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Applied Biosystems 3500, User Manual

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CHAPTER 6    

Run calibrations and install checks ................... 103

Section 6.1 

Run spatial and spectral calibrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

Run a spatial calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

Spatial calibration overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
When to perform a spatial calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Perform a spatial calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Evaluate the spatial calibration profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Example spatial profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Export spatial calibration results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
View and print a calibration report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Save historical reports (.pdf) for record keeping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Run a spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Spectral calibration overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
When to perform a spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Estimated run time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Prepare for spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Perform a spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Spectral Quality Values and Condition Numbers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Evaluate the spectral calibration data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
What you see during a spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Capillary information sharing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Example spectral calibration data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Export spectral calibration results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
View and print a calibration report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Save historical reports (.pdf) for record keeping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
View the spectral calibration history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Section 6.2 

Run an install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Run a Sequencing install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

When to perform a sequencing install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Estimated run time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Prepare for the sequencing install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Perform a sequencing install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
What you see during a sequencing install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Pass/fail criteria for the optional spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . 126
Pass/fail criteria for the sequencing install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Evaluate sequencing install standard data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Example sequencing install check results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
View previously run install standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
View and print an install check report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Save historical reports (.pdf) for record keeping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Run a fragment/HID install check   . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

When to perform a fragment/HID install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Estimated run time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Prepare for the fragment/HID install check   . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Perform the fragment/HID install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Contents

3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.1

7

Summary of Contents for 3500

Page 1: ...Only Not for use in diagnostic procedures Applied Biosystems 3500 3500xL Genetic Analyzer USER GUIDE with 3500 Series Data Collection Software 3 1 Catalog Numbers 4405186 4405187 Publication Number 100031809 Revision C ...

Page 2: ...entific reagents Indicate 8 maximum number of freeze thaw cycles for Hi Di Formamide Change Part No to Cat No Change figure callouts to numbers Correct definitions for Read Length and Basepair Accuracy Read Length Accuracy B 01 June 2017 Add Radio compliance Update conformity symbols Update licensing information Update branding A March 2015 New information for version 3 1 software Change buffer an...

Page 3: ...ring samples 18 Preparing the instrument 18 During a run 18 Results 19 Materials for routine operation 19 Instrument consumables handling usage limits and expiration 19 Buffers 19 Polymer 20 Conditioning reagent 21 Capillary arrays 21 Hi Di Formamide 22 Important notice regarding use of consumables that exceed supported limits 22 Overview of the 3500 Series Data Collection Software 3 1 23 About th...

Page 4: ... System preferences 37 User preferences 37 CHAPTER 3 Set up and run 40 Setup workflow 41 Prepare the instrument 41 Create or import a plate 42 About plate templates 42 Create a plate from a template 42 Import a plate 44 Assign plate contents 44 Access the Assign Plate Contents screen 45 Name samples and assign sample types in the plate view 47 Assign assay file name convention and results group in...

Page 5: ...use and resume 72 Abort or terminate 72 More features in Assign Plate Contents 73 Name samples in the plate view 73 Customize the plate view 74 View the capillary to plate map 75 Use the table view 75 Sort by one or multiple columns 76 Customize a table 76 Add assays file name conventions and results groups to a plate 77 Create a plate import template 77 Create a plate import file 78 Edit a plate ...

Page 6: ...e currently running fragment HID analysis plate 93 Review previously run fragment analysis HID samples 94 Review sample quality 94 Review normalized data 96 How normalization is applied 96 Normalization factor in secondary analysis 96 Review plots 97 Zoom on data 97 Change plot settings 97 Overlay samples 98 Label peaks 98 View thumbnails 99 Rename samples 99 Sort by one or multiple columns 100 Re...

Page 7: ...rt spectral calibration results 119 View and print a calibration report 119 Save historical reports pdf for record keeping 120 View the spectral calibration history 120 Section 6 2 Run an install check 121 Run a Sequencing install check 121 When to perform a sequencing install check 121 Estimated run time 121 Prepare for the sequencing install check 121 Perform a sequencing install check 123 What ...

Page 8: ...ary entry 141 View audit and e signature histories for library entries 141 Sort and search library entries 141 Customize a table 142 Plates library 142 Plate overview 142 Create a new plate 143 Define plate properties 144 Assays library 144 Assay overview 144 Create a new assay 145 Assay settings 146 File Name Conventions library 147 File name convention overview 147 Create a new file name convent...

Page 9: ...alling protocol Analysis settings 176 Basecalling protocol QV settings 178 Sizecalling protocols library primary analysis fragment 178 Sizecalling protocol overview 178 Create a new sizecalling protocol 179 Sizecalling protocol Analysis settings 181 Sizecalling protocol QC settings 184 QC protocols library primary analysis HID 185 QC protocol overview 185 Create a new QC protocol 185 QC protocol A...

Page 10: ... 227 Review the Notifications Log 228 Clean the instrument 229 Install buffers 229 Install the anode buffer container ABC 229 Install the cathode buffer container CBC 230 Replenish change flush and store polymer 232 Precautions for use 232 Replenish polymer or change polymer type 232 Store partially used polymer 234 Fill capillary array with fresh polymer 234 Change and store a capillary array 235...

Page 11: ... Run re run or re inject troubleshooting 264 Data electropherogram troubleshooting 264 Review Results troubleshooting 267 Link load plate troubleshooting 269 Assign Plate Contents troubleshooting 270 Spatial calibration troubleshooting 271 Spectral calibration troubleshooting 272 Sequencing install standard troubleshooting 274 Fragment HID install standard troubleshooting 275 Monitor Run troublesh...

Page 12: ...uencing analysis reagents and consumables 290 Fragment and HID analysis reagents 291 APPENDIX E Limitations 292 General 292 Computer 292 Instrument and consumables 293 Calibration and install checks 293 Security Audit and E sig 293 Review results 294 APPENDIX F Radio Frequency Identification RFID technology 295 Precautions for use 295 Locations of RFID read write units 296 Function 296 Specificati...

Page 13: ...n 305 Laser 306 Safety and electromagnetic compatibility EMC standards 306 Safety compliance 306 EMC 307 Environmental design 307 Radio compliance 307 Chemical safety 308 Biological hazard safety 309 Documentation and support 310 Related Documentation 310 Customer and technical support 310 Obtaining support 310 Limited product warranty 311 Additional instrument symbols 312 Index 313 Contents 3500 ...

Page 14: ...ection Software 3 1 is a fluorescence based DNA analysis instrument using capillary electrophoresis technology with 8 or 24 capillaries The 8 capillary model Cat No 4405186 and the 24 capillary model Cat No 4405187 is shipped with the following components 8 capillary or 24 capillary array and POP polymer Reagents and consumables for your application and for system qualification Computer workstatio...

Page 15: ...d or implanted active medical devices To minimize such effects do not come within 8 inches 20 cm of this instrument if you have a patient worn and or implanted active medical device 1 2 3 4 5 7 6 8 9 Figure 1 Instrument interior 1 Detection cell heater block 2 Polymer delivery pump PDP 3 Anode buffer container ABC 4 Polymer or conditioning pouch 5 Cathode buffer container CBC 6 Oven door 7 Capilla...

Page 16: ...y array port check valve fitting water trap waste container buffer valve anode electrode buffer gasket and holds the anode buffer container Polymer pouch or conditioning reagent pouch Polymer pouch Supplies polymer to the polymer delivery pump Conditioning reagent pouch Used for priming the polymer pump washing the polymer pump between polymer type changes and during instrument shut down Has adequ...

Page 17: ...ent contains validated software and settings Do not update the Windows operating system or firewall settings The computer provided with the instrument does not include antivirus software because customer preferences and network requirements vary We recommend Norton Antivirus which has been tested and approved for use with the Applied Biosystems 3500 3500xL Genetic Analyzer with 3500 Series Data Co...

Page 18: ... of the capillary array This field pulls the negatively charged DNA through the separation polymer The smaller fragments migrate faster than the larger fragments and reach the detector first To ensure optimal separation and maintain denaturation of the DNA the capillaries are thermally controlled in the oven and in the detection cell The oven has a Peltier heat unit and fan circulated air In the d...

Page 19: ...NT Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see Documentation and support on page 310 Containers and pouches are ready to use Labels include a radio frequency identification RFID tag that the instrument uses to track usage and expiration date For application ...

Page 20: ...P 6 Polymer 384 sample 4393712 POP 6 Polymer 960 sample A26073 POP 7 Polymer 96 sample 4393708 POP 7 Polymer 384 sample 4393714 POP 7 Polymer 960 sample 1 The polymer has been validated for HID applications IMPORTANT For the POP 4 and POP 7 Polymers Cat Nos A26070 4393715 4393710 A26073 4393708 and 4393714 the on instrument supported limit is 14 days only when the instrument operating temperature ...

Page 21: ...ltiple pouch installations and or excessive use of wizards reduce the number of remaining samples and injections For example if you run the total bubble remove option in the Remove Bubbles wizard more than four times the number of remaining samples and injections is reduced Conditioning reagent Cat No Description Storage 4393718 Conditioning reagent 1 pouch 2 8 C After removing from storage use th...

Page 22: ... WARNING THAT THE CONSUMABLES HAVE REACHED SUPPORTED LIMITS AND CONTINUING WITH OPERATION OF THE INSTRUMENT PLEASE READ AND UNDERSTAND THE FOLLOWING IMPORTANT NOTICE AND INFORMATION Life Technologies does not recommend the use of consumables that exceed supported limits The recommended limits are designed to promote the production of high quality data and minimize instrument downtime Reagent and c...

Page 23: ...s sample data files Sequencing ab1 Fragment analysis fsa HID analysis hid Performs primary analysis For sequencing applications Basecalling For fragment analysis and HID applications Sizecalling You can access the Dashboard from any screen by clicking the Dashboard tab Figure 2 Dashboard overview About the software Dashboard Chapter 1 Instrument and software description Overview of the 3500 Series...

Page 24: ...ndicates if any consumable is about to expire based on RFID tags Calendar reminders Displays the tasks listed in the schedule Help icon Displays a help topic specific to a screen or an area of the screen All screens include icons Click the Workflow tab at the top left of the screen to access the Workflow screen The Workflow tab contains the screens where you set up load and run plates and view res...

Page 25: ...is described in Chapter 9 Maintain the Instrument Click the Library tab at the top left of the screen to access the Library The Library contains items that you manage plates assays file name conventions and results groups that you use to acquire and process data You can click Workflow or select Dashboard or any other menu item at any time to advance from the Library workflow Maintenance workflow L...

Page 26: ... access View Run Logs for reports of instrument runs Manual Commands to troubleshoot instrument performance Select Manage in the menu bar to access Archive Restore and Purge functions License Manager to renew the software license SAE menu Tools menu Manage menu Chapter 1 Instrument and software description Overview of the 3500 Series Data Collection Software 3 1 1 26 3500 3500xL Genetic Analyzer U...

Page 27: ...ot connected to an instrument You can use this stand alone version of the software to Create plates protocols and other library items then import them into a version of the software that is installed on an instrument computer Review completed results Do not select instrument related functions in the stand alone version of the software IMPORTANT Do not rename the computer after the 3500 Series Data...

Page 28: ... power on the instrument see Start the computer and instrument on page 29 q When the green front panel indicator stops blinking log in to Windows operating system then start the software see Start the software on page 30 q Check calendar reminders on page 32 q Check consumables status on page 33 q Replenish consumables on page 35 2 28 3500 3500xL Genetic Analyzer User Guide Data Collection Softwar...

Page 29: ...oftware to malfunction 3 Inspect the instrument interior Ensure that a The oven door is closed b No objects are left inside the instrument IMPORTANT Misplaced objects left inside the instrument can cause damage 4 Close the instrument door 5 Power on the instrument Power button Tray button Light button a Press the power on off button on the front of the instrument and wait for the green status ligh...

Page 30: ... icon is displayed in the taskbar then a status bubble is displayed It takes 1 minute for the Server Monitor to start up When the Server Monitor icon is displayed go to Step two Start the 3500 Series Data Collection Software 3 1 on page 31 below IMPORTANT If the Server Monitor icon does not change to you cannot start the software See Software troubleshooting general on page 262 for more informatio...

Page 31: ...t and E signature module In the 3500 Series Data Collection Software 3 1 Login dialog box Enter your User Name and Password then click OK See your 3500 system administrator for login information Note For information on creating user accounts see Chapter 8 Use Security Audit and E Sig functions SAE Module Step two Start the 3500 Series Data Collection Software 3 1 Step three Log in Chapter 2 Start ...

Page 32: ...erences 1 Review the Calendar Reminders pane 2 Perform any scheduled tasks then click to mark it as complete or click to mark it as dismissed if you do not perform the task Actions are recorded in the Notifications Log for more information see Review the Notifications Log on page 228 3 Perform any daily monthly or quarterly tasks that are not listed in the Calendar Reminders pane see Maintenance s...

Page 33: ...aining for a consumable See Instrument consumables handling usage limits and expiration on page 19 for information When 10 of the allowed use of the consumable remains the gauge moves into the red warning range The consumable also displays in red in the Consumables pane IMPORTANT We recommend that you add a calendar reminder to the schedule for polymer and buffer replacement Set the notification t...

Page 34: ...amples 40 injections The 960 sample count limit is reached before the 50 injection count limit 1 Inspect the instrument interior 2 Wipe any spills 3 Check for leaks around the buffer pin valve check valve and array locking lever 1 2 1 Buffer pin valve 2 CV check valve fitting 4 Remove dried residue and ensure that the array locking lever is pushed securely in place Check the fill levels on buffers...

Page 35: ...er container CBC on page 230 Fill capillary array with fresh polymer on page 234 Install or change the capillary array on page 235 When you install the CBC buffer septa press firmly to seat the septa IMPORTANT Look at the CBC from the side and ensure that there is no gap between the container and the lip of the septum Replenish consumables Ensure proper installation of CBC septa Chapter 2 Start th...

Page 36: ...er Preference trigger time for calendar reminders Sequencing Export Settings Spectral Calibration number of allowed borrowing events These user preferences are saved individually for each user Library filtering Plate Setup Reports Settings Run Setup Sequencing Settings review and report settings Warning dialogs Note Users can also save user preferences while viewing tables See Customize a table on...

Page 37: ...notation tab in the sequencing trace view such as data collection time run time start finish phd 1 scf Sequencing files fsta qual seq Reference files specify Entire Sequence or Post trim Sequence Only In the Preferences dialog box click a user preference select a setting then click Apply to save the preference User preference Sets IVD Setup Workflow Not supported Library Filtering Default filter f...

Page 38: ...and ranges for pure and mixed base QVs quality values displayed in the Trace View a Click a pure base or mixed base color bar to select a new color b Place the mouse pointer over a slider then drag to set a new range We recommend that you set the following ranges for QVs Pure bases Low QV 15 Medium QV 15 to 19 High QV 20 default Mixed bases Low QV 5 Medium QV 5 to 10 High QV 10 investigate to dete...

Page 39: ...e signal in QC and Signal Strength Reports based on Average Raw Signal Intensity or Average Raw Signal to Noise Ratio Display well image by Specify the thumbnail option for Plate Reports Wider thumbnail without file name or Smaller thumbnail without file name Warning Dialogs Suppress warning messages for deleting an injection or exporting a library item Chapter 2 Start the system Set preferences o...

Page 40: ...ction list 59 Review and modify the injection list in Preview Run 62 Start the run from Preview Run 64 Monitor the run 65 Check sequence or sample quality and re inject samples 67 Review completed injections in Review Results 72 Pause resume or stop a run 72 More features in Assign Plate Contents 73 More features in Monitor Run 80 3 40 3500 3500xL Genetic Analyzer User Guide Data Collection Softwa...

Page 41: ...r consumables 2 Ensure that buffer levels are at the fill lines see Check buffer fill levels on page 34 3 Set the oven temperature then click Start Pre heat Pre heat the oven and detection cell while you prepare for a run detection cell temperature is set by the software Pre heating helps mitigate subtle first run migration rate effects The pre heat function automatically turns off after 2 hours W...

Page 42: ...parameters a plate template can also contain a list of the appropriate assays for an application For more information see Create a plate template on page 79 The software includes factory provided plate templates that you can use as a starting point to create a plate you can also create your own plate templates In addition to pre defined plate parameters a plate template can also contain a list of ...

Page 43: ...cessing program such as Microsoft Word which may include invisible non ASCII characters Non ASCII characters in plate or sample information may cause a run to stop or may prevent a run from starting 96 Select if you are using a 96 well standard reaction plate or 8 strip standard tubes with a retainer 96 Fast Select if you are using a 96 well Fast reaction plate or 8 strip fast tubes with a retaine...

Page 44: ...the navigation pane 3 Click Import then select the plate import file 4 Click Assign Plate Contents Assign plate contents You assign the following information to the wells in a plate before you can run the plate Sample names and sample types required Identifies the well positions of each sample for data collection and processing Assay required Specifies the parameters that control data collection a...

Page 45: ...ashboard by clicking the Workflow tab then selecting Assign Plate Contents in the navigation pane 2 Click Show In Wells to specify the attributes to display in wells Figure 4 shows the Plate View of the Assign Plate Contents screen Access the Assign Plate Contents screen Chapter 3 Set up and run Assign plate contents 3 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 45 ...

Page 46: ...es 2 Name samples 3 Assign assays file name conventions and results groups 4 Assign sample types and user defined fields 5 Link plate for run Chapter 3 Set up and run Assign plate contents 3 46 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 47: ...and select Fill or Fill Series to populate the selected fields Note To use Fill Series type a number as the last character of the named well The number will increment for each well in the series Note You can copy and paste sample names instead of using fill commands Name samples and assign sample types in the plate view Chapter 3 Set up and run Assign plate contents 3 3500 3500xL Genetic Analyzer ...

Page 48: ...in some software screens may cause problems with plate file folder user account and or library item names and may interfere with starting a run and or importing and exporting library items IMPORTANT If you copy paste sample or plate information into the Assign Plate Contents screen or into a plate import file copy from a plain text editor such as Notepad Do not copy from a word processing program ...

Page 49: ...to Add assays file name conventions and results groups to a plate on page 77 1 Select the wells for which to specify an assay 2 Select the checkbox next to the assay name to assign the assay to the selected wells 3 Repeat for file name conventions and results group 4 Select Save Plate 5 Go to Print the plate layout on page 50 Assign assay file name convention and results group in the plate view Ch...

Page 50: ...sults group 1 In the Assign Plates for Run screen click View Plate Grid Report 2 Select Print Preview or Print as needed 3 To save the report electronically pdf print the report and select CutePDF Writer as the printer 4 Close the report 5 Go to Prepare and load sample plates on page 50 Prepare and load sample plates IMPORTANT Do not use warped or damaged plates How file location in file name conv...

Page 51: ...e secondary analysis software GeneMapper ID X Software v1 3 or later IMPORTANT Variation in laboratory temperature can cause changes in fragment migration speed that can in turn cause sizing variation We recommend the frequency of allelic ladder injections described above to account for normal variation in fragment migration speed However during internal HID validation studies verify the required ...

Page 52: ...yout on page 50 2 Briefly centrifuge the plate 3 Verify that each sample is positioned correctly in the bottom of its well IMPORTANT If the contents of any well contain bubbles or are not located at the bottom of the well briefly centrifuge the plate remove the plate from the centrifuge and verify that each sample is positioned correctly in the bottom of its well 4 Store the plate on ice and prote...

Page 53: ...flat on the plate 2 Place the plate into the plate base 3 Snap the plate retainer cover onto the plate septa and plate base 4 Verify that the holes of the plate retainer and the septa are aligned If holes are not aligned take it apart then re assemble IMPORTANT The array tips will be damaged if the plate retainer and septa holes do not align correctly 5 If the contents of any well contain bubbles ...

Page 54: ...ormance See Appendix D Catalog numbers for plate assembly specifications and catalog numbers 1 2 3 4 5 6 Figure 5 8 strip standard tube assembly 1 Plate retainer 2 Septa 3 Retainer 4 8 strip tubes 5 96 well tray 6 Plate base Chapter 3 Set up and run Prepare and load sample plates 3 54 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 55: ...down 4 Place the tray tube retainer assembly into the plate base 5 Snap the plate retainer cover onto the plate septa and plate base 6 Verify that the holes of the plate retainer and the septa strip are aligned If holes are not aligned re assemble and then assemble the plate assembly 7 If the reagents of any well contain bubbles or are not located at the bottom of the well briefly centrifuge the p...

Page 56: ...the plate remove the plate from the centrifuge and verify that each sample is positioned correctly in the bottom of its well 1 Click the Tray button on the front panel to move the autosampler to the front position then open the instrument door 2 Place the plate in the autosampler with the labels facing you or the instrument door and the notched corner of the plate in the notched corner of the auto...

Page 57: ...un Note By default plate A position is selected 2 Go to Load plates for run and create the injection list on page 59 Link the plate Chapter 3 Set up and run Prepare and load sample plates 3 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 57 ...

Page 58: ...lick Quick Start Run to display the Select Plate from Library dialog box 2 Select a plate then click Load Plate 3 Click Start Run from the Load Plates for Run screen Note If an install check for the run application type Sequencing or Fragment has not been performed a message is displayed and the run does not start Chapter 3 Set up and run Quick Start a run 3 58 3500 3500xL Genetic Analyzer User Gu...

Page 59: ... Name or use the default run name Start Instrument Run Date Time Stamp YYYY MM DD hh mm ss SSS milliseconds for example Run 2014 06 10 17 33 46 522 where the run start date is February 5 2009 and the run start time is 15 03 42 096 Note An instrument run begins when you click Start Run on the Load Plates for Run screen and ends when the last injection on the last plate has completed For example if ...

Page 60: ...k button Figure 7 Load Plates for Run 4 If needed click Unlink then follow the steps in Link a plate if a plate is not linked on page 61 5 As needed click Switch Plates to assign the plate to the other position in the autosampler Chapter 3 Set up and run Load plates for run and create the injection list 3 60 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 61: ...run application type Sequencing or Fragment has not been performed a message is displayed and the run does not start If you access the Load Plates for Run screen from the navigation pane a plate may not be linked indicated by the active Link Plate button To link a plate 1 Click Link Plate to display the Select Plate from Library dialog box Link a plate if a plate is not linked Chapter 3 Set up and...

Page 62: ...nt Plates tab pending plates or the Recent Runs tab processed plates Review and modify the injection list in Preview Run The Preview Run screen allows you to modify the injection list before you start the run 1 Access the Preview Run screen Figure 8 from The Load Plates for Run screen by clicking Create Injection List The navigation pane by selecting Preview Run The Dashboard by clicking the Workf...

Page 63: ...n list and a plate view The injection list is linked to the plate view Click an injection to select the associated wells in the plate view IMPORTANT If the injection list is blank make sure that you clicked Create Injection List on the Load Plates for Run screen Chapter 3 Set up and run Review and modify the injection list in Preview Run 3 3500 3500xL Genetic Analyzer User Guide Data Collection So...

Page 64: ...sults group folder if specified in the results group Start the run from Preview Run When the injection list is configured click Start Run The Monitor Run screen is automatically displayed IMPORTANT You must re inject samples before the run completes unless the Pause after last injection preference is set Note If an install check for the run application type Sequencing or Fragment has not been perf...

Page 65: ...iew The injection list is linked to the plate view Click an injection to select the associated wells in the plate view A selected injection is highlighted in green in the plate view Figure 9 Monitor Run screen 1 Click the Table Settings button then specify the columns to show or hide in the injection list Chapter 3 Set up and run Monitor the run 3 3500 3500xL Genetic Analyzer User Guide Data Colle...

Page 66: ...the Flag attribute green marks are displayed for wells with Average QV values that are within range yellow marks are displayed for wells with Average QV values that are in the suspect range and red marks are displayed for wells with Average QV values that are out of range Place the mouse pointer over a well to display sample details Chapter 3 Set up and run Monitor the run 3 66 3500 3500xL Genetic...

Page 67: ...he injection is also flagged with Note If the Injection Analysis or Flag columns are not displayed you can click the Table Settings button then show them in the injection list 1 Expand the Flag pane at the bottom right of the screen Check sequence or sample quality Chapter 3 Set up and run Check sequence or sample quality and re inject samples 3 3500 3500xL Genetic Analyzer User Guide Data Collect...

Page 68: ...scription Offscale green or red red At least one data point in the analysis range has saturated the CCD camera Note In the View Results screen an offscale sample is flagged with QV Average Quality Value sequencing green yellow red yellow or red The Average Quality Value based on CRL Trace Score and QV20 results is in the Suspect or Fail range For information see Basecalling protocol QV settings on...

Page 69: ...t results groups To enable Re inject select samples that specify the same results group To collect data for all wells in an injection 1 Select the injection in the injection list 2 Click Re inject To collect data for only specific wells Note You can also re inject specific samples in Review Results 1 Select the injection 2 Select in the array view the capillary that corresponds to the well or samp...

Page 70: ...nal_Assay 1 to the re injected well only Saves the plate the software does not save the copy of the assay to the library If the Injection Number attribute is selected for display in the plate view the number of the original injection and the re injection are shown 1 2 1 Sample 1 selected for re injection 2 Re injection number listed for all samples in the re injection If you select a protocol othe...

Page 71: ... select for re injection a sample that is in injection 2 The software prompts you to select one or more allelic ladder samples to re inject The allelic ladders available to select are from the same plate and within the same results group as the original injection If the results group does not contain an allelic ladder sample the software does not prompt you to select one for re injection In the Ad...

Page 72: ...lts Pause resume or stop a run As needed click Pause Pauses the run after the current injection completes the symbol is not displayed in the injection list because the injection continues to completion Resume Resumes the run As needed click Abort Immediately aborts the current injection and pauses the instrument run You can resume the run or terminate the injection list Do not click Delete to stop...

Page 73: ... multiple samples Click a named well Click drag multiple wells Right click and select Fill or Fill Series to populate the selected fields Note To use Fill Series type a number as the last character of the named well You can also copy and paste sample names To name all wells at one time Select all wells Select assays file name conventions and results group for the plate Enter name and select sample...

Page 74: ...2 Click Select Wells to select wells with a specific attribute 3 Click Zoom In Zoom Out and Fit as needed Customize the plate view Chapter 3 Set up and run More features in Assign Plate Contents 3 74 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 75: ... Table View 2 Click the Sample Name field then type a name 3 3 Click next to each field then select a setting View the capillary to plate map Use the table view Chapter 3 Set up and run More features in Assign Plate Contents 3 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 75 ...

Page 76: ...t click another column header to sort another column Alt Shift click a third column header to sort a third column Numbers in the column headers reflect sort order Click the Table Settings button then specify the columns to show or hide Click Apply To use the settings for this session only Save to Preferences To save for future use by all users Preferences are saved for the logged in user Restore D...

Page 77: ...it to create a file that contains the correct header and column information for importing 1 In the Dashboard click Create Plate from Template 2 In the Open Plate Template from Library dialog box a Select a filter to display the plate template type of interest b Select a plate template then click Open 3 Enter a name for the plate then specify the capillary length and polymer type for the plate 4 Cl...

Page 78: ... the remaining columns Note If you specify assay results group or file name convention names the names you enter must exactly match the names of existing items in the library IMPORTANT If you copy paste sample or plate information into the Assign Plate Contents screen or into a plate import file copy from a plain text editor such as Notepad Do not copy from a word processing program such as Micros...

Page 79: ...w plate on page 143 2 Optional Add sample names and sample types see Name samples and assign sample types in the plate view on page 47 3 Optional Add the assays file name conventions and results groups appropriate for this plate template s application see Add assays file name conventions and results groups to a plate on page 77 Adding assays file name conventions and results groups to the plate te...

Page 80: ...view tab As needed Click to zoom in and out Click to detach a view and display it in a separate window that you can move around on the screen To locate a detached view click the 3500 task bar icon Specify the default plate type for the Open Plate dialog box Save electronic version of reports Review the Instrument Run views Chapter 3 Set up and run More features in Monitor Run 3 80 3500 3500xL Gene...

Page 81: ...number time for the selected capillary You can select and deselect the dye colors to display The EPT view ElectroPhoresis Telemetry shows instrument data conditions laser power temperatures electrophoresis voltage as a function of time In the legend to the right of the EPT view you can select and deselect the traces to display in the view Array view Sample view EPT view Chapter 3 Set up and run Mo...

Page 82: ...xport sequencing results 90 Modify sequence data 90 Workflow Review sequence quality on page 84 q Re inject samples from Review sequencing results on page 89 q View print and save pdf trace quality reports on page 89 q Export sequencing results on page 90 Access the View Sequencing Results screen Access the View Sequencing Results screen from The Monitor Run screen by clicking Review Results The n...

Page 83: ...click Open Trace to display the data in the Trace pane Note The basecaller version listed in the basecalling protocol is limited to a 3 digit number The version listed in sequencing results is a 4 digit number The fourth digit is an internal number used by the software Chapter 4 Review sequencing results Review results for the currently running sequencing plate 4 3500 3500xL Genetic Analyzer User ...

Page 84: ...ent run click Import then select the samples to review Review sequence quality 1 Display Metric Analysis results to review sample basecalling and trimming results 2 Click the Table Settings button then specify the columns to show or hide 3 Double click column headers to sort columns Multi column sorting is supported see Sort by one or multiple columns on page 76 Chapter 4 Review sequencing results...

Page 85: ...uality Pass fail check determined by the settings in the Basecalling protocol QV Settings tab PUP Score A measure of noise or pull up that is determined by taking the mean of the ratios of signal strength calculated for each base called peak primary peak secondary peak under the primary peak A higher value indicates less baseline or secondary noise A lower value indicates an elevated baseline or s...

Page 86: ...fied No analysis performed 6 Optional Click Minimize and Restore to collapse and expand the samples table Review traces 1 Select the samples of interest in the samples table then click Open Trace 2 Select items from the trace toolbar to manipulate the trace as needed Place the mouse pointer over a button for the description of the button 3 Optional Modify trace display Use the Tile Viewer options ...

Page 87: ...tions Place mouse pointer in trace to display QV Peak analyzed data QV bar Move slider to scale vertically Place mouse pointer in trace to zoom 6 Click the tabs at the bottom of the trace pane for different views of the data 1 Click the View Thumbnails button to display results as thumbnails Display thumbnails Chapter 4 Review sequencing results Review traces 4 3500 3500xL Genetic Analyzer User Gu...

Page 88: ...s have the same probability of error for the associated basecall 10 q 10 Note the following High quality pure bases typically have QVs of 20 or higher The distribution of quality values for mixed bases differs dramatically from that of pure bases Mixed bases have a maximum QV of 20 Review all mixed base calls QV Pe QV Pe 1 79 0 30 0 10 5 32 0 35 0 032 10 10 0 40 0 010 15 3 2 45 0 0032 20 1 0 50 0 ...

Page 89: ...ce Reports then select a report type to view and print 2 Optional Modify report settings You can specify additional report settings in Preferences see Set preferences optional on page 36 3 Double click different elements in the QC report to open the Trace view and display the associated sample 4 To print the report click Print then preview or print 5 To save the report electronically pdf print the...

Page 90: ...est 2 Select an export option Results Reports or Traces 3 Select the export options and the location for the export file then click OK The file s are exported to the specified location with the following naming conventions Results export_ReportName txt Reports ReportName is the format you selected txt xls pdf or html Traces FileName is the format you selected annotation txt phd 1 scf fsta qual or ...

Page 91: ...eview plots 97 Review sizing 100 Re inject samples from Review fragment results 101 View print and save pdf sample quality reports 101 Export sizing results 102 Modify fragment analysis or HID data 102 Workflow Review sample quality on page 94 q Re inject samples from Review fragment results on page 101 q View print and save pdf sample quality reports on page 101 q Export sizing results on page 10...

Page 92: ... Run screen by clicking Review Results The navigation pane by clicking View Fragment Results The Dashboard by clicking View Run Results Chapter 5 Review fragment HID analysis results Access the View Fragment HID Results screen 5 92 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 93: ...es table lists results for completed injections in the current run Select one or more samples in the samples table to display their data in the plot view and sizing table view Chapter 5 Review fragment HID analysis results Review results for the currently running fragment HID analysis plate 5 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 93 ...

Page 94: ...ent Samples view is selected If you are importing HID files click HID Samples Review sample quality 1 In the samples view click the Table Settings button then specify the columns to show or hide 2 Double click Offscale Pull Up fragment Broad Peak HID and SQ columns to sort suspect and failing flags to the top of the table Multi column sorting is supported see Sort by one or multiple columns on pag...

Page 95: ... normalization size standard sample Normalization Factor is not within range No Data Normalization is enabled but Sizing Quality is NO Sample was not collected with a normalization size standard N A Sample was not collected on a 3500 instrument For more information see Review normalized data on page 96 Note If the Sizing Quality is normalization is not applied even if the Normalization Factor is w...

Page 96: ...protocol 5 If the calculated Normalization Factor is within the Normalization Factor range multiplies the peak heights of the sample by the calculated Normalization Factor 6 If the calculated Normalization Factor is outside the Normalization Factor range multiplies the peak heights of the sample by the maximum or minimum Normalization Factor threshold setting for example if the Normalization Facto...

Page 97: ...ge Down in the plot view to move through the samples If the button is dimmed the Plot Settings dialog is open Click the 3500 task bar icon then select Plot Settings 4 Display multiple plots as needed In the Plot Settings Display tab select Checkerboard 5 Click a peak to label it to label all peaks see Label peaks on page 98 1 Place the pointer above the top of the plot or to the left of the plot a...

Page 98: ...g settings are not automatically applied when you access View Results or when you click Apply Not specified default labeling preferences 1 In Labels to Show select the needed labels 2 In Labeling Options Select Show Peak Labels To label all peaks with the selected labels click Label Peaks make sure All is selected To label selected peaks select the category from the Label Peaks list Height Area Si...

Page 99: ... the samples to rename or click the Sample Name column header to select the entire column 2 Click Rename 3 In the Search field enter the sample name to change 4 In the Rename field enter the new name 5 Click Search then click Rename View thumbnails Rename samples Chapter 5 Review fragment HID analysis results Review plots 5 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 99 ...

Page 100: ...umn sorting is supported see Sort by one or multiple columns on page 76 5 Selecting rows in the sizing table then click Label Selected Peaks 1 In the plot view toolbar deselect all dye colors except the size standard dye color red or orange 2 In the sizing table select the size standard peaks of interest 3 Click Label Selected Peaks to label the size standard peaks in the plot view Note If labels ...

Page 101: ...e bottom of the screen 3 Optional Modify report settings 4 To print the report click Print then preview or print Sizing One page per selected sample that shows the quality ranges set in the sizecalling or QC protocol the quality values for the sample and the electropherogram for the sample Plot zooming is not retained in the report Overlay One page for all selected samples that shows the size stan...

Page 102: ...is or HID data To edit modify or further analyze fragment analysis or HID data import the sample data files into a secondary analysis software application such as Fragment analysis GeneMapper Software 5 or later HID GeneMapper ID X Software v1 2 or later Chapter 5 Review fragment HID analysis results Export sizing results 5 102 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 103: ...ctral calibrations 104 Run a spatial calibration 104 Run a spectral calibration 107 Section 6 2 Run an install check 121 Run a Sequencing install check 121 Run a fragment HID install check 130 6 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 103 ...

Page 104: ...if you will be selecting the Fill option for the calibration fill the array with polymer 2 Select Calibration4Spatial Note The screen does not display results unless you have previously performed a spatial calibration 3 Select No Fill or select Fill to fill the array with polymer before starting the calibration 4 Select Perform QC Checks 5 Click Start Calibration During the calibration the softwar...

Page 105: ...peak No off apex markers An even peak profile all peaks about the same height 2 If the results meet the criteria above click Accept Results If the results do not meet the criteria above the Accept button is dimmed Go to Spatial calibration troubleshooting on page 271 IMPORTANT Do not log off or close the software before clicking Accept Results Spatial calibration results are not saved until you cl...

Page 106: ... then click Save The export file contains the following results Capillary Number Position pixels Spacing Intensity Example spatial profiles Export spatial calibration results Chapter 6 Run calibrations and install checks Run a spatial calibration 6 106 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 107: ... the custom dye set from the Dye Set list The AnyDye selection in the Dye Set list contains default settings It does not correspond to custom dye sets created with the AnyDye dye set template Perform a spectral calibration when you Use a dye set that you have not previously calibrated Replace the capillary array for maintenance purposes Replace the capillary when it expires the expiration date is ...

Page 108: ...y the software Pre heating helps mitigate subtle first run migration rate effects The pre heat function automatically turns off after 2 hours We recommend that you pre heat the oven for at least 30 minutes before you start a run if the instrument is cold Temperatures are displayed in red as they warm to the set points When temperatures are at the set point they are displayed in green Temperatures ...

Page 109: ...software for the install check 8 capillary 96 well plate A1 through H1 24 capillary 96 well plate A1 through H1 A2 through H2 and A3 through H3 24 capillary 384 well plate Note 384 well plates are not supported on 8 capillary instruments Columns 1 3 and 5 in rows A C E G I K M O 2 Briefly centrifuge the plate that contains the standards Chapter 6 Run calibrations and install checks Run a spectral ...

Page 110: ...nstrument 5 Prepare the plate assembly as described in Prepare the plate assembly on page 53 Load the plate in the instrument 1 Click the Tray button on the front panel to move the autosampler to the front position then open the instrument door 2 Place the plate in the autosampler with the labels facing you or the instrument door and the notched corner of the plate in the notched corner of the aut...

Page 111: ...w previous calibration data click History View 2 Select the number of wells standard and dye set 3 Select the plate position for the plate loaded in the instrument Note You do not create a plate in the software for the calibration 4 Specify the starting well for the injection position in which you loaded the standard in the plate Perform a spectral calibration Chapter 6 Run calibrations and instal...

Page 112: ...ructs the software to automatically replace information from failed capillaries with information from an adjacent passing capillary with the highest Quality value For more information see What you see during a spectral calibration on page 115 7 Click Start Run The following occurs If you used the default setting Perform run 2 and 3 if run 1 fails the instrument sets up three injections see What yo...

Page 113: ...the matrix standard contains artifacts leading to the creation of one or more extra peaks The extra peaks cause the true dye peak to be missed by the algorithm and can lead to a higher Quality Value than would be computed with the correct peak Therefore it is important to visually inspect the spectral calibration profile for each capillary Condition Number A Condition Number indicates the amount o...

Page 114: ...e blue green yellow red orange 6 dye blue green yellow red purple orange Order of the peaks in the raw data profile from left to right Sequencing matrix standard only 4 dye red yellow blue green Order of the peaks in the raw data profile from left to right Fragment analysis 4 dye red yellow green blue 5 dye orange red yellow green blue Extraneous peaks in the raw data profile intensity vs scan Non...

Page 115: ...er above the top of the plot or to the left of the plot at the start of the area you want to zoom then click to turn the pointer to 2 With the still above the plot or to the left of the plot click drag to the end of the area you want to zoom Do not drag the inside the plot area Doing so changes back to a pointer and does not zoom as expected You can also click zoom and fit buttons to zoom A spectr...

Page 116: ...njection 2 is performed Injection 2 The software evaluates the Quality Value for each capillary across injections 1 and 2 and uses the information from the capillary with the highest Quality Value If all capillaries now pass the calibration is complete and injection 3 is not performed If the same capillary fails in both injection 1 and 2 injection 3 is performed Injection 3 The software evaluates ...

Page 117: ...1 and 2 and uses the information with the highest Quality Value for each capillary If all capillaries pass the calibration is complete and injection 3 is not performed If after borrowing 1 or 3 capillaries from injection 1 or 2 do not pass injection 3 is performed Injection 3 The software evaluates the quality values between adjacent capillaries in injection 3 and for each capillary across injecti...

Page 118: ...ed from Sequencing Standard Dye Set G5 created from Matrix Standard Set DS 33 Example spectral calibration data Chapter 6 Run calibrations and install checks Run a spectral calibration 6 118 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 119: ...e date on which a capillary array is installed for the first time on the instrument Install standard reports use the most recent install date if a capillary array was removed and re installed on the instrument 1 Click View Report 2 Optional In the Report screen click toolbar options to manipulate the report Place the mouse pointer over an item for a description of the item 3 To print the report cl...

Page 120: ...inter 4 Specify a name and location for the report Only the most recent spectral calibration for each dye set is maintained in the software Select History View then select a dye set to view the associated calibration history Save historical reports pdf for record keeping View the spectral calibration history Chapter 6 Run calibrations and install checks Run a spectral calibration 6 120 3500 3500xL...

Page 121: ...les status on page 33 Ensure that Consumables are not expired Adequate injections remain for consumables 2 Ensure that buffer levels are at the fill lines see Check buffer fill levels on page 34 3 Set the oven temperature then click Start Pre heat Pre heat the oven and detection cell while you prepare for a run detection cell temperature is set by the software Pre heating helps mitigate subtle fir...

Page 122: ...he example below shows injection position 1 but you can specify the starting well for an injection position IMPORTANT You do not create a plate in the software for the install check 8 capillary 96 well plate A1 through H1 24 capillary 96 well plate A1 through H1 A2 through H2 and A3 through H3 24 capillary 384 well plate Note 384 well plates are not supported on 8 capillary instruments Columns 1 3...

Page 123: ...ove the autosampler to the front position then open the instrument door 2 Place the plate in the autosampler with the labels facing you or the instrument door and the notched corner of the plate in the notched corner of the autosampler 3 Close the instrument door to initialize the instrument 1 Access the Sequencing Install Standard screen 2 Select the chemistry type Note BDTv3 1 with POP 6 polymer...

Page 124: ...to A01 This is a display issue only the starting well you specify is used for the install check 5 Optional If you have not already run a spectral calibration select Keep Spectral Calibration Data to save the sequencing install standard run if it passes as a spectral calibration Note The spectral calibration record will only be saved if the Keep Spectral Calibration Data option is checked on the sc...

Page 125: ...e instrument performs one run then evaluates Spectral data if you specified to keep spectral data Sequence data What you see during a sequencing install check Chapter 6 Run calibrations and install checks Run a Sequencing install check 6 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 125 ...

Page 126: ... values shown in this figure are examples only Figure 10 Capillary Run Data The software evaluates the Quality Value and Condition Number for each capillary for more information see Spectral Quality Values and Condition Numbers on page 113 Borrowing is automatically enabled 1 borrowing event is allowed for 8 capillary instruments Up to 3 borrowing events are allowed for 24 capillary instruments Fo...

Page 127: ...racy is determined by basepair comparison between the base called sample sequence and the known reference sequence of the sequencing install standard within the contiguous read length region calculated as described in the CRL definition above Read Length The length of read in bases at which base calling accuracy is 98 5 The Read Length is measured only within the following regions BDTv1 1 20 619 b...

Page 128: ...d distinct Peak apexes are separate and distinct the tails will overlap 3 Optional Review the CRL Basepair Accuracy to determine discrepancies from the reference sequence If you observe large discrepancies for example 5 10 contiguous miscalled bases in the middle of a sequence review the data If you see a raw data peak larger than the adjacent peaks with baseline pull up in all 4 dye color channel...

Page 129: ...elect CutePDF as the printer b Click Reject Results For troubleshooting information see Sequencing install standard troubleshooting on page 274 IMPORTANT If you reject results the spectral calibration is not saved Select History View then select an install standard to view the associated install check information Example sequencing install check results View previously run install standards Chapte...

Page 130: ...sults Only the most recent results are maintained in the software 1 Click View Report 2 Click Print 3 In the Printer dialog box select CutePDF Writer as the printer 4 Specify a name and location for the report Run a fragment HID install check If an install check is not performed when your instrument is installed you must perform an install check before you can run plates We recommend that you run ...

Page 131: ...es may fluctuate slightly when they reach the set point as they stabilize 4 Check the pump assembly for bubbles and run the Remove Bubble wizard if needed see Remove bubbles from the polymer pump on page 237 Prepare the fragment HID install check standard Prepare the standard as described in the product insert See Appendix D Catalog numbers for catalog numbers Fragment analysis DS 33 GeneScan Inst...

Page 132: ...contents of any well contain bubbles or are not located at the bottom of the well briefly centrifuge the plate remove the plate from the centrifuge and verify that each standard is positioned correctly in the bottom of its well 4 Store the plate on ice until you prepare the plate assembly and load the plate in the instrument 5 Prepare the plate assembly as described in Prepare the plate assembly o...

Page 133: ...r of the autosampler 3 Close the instrument door to initialize the instrument 1 Access the Fragment Install Standard or HID Install Standard screen 2 Select the plate type number of wells 3 Select the plate position in the instrument Note You do not create a plate in the software for the install check Perform the fragment HID install check Chapter 6 Run calibrations and install checks Run a fragme...

Page 134: ...e number of observed allele and size standard peaks The Capillary Run Data display is updated after the run is complete The number of observed size standard and allele peaks is shown Results for each allele are shown at the bottom of the screen in the Run Information table Number of peaks per capillary Plot and allele size height for selected capillary Allele results for all capillaries What you s...

Page 135: ...expected alleles Pass Fail Alleles with a sizing precision and minimum peak height that do not meet thresholds fail For information only Nominal Size Expected allele fragment peak size bp Mean Average fragment size for the observed allele peaks Peak Height Min Percentage of observed allele peaks with a peak height above the minimum threshold Sizing Accuracy Difference between the expected allele s...

Page 136: ...eck information Example fragment install standard results Example HID install standard results View previously run install standards Chapter 6 Run calibrations and install checks Run a fragment HID install check 6 136 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 137: ...the Report screen click toolbar options to manipulate the report Place the mouse pointer over an item for a description of the item 3 To print the report click Print 4 To save the report electronically pdf print the report and select CutePDF Writer as the printer IMPORTANT Save a report electronically for record keeping The software does not save historical results Only the most recent results are...

Page 138: ...ed plate templates that you can use to create plates for each run Assays Contains factory provided assay templates that you cannot modify You can also create new assays Optional Filename Conventions Contains factory provided file name conventions that you cannot modify You can also create new file name conventions Optional Results Groups Contains factory provided results groups that you cannot mod...

Page 139: ...llowing symbols Factory provided Cannot be edited or deleted Template Locked If the SAE module is enabled on your system a locked item can be unlocked and modified by the user who created it the administrator or another user with unlock permissions For information see Chapter 8 Use Security Audit and E Sig functions SAE Module General library procedures Click the Library tab to access the Library ...

Page 140: ...en you select an item to include in a higher level item a copy of that item is included in the higher level item For example when you select an instrument protocol to include in an assay a copy of the instrument protocol is included in the assay If you delete the instrument protocol the copy of the instrument protocol in the assay remains intact For information on how deleted items are tracked in ...

Page 141: ...s enabled for e sig Note Factory provided items do not list creation date in the audit history If you duplicate a factory provided item the new item contains an audit history that starts with the duplication date listed as the creation date 3 For more information see Display audit histories on page 204 Sort by one or multiple columns Double click column headers to sort To sort by multiple columns ...

Page 142: ...al file name conventions and optional results groups to wells in the plate If you add these items from the library a copy of the items is added to the plate and can be modified independently from the original items stored in the library For information on how changes are tracked if auditing is enabled see Audit action on page 205 Plate templates The Plates library includes templates that specify t...

Page 143: ...ary filtering user preference Click Disable Filters to show all items in the list 2 Click Create The software switches to the Workflow tab 3 To create a new plate specify settings Define plate properties on page 144 To create a new plate based on an existing plate click New Plate then select an option Select a plate click Open then specify settings 4 Select a Save option Create a new plate Chapter...

Page 144: ...odule and primary analysis protocol needed to collect data and basecall or sizecall a sample Assays File Name Conventions and Results Groups may already be listed in the plate template when you create a plate from a template An assay contains One or more instrument protocols appropriate for the sample type dye set for which the assay will be used A primary analysis protocol that depends on your ap...

Page 145: ...r preference Click Disable Filters to show all items in the list 2 Select an assay 3 Click Edit Note You can also create an assay from the Assign Plate Contents screen Create a new assay Chapter 7 Manage library resources Assays library 7 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 145 ...

Page 146: ...Name Name of the assay Locked Prevents the item from being edited Color Color code for the assay when it is displayed in the Assign Plate Contents screen if Assay Color is selected for Show In Wells Application Type Sequencing or Fragment Chapter 7 Manage library resources Assays library 7 146 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 147: ...s on page 181 QC Protocol HID Protocol for primary analysis peak detection and sizing and quality determination For information see QC protocols library primary analysis HID on page 185 File Name Conventions library A File Name Convention FNC specifies the naming convention for sample data files It is an optional component in a plate If you do not specify a file name convention data files are name...

Page 148: ... tracked if auditing is enabled see Audit action on page 205 If factory provided file name conventions do not suit your needs you can create new file name conventions 1 Access the File Name Conventions library 2 Click Create Note You can also create a file name convention from the Assign Plate Contents screen Create a new file name convention Chapter 7 Manage library resources File Name Convention...

Page 149: ...ou select attributes the software displays a preview of the file name 4 To add delimiters between items in the Selected Attributes list a Ctrl click or Shift click to select two or more attributes b Select a delimiter c Select the Add between attributes check box d Click Add 5 Save the file name convention If you are creating the file name convention from the Library click Save If you are creating...

Page 150: ...vention Names must be unique Locked When enabled allows the entry to be unlocked and modified only by the user who created it the administrator or another user with unlock permissions Chapter 7 Manage library resources File Name Conventions library 7 150 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 151: ...f Run run start time Unique Time Stamp Integer numeric string in milliseconds that does not correspond to the current time User defined Fields up to 5 specified in Assign plate contents on page 44 User Name available only when security is enabled in the SAE module Well Position IMPORTANT The maximum allowed length of a file name including the path is 240 characters The software warns you if your s...

Page 152: ...d in the library For information on how changes are tracked if auditing is enabled see Audit action on page 205 To accurately genotype samples the GeneMapper ID X Software requires at least one allelic ladder sample per run folder Multiple allelic ladder samples in a single run folder can also be used for analysis We recommend that you run one allelic ladder for a set of 24 samples 8 capillary ins...

Page 153: ...ps 1 Access the Results Group library 2 Click Create Note You can also create a results group from the Assign Plate Contents screen Create a new Results Group Chapter 7 Manage library resources Results Group library 7 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 153 ...

Page 154: ... Ctrl click or Shift click to select two or more attributes b Select a delimiter c Select the Add between attributes check box d Click Add 5 Save the results group If you are creating the results group from the Library click Save If you are creating the results group from the Assign Plate Contents screen click Apply to Plate or Save to Library Chapter 7 Manage library resources Results Group libra...

Page 155: ...nique The Results Group Name is a required attribute you cannot remove this attribute from the Selected Attribute list Locked When enabled allows the entry to be unlocked and modified only by the user who created it the administrator or another user with unlock permissions Chapter 7 Manage library resources Results Group library 7 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3...

Page 156: ...de in the results group name Dash Dot Underscore _ Plus Dollar Prefix suffix text Text to display for the prefix or suffix text attribute fields Select re injection folder option Store reinjection sample files in a separate reinjection folder same level as injection folders Store reinjection sample files with original sample files same level Select folder option Location Default file location spec...

Page 157: ...G results group and the folders created when it is used This results group does not create a folder for each injection All samples for a plate are stored in the same folder If you include two plates in a run a separate folder is created for each plate Figure 14 PN_Injfolder_RG results group Figure 15 PN_RG results group Results group example 1 store files by plate name Chapter 7 Manage library res...

Page 158: ...mbers in the figure relate the elements in the results group with the elements in the file hierarchy created by a run that uses this results group see Figure 19 Figure 16 Results group example Figure 17 shows the injection list for a run that specifies duplicate and re injections Results Group example 2 store re injections in separate folders Chapter 7 Manage library resources Results Group librar...

Page 159: ...t specifies a sample name syntax of sample name primary analysis protocol name unique time stamp integer The numbers in the figure relate the elements in the file name convention with the files created by a run that uses file name convention see Figure 19 Figure 18 File name convention example Chapter 7 Manage library resources Results Group library 7 3500 3500xL Genetic Analyzer User Guide Data C...

Page 160: ...on list 6 Results group name syntax from results group 7 File name syntax from file name convention We recommend that you run one allelic ladder for each set of 24 samples see Allelic ladder location HID analysis on page 152 To store one allelic ladder per run folder on an 8 capillary instrument create one results group for each set of three injections on the plate Each results group specifies a r...

Page 161: ...nstrument during data acquisition An instrument protocol is a required element of an assay for all applications When you create an assay you add one or more instrument protocols to the assay If you add these items from the library a copy of the items is added to the assay and can be modified independently from the original items stored in the library For information on how changes are tracked if a...

Page 162: ...ou can create new instrument protocols 1 Access the Instrument Protocols library 2 Click Create Create a new instrument protocol Chapter 7 Manage library resources Instrument protocol library 7 162 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 163: ... click Save If you are creating the assay from the Assign Plate Contents screen click Apply to Plate or Save to Library Instrument protocol settings Setting Description Application Type Sequencing Fragment analysis HID Capillary Length Polymer Dye set Capillary length polymer type and dye set with which the protocol will be used Run module Factory provided modules that specify instrument control p...

Page 164: ...ch Run Voltage Voltage step interval sec Dwell time at each voltage ramp step First read out time ms The interval of time for a data point to be produced First ReadOut time should be equal to Second ReadOut time Second read out time ms The interval of time for a data point to be produced Second ReadOut time should be equal to First ReadOut time Fragment and HID protocols only Normalization paramet...

Page 165: ...0 LIZ v2 size standard used for normalization divided by the Normalization Target Samples are flagged with in results if Normalization Factor is within threshold range or with if it is out of threshold range Dye sets library A dye set defines the following for an instrument protocol Dye color s Order of the dye peaks in the standard Spectral analysis parameters When you create an instrument protoc...

Page 166: ...suit your needs you can create new dye sets 1 Access the Dye Sets library 2 Click Create Create a new dye set Chapter 7 Manage library resources Dye sets library 7 166 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 167: ...to be unlocked and modified only by the user who created it the administrator or another user with unlock permissions Useful when your system includes the SAE module described in Chapter 8 Use Security Audit and E Sig functions SAE Module Chemistry The standard for which you are creating the dye set Sequencing Standard or Matrix standard Chapter 7 Manage library resources Dye sets library 7 3500 3...

Page 168: ...ed Selection Specifies the dyes used in the samples For example if you use the 5 dye kit and have samples with only blue peaks you can reduce or deconvolute with blue and orange size standard dyes only Parameters Specifies the Quality Value Condition Number Scan and Sensitivity requirements for the dye set Notes Optional text entry If factory provided dye sets do not suit your needs you can create...

Page 169: ...er number is Blue 1 Green 2 Yellow 3 Red 4 Purple 5 Orange 6 Example 2 With the Purple dye deselected internal order number is Blue 1 Green 2 Yellow 3 Red 4 Orange 5 the internal order number of Orange changes to 5 Example 3 With the Blue Yellow and Purple dyes deselected internal order number is Green 1 Red 2 Orange 3 the internal order number of Green changes to 1 Red changes to 2 and Orange cha...

Page 170: ...in the calibration standard you are using is Orange Red Green specify for Calibration Peak Order 3 Orange 2 Red 1 Green 6 Expand the Parameters section then specify remaining settings 7 Click Save Size standards library A size standard defines the sizes of known fragments It is used to generate a standard curve The standard curve is used to determine the sizing of unknown samples When you create a...

Page 171: ...pplied to samples with failing sizing quality Select a size standard definition file appropriate for your application that accurately sizes samples For example if your application includes small fragments that may be obscured by primer peaks or large fragments that may not be present due to slower migration rates specify a size standard definition file that eliminates these fragments from sizing F...

Page 172: ...st on the left Separate sizes with a comma space or return 7 Click Add Sizes 8 Click Save Figure 22 Create New Size Standard 1 Select a factory provided normalization size standard indicated in the name with Normalization 2 Click Duplicate 3 Edit the copy of the normalized size standard The size standard peaks used to normalize the data are displayed in gray and are not editable 4 Click Save Modif...

Page 173: ... assay and can be modified independently from the original item stored in the library For information on how changes are tracked if auditing is enabled see Audit action on page 205 If factory provided basecalling protocols do not suit your needs you can create new basecalling protocols 1 Access the Basecalling Protocols library 2 Click Create 3 In the Analysis Settings tab of the Create New Baseca...

Page 174: ...ange the QV flag in Monitor Run is set to green If any values are in the suspect range the QV flag in Monitor Run is set to yellow If any values are in the fail range the QV flag in Monitor Run is set to red View Sequencing Results4Metric Analysis Results table The pass check fail status for Trace Score Quality CRL Quality and QV20 Quality results Chapter 7 Manage library resources Basecalling pro...

Page 175: ...23 Create New Basecalling Protocol Analysis Settings Chapter 7 Manage library resources Basecalling protocols library primary analysis sequencing 7 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 175 ...

Page 176: ...se Security Audit and E Sig functions SAE Module Description Optional text entry Basecaller Basecalling algorithm used to identify bases Note The basecaller version listed in the basecalling protocol is a 3 digit number The version listed in sequencing results is a 4 digit number The fourth digit is an internal number used by the software Mobility file Compensates for mobility differences between ...

Page 177: ... a necessary but not sufficient condition for the basecalling algorithm to call a mixed base Analyzed Data Scaling Determines scaling of the processed traces This parameter does not affect the accuracy of the basecalling True Profile The processed traces are scaled uniformly so that the average height of peaks in the region of strongest signal is about equal to a fixed value The profile of the pro...

Page 178: ...verage basecall Quality Value QV of bases in the clear range sequence of a trace The clear range is the region of the sequence that remains after excluding the low quality or error prone sequence at the 5 and 3 ends The clear range is calculated by the KB basecaller using QVs QV20 The total number of bases in the entire trace with Quality Value 20 Sizecalling protocols library primary analysis fra...

Page 179: ...nalysis settings on page 181 4 Click QC Settings In the QC Settings tab of the Create New Sizecalling Protocol dialog box Figure 26 specify settings Sizecalling protocol QC settings on page 184 Create a new sizecalling protocol Chapter 7 Manage library resources Sizecalling protocols library primary analysis fragment 7 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 179 ...

Page 180: ...C settings with appropriate pass fail ranges The 3500 Series Data Collection Software 3 1 does not support re analyzing data with new settings Figure 25 Create New Sizecalling Protocol Analysis Settings Chapter 7 Manage library resources Sizecalling protocols library primary analysis fragment 7 180 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 181: ... entry Size standard Size standard definition in the software that corresponds to the dye set used in the chemistry To apply normalization select a normalization size standard see Normalization size standards provided on page 171 Chapter 7 Manage library resources Sizecalling protocols library primary analysis fragment 7 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 181 ...

Page 182: ... 3rd Order Least Squares Uses regression analysis to build a best fit size calling curve 2nd Order Least Squares Uses regression analysis to build a best fit size calling curve Cubic Spline Interpolation Forces the sizing curve through all the known points of the selected size standard Global Southern Method Compensates for standard fragments with anomalous electrophoretic mobility similar to leas...

Page 183: ... Polynomial Degree Polynomial Degree cannot be greater than Peak Window Size Adjust to affect the sensitivity of peak detection You can adjust this parameter to detect a single base pair difference while minimizing the detection of shoulder effects and or noise The peak detector calculates the first derivative of a polynomial curve fitted to the data within a window that is centered on each data p...

Page 184: ... Results Results that are within the Pass range are flagged as Pass Results that are within the Fail range are flagged as Fail Results that are between the Pass and Fail ranges are flagged Check For example with a Pass Range of 0 75 to 1 0 and a Fail Range of 0 0 to 0 25 any result 0 75 is any result 0 25 is and any result between 0 25 to 0 75 is How Size Quality is determined The Size Quality alg...

Page 185: ...s enabled see Audit action on page 205 If factory provided QC protocols do not suit your needs you can create new QC protocols 1 Access the QC Protocols library 2 Click Create 3 In the Analysis Settings tab of the Create New QC Protocol dialog box Figure 27 specify settings QC protocol Analysis settings on page 187 4 Click QC Settings In the QC Settings tab of the Create New QC Protocol dialog box...

Page 186: ...tings ensure that the size standard is accurately detected and sized with the new settings IMPORTANT Normalization is not applied to samples with Size Quality flags The 3500 Series Data Collection Software 3 1 does not support re analyzing data with new settings Figure 27 Create New QC Protocol Analysis Settings Chapter 7 Manage library resources QC protocols library primary analysis HID 7 186 350...

Page 187: ...t Full to collect data points for the entire scan region including the primer peak You can specify a limited analysis range in the GeneMapper ID X Software Note If you select Partial ensure that the Analysis Range contains all size standard fragments included in the Sizing Range specified below Sizing Range Select Partial then specify 80 to 400 to limit the fragment sizes evaluated for the size st...

Page 188: ...to smooth the outline of peaks and reduce the number of false peaks detected None to apply no smoothing Best if the data display sharp narrow peaks of interest Light default to provide the best results for typical data Light smoothing slightly reduces peak height Heavy for data with very sharp narrow peaks of interest Heavy smoothing can significantly reduce peak height Baseline Window Specify a w...

Page 189: ...ase peak window size To decrease peak detection sensitivity Decrease polynomial degree increase peak window size Slope Thresholds Peak Start and End Not recommended for use with AmpFℓSTR kit data Peak Start The peak starts when the first derivative slope of the tangent in the beginning of the peak signal before the inflection point becomes equal to or exceeds the Peak Start value This threshold is...

Page 190: ...ple calculates an interim SQ a value between 0 and 1 Weighting The Broad Peak BD threshold specified in the QC Protocol QC Settings tab affects the SQ To determine the final SQ value the software Evaluates size standard peak widths in the sample in the dye color specified in the size standard definition If the width of any size standard peak in the sizing range exceeds the broad peak threshold app...

Page 191: ... auditing and specify the audit mode Provides reports for audited library items SAE changes and actions Electronic signature e sig Determines if users are permitted prompted or required to provide a user name and password when performing certain functions Can be configured so that a predefined list of functions can be performed only if the data used for the functions is signed for example you can ...

Page 192: ...ll user accounts and set up notifications when certain security events occur Access the Security screen Figure 29 Security screen Configure the security system Chapter 8 Use Security Audit and E Sig functions SAE Module Administrators 8 192 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 193: ... password field 4 Specify the required characters in passwords spaces and alpha numeric upper lower case and special characters any non space non alpha or non numeric characters 5 Specify password reuse You cannot disable the password reuse restriction Note Do not disable the Account Suspension feature 6 Click Setup Messaging Notification to specify when and how to notify the administrator of cert...

Page 194: ... In the Security screen Figure 29 click Setup Messaging Notifications to display the Setup Notifications dialog box 2 Select the events for notification Number of failed authentications over specified time interval A user attempts to log in with an incorrect password The message indicates the number of failed authentications Session timeout for a user No activity occurred in a user account for the...

Page 195: ...vents indicating the time each event occurred and the user who triggered the event The Administrator has the option of acknowledging the event which removes it from the notification list 4 Click OK Create or edit a user account The software includes a default Administrator user account with permissions defined by the account user role to perform all functions in the software Create a user account ...

Page 196: ... electronic signature state determines if a user account has permission to electronically sign objects Leave the status set to Active Note The Dx User function is not supported 6 Optional Enter email for information only phone and comments 7 Click Save If the Save button is dimmed it indicates an invalid entry in a field Click a field to display the limits for the field then enter a valid entry Th...

Page 197: ...atus from Active to Inactive 4 Click Save Determine the name of the logged in user To display the full name of the logged in user Place the mouse pointer on the Logout menu The full name of the logged in user is also displayed in the Load Plates for Run screen and the Monitor Run screen Create or edit a user role User roles determine the permissions associated with a user account Three default use...

Page 198: ...in a category select the checkbox next to the category 5 Click Save Role User role permissions Category Permissions Setup Create plate plate template Run Start plate run Edit default instrument run name Manage injection list Duplicate injection Re inject Primary Analysis Edit sample names Export sequencing results Chapter 8 Use Security Audit and E Sig functions SAE Module Administrators 8 198 350...

Page 199: ...m preferences Edit user preferences Import user preferences Export user preferences all Calibrations Perform spatial calibration Perform spectral calibration Install check Run install standard check Archiving Archive Purge Restore SAE configuration Log in to timed out user sessions Edit a user role 1 In the Roles screen select a user role then click Edit 2 Edit settings as needed You cannot edit t...

Page 200: ... over an item for a description of the item 3 To print the report click Print Close the report Save electronic copies pdf of the report To save the report electronically pdf print the report and select CutePDF Writer as the printer Chapter 8 Use Security Audit and E Sig functions SAE Module Administrators 8 200 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 201: ... the reasons available to users when audit mode is set to Prompt or Required Access the Audit Settings screen Figure 30 Audit Settings Manage auditing Chapter 8 Use Security Audit and E Sig functions SAE Module Administrators 8 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 201 ...

Page 202: ...ems that the system audits silently in addition to the configurable items listed above see Generate audit reports on page 204 2 Set the Audit Mode for each item you enable for auditing Prompt The event is audited a reason prompt is displayed but the user can cancel and continue without entering a reason Required The event is audited a reason prompt is displayed and the user must specify a reason S...

Page 203: ...defined reason in the Audit Reason dialog box displayed when a user performs an audited action select the Select a reason from the list for your change checkbox Users are not permitted to enter a reason 2 As needed click Create or select a reason then click Edit or Delete Chapter 8 Use Security Audit and E Sig functions SAE Module Administrators 8 3500 3500xL Genetic Analyzer User Guide Data Colle...

Page 204: ...guration History SAE configuration records including audit history for each user account Action log System specified audit events 3 Optional Modify the display Sort the table See Sort by one or multiple columns on page 76 Specify filters date range user name action object or record type object or record name reason then click Go Note The Reason field in System Configuration History is not used Sel...

Page 205: ... exists and asks if you want to overwrite it You click Yes This action is considered a creation of a new results group not a modification of the existing results group No Update record is created a Create record is created Create A record is created when you Create an item in the library Create an item from within another item Modify an item from within another item then overwrite the item in the ...

Page 206: ...e security Modify security policies Session timeout settings Account settings Update Modify user name settings Modify password settings Modify security policies Password expiration Account suspension Audit reason for change Update Modify reason for change Create Create reason for change Delete Delete reason for change Audit settings Update Enable auditing Disable auditing Audit type Update Modify ...

Page 207: ... user role Delete Delete user role Action log The action log lists system specified audit events All items in the action log are audited silently except for the items noted as configurable Configurable items may include comments in the action log Table 2 Audit Action log Category Action Assay Assay exported successfully Log In User logged in Login failed User logged out Maintenance Wizards Remove ...

Page 208: ...n Records Archive Purge Restore User Profile Export View and print audit reports 1 Display the records of interest 2 Filter the list to decrease the time required to generate reports IMPORTANT You cannot cancel a report after you click a view button Chapter 8 Use Security Audit and E Sig functions SAE Module Administrators 8 208 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1...

Page 209: ...ANT Font setting changes are activated after you close then reopen the report IMPORTANT If you change font settings before you generate a report the report may not be generated Generate the report again 6 To save the report electronically pdf print the report and select CutePDF Writer as the printer Chapter 8 Use Security Audit and E Sig functions SAE Module Administrators 8 3500 3500xL Genetic An...

Page 210: ...o restore system configuration or action audit records click Restore then select the asz file to restore Export audit records As needed you can export audit records to a txt file for additional manipulation and reporting outside the software 1 Display the records of interest 2 Select the records to export 3 Click Export Audit Records 4 Specify a name and location for the export txt file 5 Click Sa...

Page 211: ...oftware then log back in Access the E Signature Settings screen Access the E Signature Settings screen Manage electronic signature E sig Chapter 8 Use Security Audit and E Sig functions SAE Module Administrators 8 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 211 ...

Page 212: ...ers when they perform a function Users can sign or can continue without signing b From the bottom right of the screen select a function for example start run before which the system will check for required e sigs This selection presents an e sig prompt to users when they start a run if the required signatures have not previously been made Users must sign before they can continue For check before f...

Page 213: ...e Save Approve Sequencing Install Standard Results Accept Approve MicroSeqID Install Standard Results Accept Approve BDTv1 1 POP6 Install Standard Results Accept Approve Fragment Install Standard Results Accept Approve HID Install Standard Results Accept Table 4 E signature settings functions to check before E Signature type Function to check before Signatures and authorities required defaults if ...

Page 214: ...ig system is configured to require signatures from two users one from the user account named Administrator and the other from any user account with a scientist user role for a spatial calibration before it can be used in a run The spatial calibration has not been signed A user starts a run The following message is displayed Before the run can start the following users must sign The Administrator u...

Page 215: ...ords that are displayed if they are specified in E Signature settings are Approve Dye Set Approve Size Standard Approve Spatial Calibration Approve Spectral Calibration Approve Instrument Protocol Approve Sizecall Protocol Approve Basecall Protocol Approve QC Protocol Approve Assay Approve Plate Template Approve Plate Approve Sample Approve BDTv1 1POP6 Install Standard Results Approve Sequencing I...

Page 216: ...ave the report electronically pdf print the report and select CutePDF Writer as the printer 6 Close the report Export e sig records As needed you can export e sig records to a txt file for additional manipulation and reporting outside the 3500 Series Data Collection Software 3 1 1 Display the records of interest as described in Display e sig records on page 215 2 Select the records to export 3 Cli...

Page 217: ...ins all settings in the following screens Security Account setup and security policies Audit Objects selected for auditing audit modes and reasons E Signature Settings Objects selected for E Signature functions number of signatures and authorities User Roles All user roles and associated permissions 3 Click Export 4 Specify the name and location for the exported dat file then click Save A message ...

Page 218: ...s the following functionality System security Controls user access to the software Auditing Tracks changes made to library items actions performed by users and changes to the SAE settings Electronic signature e sig Requires users to provide a user name and password when performing certain functions Depending on the way that your administrator configures these features you may see the following dia...

Page 219: ...er account does not have permission to perform a function in the software the associated menu commands are dimmed Determine the name of the logged in user To display the full name of the logged in user Place the mouse pointer on the Logout menu The full name of the logged in user is also displayed in the Load Plates for Run screen and the Monitor Run screen Change your password when it expires Whe...

Page 220: ...unt status from Suspended to Active Note While a user is suspended a different user can click Reset then log in Session timeout If your system is configured to timeout and there is no user activity for the specified time the Log In dialog box indicates that your user session has timed out You must enter your user name and password to access the software Note The administrator or another user with ...

Page 221: ...o sign at the same time When the first signer signs the E Sig status is set to Partially Signed When the second signer signs the E Sig status is set to Signed You may also be permitted to sign objects such as plates calibrations or other library items If e sig is enabled for items any of the following may apply The E Signature button is enabled in the library or the calibration You are prompted to...

Page 222: ... or the library displays an Is signed column that reflects the e sig status of an item Chapter 8 Use Security Audit and E Sig functions SAE Module Users 8 222 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 223: ...lists the common tasks required to maintain your Applied Biosystems 3500 3500xL Genetic Analyzer in good working condition Wear appropriate protection including gloves laboratory goggles and coat whenever you work with the fluids used on this instrument or parts that may come into contact with these fluids IMPORTANT Use only the cleaning agents listed in this guide Use of cleaning agents other tha...

Page 224: ...fluid inside the anode buffer container and the cathode buffer container The fluid must line up with the fill line Install the anode buffer container ABC on page 229 Install the cathode buffer container CBC on page 230 Ensure proper installation of CBC septa on page 35 Ensure that the CBC septa are properly seated on the container Ensure that the plate assemblies are properly assembled Align the h...

Page 225: ... pump Figure 31 Restart the computer and instrument Restart the instrument and the computer on page 251 Monthly instrument maintenance tasks Task Frequency For information see Run install check Monthly or as needed Run an install check on page 121 Flush the pump trap Flush the water trap pump trap on page 238 Empty the oven condensation reservoir Figure 31 Replace cathode buffer container septa In...

Page 226: ...see Review the Notifications Log on page 228 To access the maintenance calendar click the Maintenance tab then click Schedule A set of recommended tasks are scheduled in the calendar flagged with FR Factory Repeating in the monthly view and F Factory in the daily view User specified repeating tasks are flagged with R Repeating in the monthly view Weekly factory repeating tasks in calendar Mothnly ...

Page 227: ...ents in the calendar The Month and Day tabs allow you to view your schedule in different formats Click Detach to move the calendar window 1 Click the Maintenance tab then click Schedule 2 Click Planned Maintenance Report 3 Specify the date range then click OK 4 Select Print as needed 5 To save the report electronically pdf print the report and select CutePDF Writer as the printer Create calendar e...

Page 228: ...w calendar reminders on page 223 1 Access the Notifications Log 2 View the Notification Log Report and print as needed Note Multi column sorting is supported see Sort by one or multiple columns on page 76 Chapter 9 Maintain the Instrument Review the Notifications Log 9 228 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 229: ...nt free tissue 6 Clean out the drip trays with deionized water or ethanol and lint free tissue Note The drip tray can be removed Install buffers IMPORTANT Wear appropriate protection including gloves laboratory goggles and coat whenever you work with the fluids used on this instrument or parts that may come into contact with these fluids IMPORTANT Use only the parts listed in Appendix D Catalog nu...

Page 230: ...rately by the instrument 8 Close the instrument door to re initialize 9 In the Dashboard click Refresh then check the Quick View section for updated status 1 Check the expiration date on the label to ensure it is not expired and will not expire during use 2 Allow refrigerated CBC to equilibrate to ambient temperature 3 Wipe away condensation on the CBC exterior with a lint free tissue Condensation...

Page 231: ...m on each side of the CBC a Align the buffer septum the part that is symmetrical over the 24 holes of the CBC b Push the septum lightly into the holes to start then push firmly to seat it c Align the capillary washing septum over the other chamber of the CBC d Push the septum lightly into the holes to start then push firmly to seat it IMPORTANT Look at the CBC from the side and ensure that there i...

Page 232: ... example if you remove a partially used polymer pouch from an 8 capillary instrument do not reuse that polymer on a 24 capillary instrument If you remove a polymer pouch for storage 2 8 C place a pouch cap onto the pouch then place an empty pouch or conditioning reagent on the connector to prevent desiccation of any residual polymer on the connector Follow the instructions in the wizard to ensure ...

Page 233: ...ouch fitment Note You may notice a tiny droplet of polymer inside the fitment residual from the pouch filling process This is not expected to cause any performance issues Chapter 9 Maintain the Instrument Replenish change flush and store polymer 9 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 233 ...

Page 234: ...ng reagent on the connector to prevent desiccation of any residual polymer on the connector Follow the instructions in the wizard to ensure proper operation of the pouch and the instrument 1 In the Dashboard click Wizards 2 In the Maintenance wizards screen click Fill Array with Polymer 3 Follow the prompts in the Fill Array wizard window 4 Click Refresh in the Dashboard to update the screen 5 Che...

Page 235: ...e loading end header to ensure that the capillary tips are not crushed or damaged Note The Install Capillary Array wizard takes 15 45 minutes to complete 1 In the Dashboard click Wizards 2 In the Maintenance wizards screen click Install Capillary Array 3 Follow the prompts in the Install Capillary Array wizard window 4 Check the Quick View section of the Dashboard for updated status of the capilla...

Page 236: ...e clean powder free silicone free latex gloves whenever you handle the pump assembly or any item in the polymer path The polymer delivery pump can be irreversibly damaged if Polymer dries in the polymer channels of the pump assembly which can scratch the channels in the pump and can cause blockage The pump assembly is exposed to organic solvent which can cause cracking and clouding of the acrylic ...

Page 237: ...t Cat No 4414007 in addition to the Wash Pump wizard to thoroughly clean the polymer delivery pump Polymer has dried in the channels of the lower polymer block Mechanical malfunctions may cause dried polymer to appear in the polymer delivery pump Washing with either the Wash Pump Chamber and Channels wizard or this kit may not remove dried polymer the lower polymer block may need to be replaced by...

Page 238: ...iner Dispose of the excess water inside the overflow container See Chemical safety on page 308 Note Leave the trap filled with either distilled or deionized water 1 Fill the supplied 20 mL all plastic Luer lock syringe in the Polymer Delivery Pump Cleaning Kit Cat No 4414007 with distilled or deionized water Expel any bubbles from the syringe IMPORTANT Do not use a syringe smaller than 20 mL Doing...

Page 239: ...roximately 30 seconds to flush 5 mL of either distilled or deionized water through the trap Note Because the water trap volume is approximately 325 μL a relatively small volume of water is adequate for complete flushing However a larger volume improves flushing as long as force and flow rate are kept within the limits given above 6 Remove the syringe from the Luer fitting Hold the fitting with one...

Page 240: ...onto the instrument before performing instrument shutdown If the instrument will be unattended for Perform this shutdown procedure 1 week No action is required 1 to 2 weeks Keep the load end of the capillary array in 1X buffer to prevent the polymer from drying in the capillaries If fluid level is low add DI water to buffer solution Install the new CBC when ready to resume runs 2 weeks 1 Run the I...

Page 241: ...y Click Shutdown the Instrument in the Maintenance Wizards See Shutdown the instrument on page 240 Anode buffer reservoir 2 Switch off the circuit breaker on the back of the instrument 3 Disconnect the power cord and the Ethernet cable IMPORTANT While moving the instrument avoid any shock or vibration 4 Move the instrument 5 Turn the instrument legs to level the instrument To move the instrument c...

Page 242: ...MPORTANT Do not uninstall the software unless instructed to do so by Thermo Fisher Scientific When you uninstall the software you are prompted to back up the datastore the directory that contains all library items you created such as plates and protocols Select a location other than the install directory for the datastore backup IMPORTANT Do not back up the datastore to the installation directory ...

Page 243: ...ystem IMPORTANT These functions affect items stored in the library datastore These functions do not affect sample data files Frequency We recommend that you purge the library objects once every three months Archive library items 1 Access the Archive screen 2 Specify the date category specify a date range that is earlier than the date on which you made the duplicates of the library items you want t...

Page 244: ...export audit records for samples that are not in their original location samples have been deleted or moved an error message is displayed Return sample data files to their original location then export again 2 Copy the archive to a network or external drive Restore This function restores items archived from the library To restore audit records see Archive purge and restore audit records on page 21...

Page 245: ...es in the Purge warning message A message is displayed when all records are deleted Ensure that you have sufficient drive space by regularly Archiving data Deleting unneeded files Emptying the trash Defragmenting the drives Automatic disk space check before a run Before a run the software checks free disk space and displays a message when the hard disk is 70 75 full At 78 full the software will no...

Page 246: ... your license for the new network card or computer You must use the same email address to activate and renew software licenses Create an email address that is routinely monitored by your team rather than a single individual Use this email address to activate and renew software licenses The 3500 Series Data Collection Software 3 1 Software Activation dialog box is displayed when you start the softw...

Page 247: ...e license when it expires 5 Obtain the software license file from your email 6 Make a copy of the software license file and keep in a safe location 7 Copy the software license file to the desktop of the 3500 Series Data Collection Software 3 1 computer 8 If the Software Activation dialog box has closed start the 3500 Series Data Collection Software 3 1 to open it see Step two Start the 3500 Series...

Page 248: ...ate and renew the software license If you do not have the activation email address available enter any email address click the licensing link in the Software Renewal dialog box then click Contact Support in the License Renewal web page 4 Request the renewed software license file by performing step 1c as listed on the renewal screen 5 Obtain the renewed software license file from your email 6 Copy ...

Page 249: ...he license is installed and validated 10 Click Close Service Log and Usage Statistics The Service Log and Usage Statistics functions are for use by Thermo Fisher Scientific service engineers at the time of service Chapter 9 Maintain the Instrument Service Log and Usage Statistics 9 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 249 ...

Page 250: ...ooting 272 Sequencing install standard troubleshooting 274 Fragment HID install standard troubleshooting 275 Monitor Run troubleshooting 276 Audit troubleshooting 277 Electronic signature troubleshooting 277 Manual commands troubleshooting 278 Troubleshooting procedures 278 If you encounter any unforeseen and potentially hazardous event while operating the instrument turn off the power unplug the ...

Page 251: ... then power off the instrument 4 When the computer is completely powered off wait 60 seconds then power on the computer Wait until the Windows login screen is displayed Do not log in 5 Power on the instrument and wait until the green status light on the front panel is on and not flashing before proceeding 6 Log in to Windows operating system 7 Look in the Windows taskbar at the bottom right of the...

Page 252: ...rior 1 Detection cell heater block 2 Polymer delivery pump PDP 3 Anode buffer container ABC 4 Polymer or conditioning pouch 5 Cathode buffer container CBC 6 Oven door 7 Capillary array 8 Oven condensation reservoir 9 Autosampler Appendix A Troubleshoot Instrument components A 252 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 253: ...e 3 Lower polymer block 4 Anode buffer container ABC 5 Array locking lever 6 Water trap waste container 7 Check valve fitting 8 Polymer pouch lever 9 Polymer pouch 10 Drip tray Appendix A Troubleshoot Instrument components A 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 253 ...

Page 254: ...ion of CBC septa on page 35 Autosampler does not move the plate to a higher position Array electrodes are bent The plate is not aligned correctly resulting in the array tips missing center of septa The plate retainer may not be snapped onto the plate base Ensure that the plate retainer plate or tube strip and plate base are assembled correctly Listen for a snap when the plate retainer and the plat...

Page 255: ...emely noisy and vibrating while running any wizard The array locking lever is not in the correct position IMPORTANT If the lever is not in the correct position you will receive Leak error message Lock the lever in the correct position If this is not possible contact Thermo Fisher Scientific Polymer delivery pump block is not pushed back into position after capillary array change Gently push the bu...

Page 256: ...ogged Crystals present in polymer delivery pump path Run the Wash Pump and Channels wizard See Flush the water trap pump trap on page 238 and Wash the pump chamber and channels on page 237 If the problem persists contact Thermo Fisher Scientific 1 2 Figure 35 Pump chamber and valve fitting 1 Debris in chamber 2 Check valve fitting Appendix A Troubleshoot Instrument troubleshooting A 256 3500 3500x...

Page 257: ...ce schedule on page 223 Fluid does not move through the polymer delivery pump and into the ABC from polymer or conditioning pouch Blockage in fluid path or problem with polymer delivery pump Contact Thermo Fisher Scientific Poor signal and resolution after replenishing polymer The Check Valve is clogged see Figure 35 Wash the channels using the Polymer Delivery Pump Cleaning Kit Cat No 4414007 If ...

Page 258: ...ou remove the heat seal from a new pouch some residual seal remains on top of the pouch The top seal of the pouch has become delaminated and left the polyethylene behind on the pouch cap Use a pipette tip to remove the entire seal from the pouch cap before installing on the instrument RFID troubleshooting Symptom Possible cause Action Unable to read RFID information Failure to Read from RFID tag C...

Page 259: ...or Restart the instrument and the computer see Restart the instrument and the computer on page 251 Electric discharge message during runs The ABC buffer may be low Replace the ABC Ensure that the ABC is being replaced per calendar notifications Leak error message The array locking lever is not in the correct position Secure the array locking lever see Figure 32 Leak error occurs when capillary arr...

Page 260: ...Run the Remove Bubbles wizard to clear bubbles Leak in the polymer system Check for evidence of leaks If polymer leak occurred conduct a water wash and wash the pump trap using the Polymer Delivery Pump Cleaning Kit Cat No 4414007 supplied with the instrument Buffer valve leakage Check the buffer pin valve and see if it closes correctly Clean the buffer pin valve Ensure that the maintenance schedu...

Page 261: ...sage Dashboard troubleshooting Symptom Possible Cause Action When you click Refresh on the Dashboard and consumables information is listed as Unknown Bad connection between the computer and instrument Check the connection between the instrument and computer Consumables status in the Dashboard is not updated Dashboard does not update automatically Click Refresh After installing new CBC or ABC the c...

Page 262: ...erver Monitor Right click the status icon then select Services If any item does not display a checkmark click the item to start the service Print dialog box is not displayed when you select or click Print Dialog boxes are sometimes displayed behind the main screen Minimize the main screen The Load plate for run message does not display correctly The window is not refreshing properly Click OK to di...

Page 263: ...ed the instrument only not the computer Restart the instrument and the computer see Restart the instrument and the computer on page 251 Note Restart the instrument and the computer as part of weekly maintenance Software operations are taking longer than expected Communication problem between the computer and instrument Restart the instrument and the computer see Restart the instrument and the comp...

Page 264: ...tocols are not retained for re injections Before re running a plate examine the protocols specified for re injections and change as needed Data electropherogram troubleshooting Symptom Possible cause Action Signal too high Sample concentration is too high Dilute the sample Decrease the injection time Too much DNA added to the reaction resulting in uneven signal distribution Optimize reaction condi...

Page 265: ...olymer Capillary array used for more than 160 injections Replace the capillary array Run the Install Capillary Array wizard Degraded formamide Prepare fresh Hi Di Formamide see Hi Di Formamide on page 22 for storage conditions for sample preparation Sample has high salt concentration Dilute or desalt samples Poor resolution in some capillaries Insufficient filling of capillary array Tighten the co...

Page 266: ... used for fewer than 100 runs Poor quality samples possible cleanup problems Desalt samples Improperly stored formamide Prepare fresh Hi Di Formamide see Hi Di Formamide on page 22 for storage conditions for sample preparation Leak in system Tighten the connectors and array locking lever Migration time becomes progressively slower Leak in system Tighten the connectors and array locking lever Impro...

Page 267: ...e contaminant in sample Re amplify the DNA Sample re naturation Heat denature the sample in good quality formamide and immediately place on ice Review Results troubleshooting Symptom Possible Cause Action Zoom errors in electropherogram graphical displays Monitor Run Review Results Spectral Calibration and Install Check The zoom feature does not re baseline the sample data view or The X axis of th...

Page 268: ... broad peaks when determining sizing quality for fragment analysis data therefore the sizing quality result reported in the 3500 Series Data Collection Software 3 1 will differ from the sizing quality result reported in the GeneMapper ID X Software which considers broad peaks in sizing quality No action Sizing Overlay report displays data for all capillaries including those that fail sizing Plots ...

Page 269: ...nk the plate on page 57 You selected Quick Start Note Quick Start expects the plate to be in position A Do not use Quick Start instead open plate and link via the main workflow The Autosampler has not completed initialization Wait for the green light to light on the front panel before linking the plate It takes approximately 10 seconds for the instrument to initialize after the instrument door is ...

Page 270: ...Last Injection preference is set and the instrument is paused Go to Monitor Run and resume the run When the run is complete Create Injection List and Start Run buttons are active Assign Plate Contents troubleshooting Symptom Possible Cause Action Error message is displayed when you export a newly created plate from the Assign Plate Contents screen Plate is not saved Save the plate close the plate ...

Page 271: ...Persistently bad spatial calibration results Bad capillary array Replace the capillary array using the Install Capillary Array wizard then repeat the calibration If the problem persists contact Thermo Fisher Scientific Spatial Calibration Error message Conditioning reagent is installed The instrument cannot perform Spatial Calibration with Array fill Replace the conditioning reagent with polymer S...

Page 272: ...act Thermo Fisher Scientific Peak heights in the Spectral report are different from the values seen when viewing the spectral data in the electropherogram display The raw data electropherogram display in the software does not have the Run Scale Divisor applied to the data The final peak height values displayed in the Spectral report have the Run Scale Divisor applied No action The Spectral peaks i...

Page 273: ... clear blockage Insufficient filling of array Check for broken capillaries Run the Fill Array with Polymer wizard Expired calibration standards or old reagents Check the expiration date and storage conditions of the calibration standards and or reagents If necessary replace with a fresh lot Data Error One or more peaks fall below the minimum required amplitude of 750 One or more peaks fall below t...

Page 274: ...hermo Fisher Scientific The capillary tips may be hitting the bottom of the wells Autosampler not correctly aligned Contact Thermo Fisher Scientific The Sequencing install check fails Failed capillaries One or more for 8 capillary Three or more for 24 capillary Accept button is not active Reject button is active Blocked capillary Run the Fill Array with Polymer wizard Install a new capillary array...

Page 275: ... Replace samples with fresh samples prepared with fresh Hi Di Formamide Bubbles in sample wells Centrifuge samples to remove bubbles The capillary tips may not be touching the samples Check the volume of your samples If no results call your Thermo Fisher Scientific representative The capillary tips may be hitting the bottom of the wells Autosampler not correctly aligned Call your Thermo Fisher Sci...

Page 276: ...tween the instrument Restart the instrument and the computer see Restart the instrument and the computer on page 251 Estimated Time Remaining in Monitor Run is longer than expected Estimated Time Remaining is the time remaining in the instrument run This estimate is adjusted after the completion of every step in an injection To view time remaining per injection scroll to the Time Remaining column ...

Page 277: ...port did not complete successfully You exported records for samples that are not in their original location samples have been deleted or moved Return sample data files to their original location then export again Audit report does not print after you change font settings Font settings are not activated until you close the report Close the report reopen it then print Electronic signature troublesho...

Page 278: ...yyyy mm dd hh mm ss mmm tslog file to your Thermo Fisher Scientific representative Use a text editor such as Wordpad to view the 3500 Series Data Collection Software 3 1 generated log files Log file Description Location 3500UsageStatistics txt Provides a summary of the number of plates run and number of run types install drive Applied Biosystems 3500 LogFiles You can also view this log from the Ma...

Page 279: ...t information Figure 36 Instrument sensor details Run status of the instrument is displayed while a run is in progress View instrument sensor details Appendix A Troubleshoot Troubleshooting procedures A 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 279 ...

Page 280: ...the red status light The instrument does not respond to the Data Collection software 1 Shut down the computer 2 Close the instrument doors 3 Reset the instrument with the Reset button as shown Note The Reset button is accessible through a small hole to the left of the Tray button Reset button Review error message details Reset the instrument Appendix A Troubleshoot Troubleshooting procedures A 280...

Page 281: ...sequencing RapidSeq50_POP6 RapidSeq50_POP6xl 50 POP 6 65 168 504 450 RapidSeq50_POP7 RapidSeq50_POP7xl 50 POP 7 40 280 840 500 Rapid sequencing RapidSeq36_POP4 RapidSeq36_POP4xl 36 POP 4 45 240 720 400 RapidSeq36_POP6 RapidSeq36_POP6xl 36 POP 6 65 168 504 600 RapidSeq36_POP7 RapidSeq36_POP7xl 36 POP 7 30 368 1104 600 Rapid sequencing BigDye XTerminator BDxRapidSeq50_POP6 BDxRapidSeq50_POP6xl 50 PO...

Page 282: ...0_POP7 BDxFastSeq50_POP7xl 50 POP 7 65 168 504 700 Fast sequencing BigDye XTerminator BDxFastSeq36_POP7 BDxFastSeq36_POP7xl 36 POP 7 60 240 552 750 Standard sequencing StdSeq50_POP6 StdSeq50_POP6xl 50 POP 6 135 80 240 600 StdSeq50_POP7 StdSeq50_POP7xl 50 POP 7 125 88 264 850 Standard sequencing BigDye XTerminator BDxStdSeq50_POP6 BDxStdSeq50_POP6xl 50 POP 6 140 80 240 600 BDxStdSeq50_POP7 BDxStdSe...

Page 283: ...zing Precision 3 50bp 400bp 401bp 600bp 601bp 1200bp Frag analysis FragmentAnalysi s50_POP7 FragmentAnalysi s50_POP7xl 50 POP 7 40 280 840 40 to 520 0 15 0 30 NA 4 FragmentAnalysi s50_POP6 FragmentAnalysi s50_POP6xl 50 POP 6 100 112 336 20 to 550 0 15 0 30 NA 4 FragmentAnalysi s36_POP4 FragmentAnalysi s36_POP4xl 36 POP 4 35 312 936 60 to 400 0 15 NA 4 NA 4 FragmentAnalysi s36_POP7 FragmentAnalysi ...

Page 284: ...d deviation of sizes for one allele in the DS 33 install standard sized with the GS600 LIZ size standard across multiple capillaries in the same run For one injection to pass 100 of the alleles in that injection must meet the intra run sizing precision specifications The table shows the sizing precision of 100 of alleles in 90 of samples 4 Not applicable because of the size of the fragments collec...

Page 285: ...e kits F 5 dye Identifiler Identifiler Direct Identifiler Plus MiniFiler NGM NGM SElect NGM SElect Express SEfiler Plus Sinofiler Yfiler Yfiler Direct Other 5 dye kits G5 6 dye GlobalFiler GlobalFiler Express NGM Detect J6 HID analysis dye sets Appendix B Run modules and dye sets Dye sets B 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 285 ...

Page 286: ...ns and weight Parameter Computer Monitor Keyboard Depth 41 7 cm 16 42 in 19 3 cm 7 6 in 44 7 cm 17 5 in Width 17 5 cm 6 89 in 44 7 cm 17 5 in 15 25 cm 6 in Height 37 4 cm 14 7 in 36 6 cm 14 4 in 5 cm 2 in Weight 9 6 kg 21 lbs 6 9 kg 15 2 lbs 0 09 kg 0 2 lbs Table 12 Applied Biosystems 3500 3500xL Genetic Analyzer operating specifications Component Specification Laser Long life single line 505 nm s...

Page 287: ...A Maximum current 15 A Maximum power dissipation 417 VA 371 W approximately not including computer and monitor Computer AC 100 240 V 10 50 60 Hz 2 1 A power rated 125 VA Monitor AC 100 240 V 10 50 60 Hz 1 5 A power rated 65 VA Mains AC line voltage tolerances Up to 10 percent of nominal voltage Transient category Installation categories II Pollution degree 2 Operating conditions 15 30 C 59 86 F Ro...

Page 288: ...rnet port for computer instrument connection 2 Circuit breaker 3 Connector for instrument power cable Appendix C Instrument specifications Power and communication connections C 288 3500 3500xL Genetic Analyzer User Guide Data Collection Software v3 1 ...

Page 289: ...aps 8 tube PCR strip caps domed standard or optical grade polypropylene for 0 1 mL or 0 2 mL 8 strip PCR tubes Plate 384 well General purpose supply obtain from any laboratory supplier 384 well PCR microtiter plate standard or optical grade polypropylene 0 02 mL fully skirted design with or without barcode Table 15 Bases retainers and septa Part Description Cat No Retainer and base 8 tube RUO 4410...

Page 290: ... POP 4 960 1 4393710 Polymer POP 4 384 1 4393715 Polymer POP 4 96 A26070 Hi Di Formamide Four 5 mL bottles 4440753 1 Validated for HID applications Sequencing analysis reagents and consumables Name Cat No BigDye Terminator v1 1 3500 3500xL Sequencing Standards BigDye Terminator v1 1 4404314 BigDye Terminator v1 1 Matrix Standard Kit 4336824 BigDye Terminator v1 1 Cycle Sequencing Kit 4337449 BigDy...

Page 291: ...4425042 DS 37 Matrix Standard Kit Dye set J6 T 6 dye A31234 DS 33 GeneScan Installation Standards with GeneScan 600 LIZ Size Standard v2 0 G5 dye set 4376911 GeneScan 120 LIZ Size Standard 4324287 GeneScan 500 ROX Size Standard 401734 GeneScan 600 LIZ Size Standard v2 0 4408399 GeneScan 1200 LIZ Size Standard 4379950 AmpFℓSTR Identifiler Allelic Ladder For HID install check Contact Thermo Fisher S...

Page 292: ...reate a plate import template on page 77 The following settings are not retained after data migration from v1 0 to v3 software Manually specify the settings after migration Plate setup report settings and sequencing settings preferences Notification log in Calendar Reminders If you re run a plate that specifies a re injection and the re injection specifies a protocol other than the protocol used f...

Page 293: ... Checks as described in page 104 Refer to examples of passing spatial calibration as shown in Example spatial profiles on page 106 When performing a spectral calibration select the dye set appropriate for your application as described in Perform a spectral calibration on page 111 If you navigate away from the Install Check screen after you start the install check the starting well may be reset to ...

Page 294: ...le Dye Sample Peak column values are split in to two separate columns the column name containing the Sample Peak values is incorrectly named and the column headers after Dye Sample are shifted one column to the right You can edit the exported file Cut paste column headers one column to the right enter correct headers for the Dye and Sample Peak columns Appendix E Limitations Review results E 294 3...

Page 295: ...ces To minimize such effects do not come within 8 inches 20 cm of this instrument if you have a patient worn and or implanted active medical device WARNING Radio frequency identification RFID signals from external devices could possibly disrupt the operation of the 3500 RFID read write units RFID signals from the 3500 RFID read write units could possibly disrupt the operation of external RFID devi...

Page 296: ...buffer container CBC 9 Autosampler Function The RFID read write units 1 Read up to 256 bytes from the RFID consumables tags 2 Write up to 256 bytes to the RFID consumables tags 3 Re read the written data on the tags to confirm that it is accurate using a checksum to verify data integrity The RFID read write units perform the functions listed above at the start of each 3500 Series Data Collection S...

Page 297: ...FID tag and internal RFID read write units ABC tag 3 cm CBC tag 4 cm Capillary tag 3 cm Polymer tag 3 cm Typical use range between RFID tag and internal RFID read write units 0 5 cm Minimum separation distance of the instrument from external RFID read write units 10 cm Minimum separation distance of the instrument from other wireless technologies 3 feet Wireless security RFID tag read write re rea...

Page 298: ...e install pouch then click Refresh on the Dashboard Restart the instrument and the computer see Restart the instrument and the computer on page 251 Install a new consumable if available If problem persists contact Thermo Fisher Scientific Malfunctioning RFID label or reader Place a used CBC ABC pouch or array on the instrument If the instrument can read the RFID label install a new CBC ABC pouch o...

Page 299: ...sample salt load and injection variability between capillaries and instruments Normalization can be applied during primary analysis of the data To use the normalization feature prepare each sample with the GS600 LIZ v2 size standard then specify the appropriate normalization size standard for file primary analysis The GS600 LIZ v2 reagent can function as an internal standard for signal height norm...

Page 300: ...nd J6 dye sets Fragment POP 6 and POP 7 polymer GS600LIZ Normalization GS600 60 600 LIZ Normalization For applications that have primer peaks that obscure the 20 and 40 mer peaks of the GS600 LIZ v2 size standard Fragment POP 4 polymer GS600 80 400 LIZ Normalization HID POP 4 polymer GS600 80 400 LIZ Normalization Appendix G Normalization When to use the normalization feature G 300 3500 3500xL Gen...

Page 301: ...ymbols may be found on the instrument to warn against potential hazards or convey important safety information In this document the hazard symbol is used along with one of the following user attention words CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentia...

Page 302: ...nances for proper disposal provision and contact customer service for information about responsible disposal options Ne pas éliminer ce produit avec les déchets usuels non soumis au tri sélectif MISE EN GARDE Pour minimiser les conséquences négatives sur l environnement à la suite de l élimination de déchets électroniques ne pas éliminer ce déchet électronique avec les déchets usuels non soumis au...

Page 303: ...ques FS et la réglementation locale associées à la manipulation et à l élimination des déchets DANGER Class 3B III visible and or invisible laser radiation present when open and interlocks defeated Avoid exposure to beam DANGER Rayonnement laser visible ou invisible de classe 3B III présent en position ouverte et avec les dispositifs de sécurité non enclenchés Éviter toute exposition au faisceau L...

Page 304: ...terlock devices you may be exposed to serious hazards including but not limited to severe electrical shock laser exposure crushing or chemical exposure CAUTION Moving Parts Moving parts can crush pinch and cut Keep hands clear of moving parts while operating the instrument Disconnect power before servicing General Physical injury Appendix H Safety Instrument safety H 304 3500 3500xL Genetic Analyz...

Page 305: ...It is the responsibility of the operator or other responsible person to ensure the following requirements are met No decontamination or cleaning agents are used that could cause a HAZARD as a result of a reaction with parts of the equipment or with material contained in the equipment The instrument is properly decontaminated a if hazardous material is spilled onto or into the equipment and or b pr...

Page 306: ... cooling fan an overheated laser can cause severe burns on contact Safety and electromagnetic compatibility EMC standards The instrument design and manufacture complies with the standards and requirements for safety and electromagnetic compatibility as noted in the following table Reference Description EU Directive 2006 95 EC European Union Low Voltage Directive IEC 61010 1 EN 61010 1 UL 61010 1 C...

Page 307: ...diagnostic IVD medical equipment FCC Part 15 47 CFR U S Standard Industrial Scientific and Medical Equipment AS NZS 2064 Limits and Methods of Measurement of Electromagnetic Disturbance Characteristics of Industrial Scientific and Medical ISM Radiofrequency Equipment ICES 001 Issue 3 Industrial Scientific and Medical ISM Radio Frequency Generators Reference Description Directive 2012 19 EU Europea...

Page 308: ...l leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet ...

Page 309: ...ution requirements before working with potentially biohazardous materials Follow all applicable local state provincial and or national regulations The following references provide general guidelines when handling biological samples in laboratory environment U S Department of Health and Human Services Biosafety in Microbiological and Biomedical Laboratories BMBL 5th Edition HHS Publication No CDC 2...

Page 310: ...ermofisher com support for the latest in services and support including Worldwide contact telephone numbers Product support Order and web support Safety Data Sheets SDSs also known as MSDSs Additional product documentation including user guides and Certificates of Analysis are available by contacting Customer Support Obtaining support For service and technical support Call Toll Free in US 1 800 95...

Page 311: ...duct support documents Obtain information about customer training Download software updates and patches Limited product warranty Life Technologies Corporation and or its affiliate s warrant this product as set forth in the Applied Biosystems 3500 3500xL Genetic Analyzer Instrument Limited Warranty If you have any questions please contact Life Technologies at thermofisher com support Documentation ...

Page 312: ...CH CODE CATALOG NUMBER SERIAL NUMBER FRAGILE HANDLE WITH CARE LOWER LIMIT OF TEMPERATURE PROTECT FROM LIGHT UPPER AND LOWER LIMITS OF TEMPERATURE UPPER LIMIT OF TEMPERATURE DO NOT REUSE BIOLOGICAL RISKS CAUTION CONSULT ACCOMPANYING DOCUMENTS CONSULT INSTRUCTIONS FOR USE UPPER AND LOWER LIMITS OF HUMIDITY OBSERVE PRECAUTIONS FOR HANDLING ELECTROSTATIC SENSITIVE DEVICES 312 3500 3500xL Genetic Analy...

Page 313: ...on 71 requirements for accurate genotyping 51 71 results group for one allelic ladder per run folder 52 run requirements 152 amber indicator light 17 amplicon specifying 47 Analysis Range fragment analysis 181 HID analysis 187 anode buffer install 229 location 15 on instrument limits 19 20 antivirus software 17 AnyDye create dye set 168 archive audit records 210 data files 244 library items 243 ar...

Page 314: ...assay 146 Basepair Accuracy 127 Basic setup preference 37 BigDye Terminator kits and standards 290 biohazard safety 309 borrowing defined 116 disabled 116 enabled 116 preference 37 Broad Peak BD and SQ 190 flag in view fragment HID results 94 threshold 190 buffer See anode buffer cathode buffer Buffer Pin Valve 32 buffers part numbers and limits 19 C calendar create entries 227 maintenance 226 rev...

Page 315: ...g install or uninstall 242 date format setting 37 default settings specifying 37 define plate properties 42 detection cell location 252 disk space computer 245 documentation related 310 duplicate injection See also re injection defined 62 preview run 62 See also re injection duplicate library item 140 dye sets create 166 custom create 168 fragment analysis 284 HID analysis 285 overview 165 sequenc...

Page 316: ... analysis dye sets 284 reagents part numbers storage conditions 291 results review 91 run modules 281 sizecalling protocol 178 fragment install check during a run 134 evaluate data 135 plate prepare 109 122 131 recommended monthly schedule 226 results example 136 results example HID 136 run 130 133 signing 221 troubleshoot 275 when to perform 130 front panel indicators 17 G green indicator light 1...

Page 317: ...st injection 37 preview 64 start 64 start with existing plate 58 suppress consumables 37 suppress delete injection warning message 37 instrument run name See also run name default 59 folder name 155 See also run name instrument run views 80 instrument sensor details 279 Instrument Shutdown maintenance wizard 240 is signed field 221 L label peaks 98 100 laser specifications 286 leaks and spills 34 ...

Page 318: ...items archive purge and restore 243 uninstall software 242 maintenance consumables anode buffer change 229 capillary array change 235 capillary array check stored 236 cathode buffer change 230 polymer fill array 234 polymer replenish 232 polymer store partially used pouch 234 maintenance instrument annual planned 226 as needed 226 daily 224 monthly 225 move and level the instrument 241 pump flush ...

Page 319: ...ow Size 181 peaks label 98 pending plates 62 permissions user account 197 219 planned maintenance schedule 226 planned maintenance tasks 227 plate 384 well assembly 56 8 tube strip assembly 54 96 well assembly 53 assay 77 assembly 53 base standard 53 create from template 42 defined 142 edit 79 export 79 file name convention 77 import 44 79 link 57 59 61 load in instrument 56 110 123 133 load in so...

Page 320: ...l check eport 130 137 plate layout 50 sample quality reports 101 spatial calibration report 107 119 spectral calibration 107 120 130 137 trace quality reports 89 user report 200 probablility of error 88 processed plates 62 Pull Up peak flag 94 settings 184 pump avoid damage 236 bubbles remove 237 flush water trap 238 maintenance 236 parts of 252 remove bubbles 237 trap flush 238 troubleshoot 254 w...

Page 321: ...ts 101 install check 130 137 plate grid 50 sample quality 101 sequencing results 89 spatial calibration 107 119 spectral calibration 107 119 system configuration history 206 trace score 89 user 200 requirements environmental 287 reset button on front panel 280 restore audit records 210 library entries 244 library items 243 results review for previously run samples 84 94 See also review results seq...

Page 322: ...See plate sample quality See also review results fragment in monitor run 67 in review results 94 See also review results fragment sample type plate view 47 table view 75 sample view 81 samples review previously run 84 94 scale plots 97 scheduled maintenance notifications 223 tasks 223 scientist user role 197 security export settings 217 import settings 218 security policies 193 security administra...

Page 323: ...e activate 246 248 obtain 246 248 See also license software sort tables multi column 76 100 141 spatial calibration export 106 historical reports 107 120 130 137 perform 104 purpose 104 signing 221 troubleshoot 271 when to perform 104 specifications 286 specimen specifying 47 spectral calibration borrowing disabled 116 borrowing enabled 116 capillary sharing 115 condition number 113 estimated run ...

Page 324: ...eshootingUtility bat 278 True Profile 176 U uninstall the software 242 unlink plate 59 usage statistics 249 user account activate suspended 197 create or edit 195 delete 197 export 217 import 218 inactivate 197 permissions 197 user role permissions 198 user role create 197 user defined fields displayed in table view only 47 including in file name 150 specifying for samples 47 V validation allelic ...

Page 325: ......

Page 326: ...thermofisher com support thermofisher com askaquestion thermofisher com 25 October 2018 ...

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