with the PSQ 96 Instrument and how they should be set up Equipment includes the operator s computer to be used with the PSQ 96 Instrument the computer to be used for analysis of data network connections etc 2 1 System requireme
79. sage window will show a text confirming the test CG PYROSEQUENCING 6 80 6 2 Troubleshooting guide Chapter 6 Troubleshooting Result Cause Comments No result in any of the wells in the plate Reagent cartridge cover is not closed Reagent cartridge is not correctly filled with reagent and or nucle otides Reagents dissolved in an incor rect solution Obstructed reagent cartridge nee dles Insufficient sample or incorrectly conditioned sample e g too much dsDNA or salt present Check the reagent cartridge Check the PCR samples using a gel technique to check that you only have one spe cific band No result in some wells results in others Insufficient sample or incorrectly conditioned sample The marked wells in the software do not agree with sample place ment in the microtiter plate See above Some peaks appear but then the sequence stops One of the nucleotide chambers in the reagent cartridge is not filled with nucleotide One of the needles in the reagent cartridge the one that has not dis pensed is blocked or damaged Contaminated sample has led to consumption of substrate at sub strate addition high pre sequencing signal No light is pro duced upon nucleotide dispensa tion Change the cartridge or rinse it see the chapter on Mainte nance in the PSQ 96 Instru ment Reference Manual Check sample and sam
80. ser Keyed slot in reagent cartridge Figure 4 13 Reagent cartridge 9 Close the cartridge cover Secure the bayonet catch on the cartridge cover by pressing down and turning the black knob clockwise 10 Close the lid of PSQ 96 Instrument Make sure that it is completely closed There is a magnetic switch fitted to the lid and if this switch is not activated by a completely closed lid a warning signal will sound when nucleotides are dispensed 4 5 3 Starting a run This assumes that you have already entered all the information in the Process Identification and Process Parameters tabs When the instrument is ready to run the word Idle appears in the status bar To start a run click on the Run button in the status bar PSQ 96 SNP Software will then check that the process parameters are valid If they are the parameters are sent to PSQ 96 Instrument and the run process will begin If the parameters are not valid a warning dialog will be shown The text Preparing for run will appear in the status bar Set parameters for pressure and temperature etc will come to preset 9 PYROSEQUENCING 4 52 t PYROSEQUENCING Chapter 4 Running PSO 96 Instrument values The text Adding reagents will appear in the status bar and reagent dispensation begins When reagent dispensation is finished thetext Preparing for run will appear and the block temperature chamber temperature mixer speed and pressure will come to preset val
81. sults in the database after analysis and or after changes have been made the results must be saved before opening a new run or exiting from the Evaluation software Click on the Save button You will be asked to enter your user name and password the first time results are saved during a session 5 4 Algorithm for analysis The analysis algorithm is based on pattern recognition and treats the incoming raw data in the following way 1 The three theoretical outcomes are determined from the sequence entered in SNP entry 2 After noise determination raw data is analyzed and the peak heights are defined 3 The intensity values for the A peaks are reduced by a constant factor 4 Normalization is performed in order to determine the peak height corresponding to the incorporation of a single nucleotide 5 The genotype is determined by comparing the peak heights for the polymorphic position with the three different theoretical outcomes The one with the best match is selected The algorithm also calculates a quality value that indicates the certainty of the genotype assignment 6 The degree of agreement between the sequence within the quality control window and the expected sequence is calculated 7 Peak heights within the defined quality window are compared with the theoretical values for the respective peak If the values are above or below certain limits the user is prompted to check the data The signal to noise ratio is calcu
82. t Method v Method note r Dispensation SNP name Collection v UpdateSNP SNP panel E Store SNP Panel SNP dispensation 1 2 3 4 5 6 7 8 3 10 1 12 B 0L 0L Z ER ER ER 0L I _ I Figure 4 9 Process Parameters This screen is used to select the Method and SNPs to be used for the analysis It is also used to define new Methods see Appendix 1 1 Defining Methods Proceed as follows 1 Select the method to be used from the list of stored methods by clicking on the down arrow to the right of the Method field and selecting the method required from the list It is normally not necessary to alter the method definition How ever if adjustments to the stored methods are necessary click on the Method Advanced button to reveal the method parameters screen See Appendix 1 1 1 Defining a new SNP method for instructions on defining methods Choose the Collection under which the desired SNP is stored by clicking on the down arrow to the right of the Collection field and selecting from the list Select the SNP to be used from the list of stored SNPs by clicking on the down arrow to the right of the SNP Name field and selecting the SNP from the list Chapter 4 Running PSQ 96 Instrument 4 49 Note If the SNP that you need is not in the list enter the new SNP by using the SNP Entry module see page 27 Once you have entered the SNP start again at the beginning of step 1 4 Update SNP button
83. t in an empty cartridge Close and lock the catridge holder Close the cover of PSQ 96 Instrument and click on the Run button on the status bar The Process Output tab will open The instrument status will change to Preparing for run The chamber and heating block temperatures and the pressure will stabilize to the preset values When these values are reached the instrument status will change to Adding reagents When this is complete the text states Preparing for run The pressure will increase to the Nucleotide pressure value defined in the SNP Method used The status will change to Running and the process will begin The yellow light on PSQ 96 Instrument will illuminate indicating that the instrument is busy 10 Function check Open the cover of PSQ 96 Instrument Open the heating block lid Close the cover There will be an audible warning signal Lid open during run will appear in the event log The red Message light will illuminate The X Y table should move at a rate of about one well every 0 6 seconds The pneumatic dispensation system should deliver an air pulse every 0 6 seconds The heating block should oscillate vibrate The intensity value for all wells should be about 4 000 4096 The audible warning signal will cease The intensity will be between 5 and 5 units with the noise between 0 5 and 0 5 units The Messag
84. t settings 5 35 Analyze Results will be analyzed when you click on the Analyze button The dialog will open Analysis Bisi ES Analyze All Wels Analyze Selected wl Figure 5 10 Analyze Analyze All Wells To analyze all wells on the microtiter plate click on the Analyze All Wells button Analyze Selected Wells Clicking on the Analyze Selected Wells button presumes that wells have been marked in the Well Overview dialog For analyses to be permanently stored in the database file they have to be saved before opening a new run or exiting the PSQ 96 Evaluation module 5 3 6 Displaying overall results After the sequence data have been analyzed the Well Overview tab will change appearance and show the results of the analysis CG PYROSEQUENCING 5 68 9 PYROSEQUENCING Chapter 5 Evaluating results Well Overview if Well Result gt Well Overview Passed Check Failed Edited I nmONGEr Sample Details History Log Eu3 1 Administrator SNP Creation Date 7 3 00 12 22 26 PM Sequence to analyze A GGGCAGATCCTT Result G Quality Passed Figure 5 11 Analyzed results Legend The Legend field describes the color coding for the quality classification shown in the wells Passed light blue with the set criteria the quality of the sequencing data is very good yielding a reliable genotype assessment Check yellow with the set criteria the quali
85. the instrument and the operator s computer established the flashing message light will turn off 4 2 3 Starting the PSQ 96 Instrument Control Start the computer containing PSQ 96 Software and log on as normal When you reach the Windows NT desktop double click on the PSQ 96 SNP Instrument Control icon to open the software 9 PYROSEQUENCING Chapter 4 Running PSQ 96 Instrument 4 41 b PSG 36 Instrument Control Figure 4 3 PSQ 96 Instrument Control icon on desktop It is also possible to open the PSQ 96 Instrument Control via the Start menu From the Start menu choose Programs PSQ 96 Software PSQ 96 Instrument Control 4 3 PSQ 96 Instrument Control Software start screen description The PSQ 96 Instrument Control start screen then appears Figure 4 4 PSQ 96 Instrument Control start screen The PSQ 96 Instrument Control start screen is divided from the top into a menu bar a status bar and an entry field area 4 3 1 Menu bar There are four different pull down menus on the menu bar each containing several undermenus These are described below The status bar is only visible when contact has been established with an instrument 9 PYROSEQUENCING 4 42 9 PYROSEQUENCING Chapter 4 Running PSO 96 Instrument Instrument Modules Window Help Figure 4 5 Menu bar Instrument The undermenus are System administration For carrying out administrative functions Export Data The Export Data
86. tion button and then click on the System Information button The following window will open m General iH Pressure I Lamps Speaker Lid Temperatures I X Y Motors Mixer Software Versions Log Files Events Instrument Control 1 1 15 Instrument Control Tine s KL e I C AWINNTYPSQ9BL0g InstrumentContolb ew state Stop fat Database 50 Reset 0000 2105 Newstate Service PSQuil dl 1 01 Embedded Contrat CAPSQ96 Log PSQS6Log txt Embedded Conto 1 1 9 CAPSO96 Log PSQ96PrevLog txt PSQ96hw dll 1 01 Time s Plot Block 150 C Environment C Chamber G Pressure 100 C Miser C No plot 50 E Auto lime interval Time range o 500 z i 100 200 300 400 500 50 Set Auto Scale Max auto MinY auto 1 zl Figure 6 1 System Information General tab There are several functions that are common to all of the tabs shown in the System Information window Close Used to close the window When you click on the button there will be choice of different actions see 4 6 3 Closing down PSQ 96 Instrument Control Report Clicking on this button will generate a text file that can be sent to Pyrosequencing AB to help identify problems CG PYROSEQUENCING 6 78 9 PYROSEQUENCING Chapter 6 Troubleshooting Time Time that the System Information window has been open Plot Displays a graphical representation of the function selected Select a function
87. ty of the sequencing data permits genotype assessment with some uncertainty Manual confirmation is advisable Failed red with the set criteria the quality of the sequencing data is too poor for reliable genotype assessments Edited black ring around well either the quality score or genotype set by the software has been manually changed for this well by a user Sample Details The Sample Details field shows the full list of details for the boxed well The information includes well ID sample note and information about the SNP the genotype assignment and the sequence quality There will also be an indication whether the well data have been edited or not If the quality has been set to Check or Failed an explanation of the quality assignment is shown One or more of the following explanation messages may be shown lt Uncertain gt lt Failed gt genotype determination The algorithm is unable to clearly discriminate between the theoretical genotype outcomes Chapter 5 Evaluating results 5 69 lt Uncertain gt lt Failed gt reference sequence pattern dNTP disp disp number s gt The sequence pattern within the set quality control window deviates significantly from the expected sequence pattern in general and or at indicated nucleotide dispensations Uncertain Failed due to low signal to noise ratio Too low signal to noise ratio will result in an uncertain or failed analysis Wide peaks Pe
88. ues When all these parameters are within the specified tolerance ranges Running will appear in the status bar and the nucleotide dispensation will begin The exact values for the parameters are displayed in the Process Output tab Process Parameters tab 4 5 4 Monitoring a run When the run is underway the Process Output tab will be displayed on screen process progress scale in the status bar will indicate the progression of the run The word Running will appear in the status bar The Process Output screen appears as shown below fr Powe see Running 2min HRRRRRRRRN Process Qutput Well Overview 123 4 SE EE ELEM Mixer Block Envir E 34 300 280 250 Time s 246 200 fes 27 8 E Plot G WelG8 Process Parameters Events C Environment T Auto time interval Time range 1000 E Set Auto Scale Maky auto Miny Jauto ronmoocor 4 ESGCTAGAGC Figure 4 14 Process Output screen Plot field area Well the well view function has been selected The code beside the text indicates the well currently selected To move to another well click on the well desired in the well overview area or use the arrow keys to move to the well The graphical representation shows the pyrogram Chapter 4 Running PSQ 96 Instrument 4 53 for the selected well displayed in real time Environment offers displays of the following parameters Chamber t
89. w currently open Click on the Preferences tab Information about the current result directory and database as well as the path to the database can be found above the Result directory field System Administration e Users Instruments Preferences Current result directory h ULNULB047 Pyro SNP 1 1 Current database DSN PSQ 96 Path to database E PROGRA 1 PYROSE 1 PSQ96S 1 DB psq36 Result directory XUL CXULBO47 Pyro SNP 1 1 C Algorithm C3 Diski C3 Doc hAULAULB047 Pyro SNP 1 1 Change Database Figure 2 6 System Administration Preferences dialog To change the current default directory where the results files dat are stored proceed as follows 1 Select the desired directory by choosing the appropriate drive in the drive list on the right hand side and then the directory in the Result directory field Select a different directory by clicking to the desired directory in the usual Windows fashion Then double click on the directory to be used The path to the result directory will appear as a text string under the Result directory field 2 Click on the Change result directory button A warning dialog will appear asking you to confirm the change of result directory 9 PYROSEQUENCING 2 20 Chapter 2 System Installation and Administration To change the database used proceed as follows 1 Click on the Change Database button The following dialog will open
90. y extended However this additional dispensation is only intended for visual examination and a peak generated by this addition will not be considered by the PSQ 96 Evaluation software 3 6 2 Longer extension If it is desired to dispense more bases than those suggested by PSQ 96 SNP Entry in order to confirm the sequence following the polymorphic position this sequence must be added manually 3 6 3 Out of phase analysis Out of phase analysis I The dispensation order suggested usually contains two informative peaks for the SNP position This is due to the fact that the simplest possible dispensation order is chosen and that the dispensation order generally follows the actual sequence In certain cases it is possible by changing the dispensation order for the polymorphic position to obtain three informative peaks for the SNP and thereby generate a result that might be easier to evaluate e g AGCTGAT to AGTCTGAT for the analyzed sequence AGC TTGAT Certain SNPs have a repetitive sequence combination e g AGG TGTGTGTCAG that gives a complicated pattern In this case more than two dispensations are required to get past the polymorphic position and get the sequence for the two alleles into phase again It is not possible to avoid these by manipulating the dispensation order Out of phase analysis IT For analyzing a heterozygous sample it is possible to manipulate the dispensation order so that the resulting sequence for the two allel
91. y in which the file is stored Mark the desired file and click on Open The file will then be found in the IMPORTED category 9 PYROSEQUENCING 5 64 Chapter 5 Evaluating results 5 2 6 Delete Run button The Delete Run function makes it possible to delete a run file and remove all information associated with this file from the database The user will be asked twice to confirm the deletion Note The raw data and results will be irreversibly deleted 5 3 Analysis of run data 9 PYROSEQUENCING 5 3 1 Selecting a run From the Evaluation start screen select a run to be analyzed as follows Fields containing an asterisk indicate that no choice has been made and by default all choices will be displayed The asterisk can also be used as a wild card in combination with any other character 1 There are several methods that can be used to produce a list of runs from which to make a selection a Click on a category of run files in the Category field This will display run files stored in the database ALL means all results IMPORTED means that the run files have been imported but not necessarily analyzed UNANALYZEDDATA means that the run files has neither been imported nor analyzed User specified categories might also exist see section 5 2 3 Set category button b Selecting any of the above mentioned methods may display a list in the Run Information field that can be very long if you have performed many

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