ABI PRISM 3100-Avant and 3100 Data Collection v2.0 Software Frequently Asked Questions (FAQ)

Transcription

1 ABI PRISM 3100-Avant and 3100 Data Collection v2.0 Software Frequently Asked Questions (FAQ) Table of Contents General Information...2 Access Control/Auditing Features...2 Instrument/Plate Setup...3 Spectral Calibration...5 Run Modules...6 Analysis Protocol...6 Run History...6 Error Messages...7 Results Group and Autoanalysis...7 Autoanalysis with Sequencing Analysis Software...8 Autoanalysis with SeqScape Software...8 Autoanalysis with GeneMapper Software...8 NOTE: This FAQ supplements the ABI PRISM 3100/3100-Avant Genetic Analyzers User Guide (P/N Rev.A). For Research Use Only. Not for use in diagnostic procedures. The ABI PRISM 3100-Avant and 3100 Genetic Analyzers include patented technology licensed from Hitachi, Ltd.. as part of a strategic partnership between Applied Biosystems and Hitachi, Ltd.., as well as patented technology of Applied Biosystems. ABI PRISM, Applied Biosystems, GeneMapper and SeqScape are registered trademarks and AB (Design), Applera, GeneScan, GenoTyper, POP-4, and POP-6 are trademarks of Applera Corporation or it s subsidiaries in the US and certain other countries. All other trademarks are the sole property of their respective owners Applied Biosystems. All rights reserved. Applied Biosystems Page 1 of 8

2 General Information 1. What Operating System does Data Collection v2.0 Software run on?» Windows 2000 (Service Pack 3 and Service Pack 4). 2. What computers are supported by Data Collection v2.0 Software?» Dell 340 and Dell GX270 Workstation. 3. Can fragment analysis data generated with Data Collection v2.0 Software be used with GeneScan Analysis Software and/or GenoTyper Software?» No, Data Collection v2.0 Software data files are not supported with GeneScan Analysis Software or GenoTyper Software. 4. I have opened Data Collection Software from the Start Menu or Desktop icon why can t I see the Data Collection Viewer right away?» It may take about a minute for the Service Console to completely launch each Data Collection Software component. 5. What are the invalid alphanumeric characters for user or file names?» The invalid characters are Spaces \ / : *? < > I Access Control/Auditing Features 1. What is the default administrator username and password for the Access Control Administration tool?» Username: Administrator» Password: Administrator 2. What actions are auditable?» Create, edit or import a plate record» Create, edit or import a results group» Create, edit or import a run module 3. How do I turn the Data Collection Software logon prompt on/off?» Launch the AB Navigator application (Start menu \ Applied Biosystems \ Administrator), select Access Control Administration, and select the FoundationViewerApp Application and check/uncheck the Challenge box. 4. How do I turn the audit features on/off?» Launch the AB Navigator application (Start menu \ Applied Biosystems \ Administrator), select Audit Map Configuration and enable/disable the DC Plate Record, DC Results Group and DC Run Module Audit Map Objects 5. How do I turn off the reason for change dialog box?» From the Audit Map Configuration application, select the Audit Map Object of interest, and under the Attributes panel change the State to either Off or Silent: o On reason for change dialog box displays and requires input; old and new values are recorded o Off reason for change dialog box does not display; old and new values are not recorded o Silent reason for change dialog box does not display; old and new values are recorded 6. What are the default user profiles?» Administrator complete access to Instrument Protocols, Instrument Operations, Instrument Maintenance and AB Navigator» Scientist complete access to Instrument Protocols, Instrument Operations and Instrument Maintenance» Technician access to Instrument Operations and Instrument Maintenance 7. What actions are within Instrument Protocols?» Access to Run Module operations, Results Group operations, Analysis Protocol operations, Instrument Protocol operations and Reextraction. 8. What actions are within Instrument Operations?» Access to Plate operations, Event Log and Instrument Control operations. 9. What actions are within Instrument Maintenance?» Access to Spatial Calibrations, Manual Control and Wizards. Applied Biosystems Page 2 of 8

3 10. Can I access and edit objects created by another user?» A particular user does not own a data collection object. Instead, you are able to perform certain data collection operations according to your assigned user profile. Check with your laboratory Administrator. 11. Why can t I access certain features of Data Collection Software?» Your assigned user profile does not have the correct access privileges. Check with your laboratory Administrator. 12. Can I export and import Access Control Administration settings?» Yes. Launch the AB Navigator application (Start menu \ Applied Biosystems \ Administrator). After creating the users, applications and group profiles settings on one computer, select Access Control Administration, and select File \ Export Database. Enter a filename and click Save. Transfer the file to another computer and select File / Import Database. Locate the file and click Open. NOTE: Access Control Administration settings can only be exported/imported by a user with Administrator privileges. Instrument/Plate Setup 1. What are the available instrument operation wizards?» Install capillary array wizard» Change polymer wizard» Autosampler calibration wizard» Fill capillary array wizard» Update capillary array information wizard 2. Why is my plate unlinked when starting a wizard?» Plate information may change depending on the wizard to be used; therefore, any use of the wizards will automatically unlink any plates linked in the autosampler. 3. Why can t I link my plate?» Either there is no physical plate on the instrument, or no spatial calibration has been performed. 4. Why has my plate failed validation after attempting to start a run?» Either the plate is already linked or the run validation has failed (i.e. plate record information does not match the current instrument configuration). Make sure the plate record has the correct array length, polymer type and dye set specified, i.e. all of these parameters match the installed instrument configuration. Additionally, a plate cannot be validated if an active spectral has not been specified. A failed run validation error state will be represented by a flashing red light on the bottom of the software interface, with more information as to what caused the error listed in the Events View window. 5. How do I add another run to a plate record?» From the Plate Editor select the Edit menu item Add Sample Run. Fill in the appropriate Results Group, Instrument and Analysis Protocols. 6. How do I change the default run order for my plate?» In the Plate Editor of your plate record, alter the default Priority Value of 100 to a lesser number for at least one sample well within an injection set (run). The Priority Value scale ranges from 1-100, with lower values having higher priority. The software will use the lowest Priority Value within a run for scheduling, and samples will be run in ascending numerical order by the run Priority Value. 7. Can I reset the run order for my plate while it is processing?» A sample plate s run order is taken from the assigned Priority Values in the linked plate record. The Priority Values for the current run cannot be changed. Priorities of pending runs in a currently running plate can be modified and these changes will be reflected in the run list. Applied Biosystems Page 3 of 8

4 8. Will data objects (Plate records, Analysis Settings, Run Modules, etc.) from previous Data Collection Software versions (3100 v1.0.1 or v1.1, 3100-Avant v1.0) be importable?» No. Due to the differences in Database structure, all previous Data Collection Software calibrations and object settings are not compatible with the new Data Collection v2.0 Software and will have to be reconfigured using the new system software. 9. Can I run fragment analysis and sequencing samples within the same run using the Mixed Plate Record feature?» Within-run application combination is not possible due to differences in spectral dyes and run module parameters. However, the Mixed Plate Record feature does allow both sequencing and fragment analysis runs to be set up in the same plate. 10. Can I replace the currently running or processed plate on the autosampler with a new plate (continuous operation)?» This can be performed carefully by pausing the current run and adding a new plate after the samples have been injected. If the samples have not been injected for the current running plate, the paused run will abort once the plate is removed and samples will be injected from the newly placed plate instead.» After pausing the instrument during a run, the following message will appear:» Clicking OK will generate the following message:» To unmount a processed plate: open the instrument door, unmount the processed plate, add a new plate, and close the instrument door. The following will then occur: o The instrument resumes and the above Resume Run message will disappear automatically. o The processed plate is automatically unlinked all runs associated with the processed plate are removed from the run list. o The current run and any pending runs associated with running plate continue o There is no unmounting warning. o If a new plate is mounted, that plate is now available for plate record linking. Applied Biosystems Page 4 of 8

5 Spectral Calibration 1. Can I import spectral calibrations created in previous Data Collection Software versions (3100-Avant v1.0, 3100 v1.0.1 or v1.1)?» No. Due to the differences in Database structure, all previous Data Collection Software calibrations and object settings are not compatible with the new Data Collection v2.0 Software and will have to be reconfigured using the new system software. Therefore, new spectral calibrations must be performed when upgrading from any previous versions of 3100-Avant or 3100 Data Collection Software. 2. How do I delete spectral calibrations from the Data Collection Software database?» To enable users to activate any previous spectral calibration, Data Collection v2.0 Software saves all spectral calibration files including the Spectral Plate Record. The Cleanup Database Utility will delete all run data and Plate Records in the database but will not delete spatial and spectral calibrations. Deleting the Spectral Plate Record will delete the spectral calibration from the database. Note that all sample Plate Records must be removed from the database first in order to delete any Spectral Plate Record. 3. Why did the spectral calibration fail?» Incorrect chemistry or run module selected for the calibration run» Signal too low/high» Wrong raw data profile peak order. The correct order is (from left to right): o Sequencing 4-dye: red-yellow-blue-green o Fragment analysis 4-dye: red-yellow-green-blue o Fragment analysis 5-dye: orange-red-yellow-green-blue» Wrong spectral profile color order. The correct order is (from left to right): o 4-dye: blue-green-yellow-red o 5-dye: blue-green-yellow-red-orange» Extraneous data spikes interfering with Matrix Standard color peaks 4. Why can t I see my spectral calibration (and also the List of Calibrations pull-down menu is initially blank)?» Changing array lengths will deactivate the current active spectral calibration. Before running any sample plates, you must set an active spectral calibration or run a spectral calibration if there is no calibration available. NOTE: You cannot set the active spectral while a run is in progress. 5. Why is there an asterisk next to the name of a spectral calibration in the Spectral Viewer?» This denotes that all capillaries for a spectral calibration have failed, however the raw data can still be viewed by going to Spectral Viewer, selecting a dye set and selecting a calibration run via the List of Spectral Calibrations for Dye set pull-down list. 6. Where can I find the pass/fail status of each capillary after my spectral run is complete?» Under the Event Log sub-section of Instrument Status, go to the Event Messages window to view the spectral quality parameters generated for each capillary. 7. What is the difference between the Spect36_POP4 and SpectSQ36_POP4 spectral calibration run modules?» There are application chemistry-dependent differences in data acquisition for spectral calibrations on the 36cm array length using POP-4 polymer. As a result, fragment analysis spectral calibrations on this array length and polymer combination have been optimized to run with the Spect36_POP4 module. Sequencing spectral calibrations (whether injecting from matrix standards or sequencing standards) on this array length and polymer combination have been optimized to run with the SpectSQ36_POP4 module. Applied Biosystems Page 5 of 8

6 Run Modules 1. What is the appropriate module selection for an instrument configuration? (Shaded cells = new run modules; Green = fragment analysis, Blue = Sequencing) Capillary Length Polymer Application Run Module Run Time Resolution Performance KB Q20 LOR** 22 cm 36 cm 50 cm 80 cm POP-4 POP-6 POP-4 POP-6 POP-4 High throughput SNP analysis High throughput, small size fragment analysis Standard SNP analysis Standard fragment analysis Ultra rapid sequencing Rapid sequencing Standard sequencing Long fragment analysis Long fragment analysis Standard sequencing Long read sequencing SNP22_POP4 FragmentAnalysis22_POP4 SNP36_POP4 FragmentAnalysis36_POP4 UltraSeq36_POP4 RapidSeq36_POP6 StdSeq50_POP4 FragmentAnalysis50_POP4 FragmentAnalysis50_POP6 StdSeq50_POP6 LongSeq80_POP4 15 min 20 min 30 min 45 min 40 min 1 hr 40 min 1 hr 5 min 1 hr 30 min 2 hr 30 min 3 hr 40 min 250 bp 400 bp 250 bp 400 bp 500 bp 500 bp 650 bp 950 bp 1bp resolution at 99.99% accuracy, *Basecalling accuracy using ABI basecaller, **Resolution using KB Basecaller (trimmed Q20 length of read) 2. Why are shorter read length specifications being generated with the KB basecaller vs. the ABI Basecaller?» Applied Biosystems improved basecalling algorithm (the KB Basecaller) is designed to accurately exact more bases out of sequencing data, and reduce manual data review time by providing per-base quality value (QV) assignment, which is a measure of basecaller accuracy. The KB Basecaller v1.1 release supports all chemistries and run modules available on the ABI PRISM 3100-Avant and 3100 Genetic Analyzers running Data Collection v2.0 Software, and is also integrated in the Applied Biosystems Sequencing Analysis v5.1 Software and SeqScape v2.1 Software. In previous versions of Data Collection Software, resolution performance for all run modules was calibrated with a 98.5% basecalling accuracy (1.5% probability of error) specification using the ABI Basecaller. Utilizing the KB Basecaller v1.1 release for Data Collection v2.0 Software, resolution performance for the new StdSeq50_POP4 run module and all existing run modules was calibrated with a quality score cutoff of QV20 (99% basecalling accuracy or a 1% probability of error). Therefore, while shorter read length specifications are generated with the KB Basecaller, the calibrations are of higher basecalling accuracy. Analysis Protocol 1. Why am I unable to see any default analysis protocols in the Data Collection Software?» Analysis software must be installed and registered in order to import any analysis protocols into the Data Collection Software. 2. What are the available basecaller selections in the Sequencing Analysis Protocol Editor?» KB Basecaller (available for all sequencing run modules).» ABI Basecaller (not available for the new StdSeq50_POP4 sequencing run module). Run History 1. How do I search for previous runs?» To search for a particular plate record in the database, perform a Plate Search. Go to the Plate Manager node of Data Collection Software and choose Advanced search from the Type of search drop-down menu; enter specific plate record attributes to search on in the table and Click Search. To view data from a processed plate record, go to the Run History node of Data Collection Software and choose a run from the pull-down list of runs returned from a plate record search. 1 hr 0.50 SD 0.15 SD 98.5% * 0.15 SD 98.5% * 400 bp 500 bp 600 bp 600 bp 700 bp Applied Biosystems Page 6 of 8

7 2. What barcode reader(s) are supported?» The KEYENCE BL-80VE external barcode reader that connects to the instrument computer keyboard has been tested with 3100 and 3100-Avant Data Collection v2.0 Software. With this reader, users are able to scan barcodes into any text box in the Data Collection software. All Applied Biosystems barcoded PCR plates use the code 128 format, which is compatible with this reader model. There is also currently functionality for plate record searches based on barcode information with Data Collection v2.0 Software. Error Messages 1. Where can I view errors that occurred during an instrument run?» The error log file can be accessed from the Data Collection Software by navigating to GA instrument \ GA3100 \ <Instrument Name> \ Event Log. 2. During plate record setup an error occurred with the text: populated %java.lang.nullpointerexception.» The software has limited cut and paste functionality in the plate record. Access to this functionality will be documented in the user manual. 3. What does E drive full message mean?» The software has failed run validation due to not enough memory in the E drive to perform the requested run(s). There is active database monitoring in the Data Collection Software for drives C, D, E and F. Before starting a batch of runs, the software checks the C and E drives to make sure there is enough memory to process the pending linked plate records. If the minimum amount of disk space is not met, the software will fail validation when the run is started. Additionally, a reminder message will appear when the database (on D and F drives) is 80% full. Applied Biosystems recommends that you monitor your disk space and archive sample files weekly for maximum processing performance. Additionally, since the disk space and database space checks only happen when you click start to start a run, if you are taking advantage of the continuous operation of the ABI PRISM 3100-Avant or 3100 Series Systems, you should monitor your disk space more regularly. Results Group and Autoanalysis 1. What is the location of extracted data files/folders?» The default location for extracted sample files is: E:\AppliedBiosystems\UDC\DataCollection\Data\ga3100\<instrument name> If you have created a customized Run Folder name in the Results Group settings, the sample files will be located in: E:\AppliedBiosystems\UDC\DataCollection\Data\<custom run folder name> 2. Why are my runs not analyzed?» Make sure Autoanalysis Manager is running for GeneMapper Software and SeqScape Software data runs. Since Autoanalysis Manager does not start automatically, it must be open to receive messages from Data Collection Software v2.0. To launch Autoanalysis Manager, select Start \Programs \ Applied Biosystems \ Autoanalysis Manager \ Autoanalysis Manager 2.0.» Make sure the Do Autoanalysis box is checked in the Analysis tab of the Results Group. If not, you must either re-extract the data with a Results Group modified such that Do Autoanalysis is checked or manually analyze the collected samples with the appropriate analysis software.» Maker sure the Login ID and Password fields in the Analysis tab of the Results Group have been entered correctly for the analysis software being accessed by Data Collection Software. NOTE: Both the Login ID and Password are case-sensitive. 3. After entering the Sample File Format in the Results Group an error generates INVALID NAME. Why is my sample format unacceptable?» You must select a Sample File Format option that creates unique sample names. For example, the capillary number is unique while the instrument name is not. Applied Biosystems Page 7 of 8

8 4. How do I configure Autoanalysis with Sequencing Analysis Software, SeqScape Software or GeneMapper Software?» See section on Autoanalysis below. Autoanalysis with Sequencing Analysis Software 1. My samples were analyzed with the wrong settings.» Check the Analysis Protocol and make sure the correct mobility and base caller files were selected. If this is incorrect, either manually re-analyze or re-extract with a correctly modified Analysis Protocol. Autoanalysis with SeqScape Software 1. Jobs do not appear in the Autoanalysis Manager.» For run-by-run analysis, make sure the Do Autoanalysis box is checked in the Analysis tab of the Results Group Editor and When every run completes is checked in the Automated Processing tab.» If performing analysis by Results Group, make sure both the Do Autoanalysis and Results Group Entry Completed boxes are checked in addition to Only when the results group is complete in the Automated Processing tab. Using these settings, a job will only be queued in the Autoanalysis Manager if all of the samples associated with that Results Group have been run.» If all of these recommended parameters are correct, try re-launching the Autoanalysis Manager. 2. A job has failed with an invalid job information error message.» Under the SeqScape tab of the Autoanalysis Manager, click Edit Properties and retype the SeqScape Login ID and Password. Autoanalysis with GeneMapper Software 1. How do I perform Autoanalysis of Fragment Analysis samples?» When creating a Results Group under the Analysis tab, select GeneMapper<Instrument Name> from the pull down tab and select the Do Autoanalysis check box. Type the GeneMapper Login ID and Password. 2. Jobs do not appear in the Autoanalysis Manager.» Make sure the Do Autoanalysis box is checked. If performing analysis by Results Group, make sure Results Group Complete is checked, and under Automated Processing, the When every results group is complete is checked. For run-by-run analysis, make sure When every run completes is checked. If all of these parameters are correct, try re-launching Autoanalysis Manager. 3. A job has failed with an invalid job information error message.» Under the GeneMapper tab of the Autoanalysis Manager, click Edit Properties and retype the GeneMapper Login ID and Password. 4. Can Autoanalysis be performed using a separate computer workstation?» Remote analysis is only supported by GeneMapper Software v3.5 as long as the analysis computer can be seen on the network. Before auto-analysis commences for the first time, make sure that in addition to creating a customized Run Folder name in the Results Group settings that you also perform the Test function in the Analysis tab to ensure the Path Name and connection can be verified. 5. How do I analyze GeneMapper Software sample files on another computer workstation?» In order to analyze GeneMapper Software sample files on a separate computer workstation the following files must be transferred to the other GeneMapper Software database: GeneMapper project, analysis method, size standard, panel and bin set information. All components need to be exported and imported individually. Applied Biosystems Page 8 of 8