Applied Biosystems 3500/3500xL Genetic Analyzer User ......running any wizard. The PDP block is not...

User BulletinApplied Biosystems 3500/3500xL Genetic Analyzer

Stock #: 133UB13-01 -- 12/14/2009

SUBJECT: Applied Biosystems 3500/3500xL Genetic Analyzer: Solutions to issues related to software, data, hardware, and consumables

In this user bulletinThis user bulletin covers:

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

Section 1 Troubleshooting issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2

Hardware and consumables issues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Software and data issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

Section 2 Amendments to the information presented in the user guide . . . . . . .23

Amendment to Chapter 1 Instrument and Software Description . . . . . . . . . . .24

Amendment to Chapter 3 Set Up and Run . . . . . . . . . . . . . . . . . . . . . . . . . . . .25

Amendment to Chapter 5 Calibrate and Check Performance . . . . . . . . . . . . . .33

Amendment to Chapter 6 Manage Library Resources . . . . . . . . . . . . . . . . . . .34

Amendment to Appendix B Secondary Analysis: Sequencing . . . . . . . . . . . . .38

Amendment to Appendix C Secondary Analysis: Fragment. . . . . . . . . . . . . . .39

Related documentation • Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (PN 4401661)• Software Release Notes, 3500 & XL Data Collection V1.0 (4415398)

OverviewThis user bulletin is divided into two sections. Section one identifies the known hardware, consumables, software, and data issues. Section two provides amendments to the information presented in the Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (PN 4401661).

2 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin

Applied Biosystems 3500/3500xL Genetic Analyzer: Solutions to issues related to software, data, hardware, and consumables

Section 1 Troubleshooting issues

Hardware and consumables issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Software and data issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

3Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin

Hardware and consumables issues

Hardware and consumables issues Table 1 shows the hardware issues and Table 2 shows the consumables issues.

In both tables, the "More information" column references the Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (PN 4401661).

Table 1 Hardware issues

Symptom Possible cause Action More information

The 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 plate base are clipped together.

IMPORTANT! If array tips are bent, replace array.

See “Prepare sample plates” in Chapter 3, Set Up and Run.

The plate base is not sitting properly on the autosampler.

The plate base should sit flat on the autosampler. When placing the plate on the autosampler, ensure that the pins in the autosampler are properly aligned with the holes at the bottom of the plate base, and that the left and right sides are latched.

The plate retainer is lifted off the plate base by array.

Securely clip the plate retainer and plate base together.

The septa is stripped off the CBC.

Ensure that the septa is completely inserted into position. Listen for the light clicking sound that occurs when the septa is pressed down firmly into position.



Miscommunication between the software and instrument.

The instrument's door is left open for an extended period of time at the beginning of a run.

It takes approximately 10 seconds for the instrument to initialize after the instrument door is closed.

Do not start a run until the instrument status light is green.

See Software Release Notes, 3500 & XL Data Collection V1.0 (4415398) for information related to miscommunication issues.

The RFID read fails. Click Refresh from the Consumable Information section of the Dashboard.

See “Quick Start a run” in Chapter 3, Set Up and Run.

The user closes the Daemon only.

Never close the daemon separate from the data collection software. Follow the recommended shut down procedures.

See “To shutdown the instrument” in Chapter 8, Maintain the Instrument.

The user restarts the instrument only.

Restart the instrument first and then the computer.

Restart the computer and the and instrument on a weekly basis as part of maintenance.

See:

• “Start the instrument” and “Start the computer” in Chapter 2, Start the System.

• “Weekly instrument maintenance tasks” in Chapter 8, Maintain the Instrument.

A delay in communication between the instrument and software when performing flush array.

The instrument and computer did not complete the wizard in a timely fashion.

Wash the channels using the Pump Cleaning Kit supplied with the instrument. It contains a syringe, a tube, an adaptor, and instructions.

If the problem persists, contact Applied Biosystems.

See “Change polymer type” in Chapter 8, Maintain the Instrument.

Contact Applied Biosystems.

Table 1 Hardware issues (continued)




Polymer Delivery Pump (PDP) is extremely noisy and vibrating while running any wizard.

The PDP block is not pushed back into position.

Check the position of the Buffer-Pin Valve Lever (Yoke). If the lever does not move up and down freely, close the door and restart the instrument. After the instrument has restarted, check the lever movement. If the lever does not move up and down freely, contact Applied Biosystems.

If the lever moves up and down freely, push the PDP block all the way back against the wall. There should be a 0.5mm gap between the edges of block and back wall.

No additional information is required.

The array locking lever is not in the correct position.

IMPORTANT! If it the lever is not in correct position, you will receive “Leak error” message.

Lock the lever in the correct position. If this is not possible contact Applied Biosystems.





A leak detection error occurs when capillary arrays are filled with fresh polymer or when the polymer type is changed, causing the wizard to fail to complete.

Debris is clogging the Check Valve (CV) Fitting.

See the figure below.

While wearing gloves, use a lint-free cloth and water to wipe the CV Fitting.

Always install the Conditioning Reagent Pouch after removing a used or a partially used pouch.

Completely remove the top seal of the Polymer pouch or Conditioning Reagent Pouch before use.


See:

• “Instrument reagents and consumables” in Chapter 1, Instrument and Software Description.

• “Use the Maintenance Wizards to perform operations” in Chapter 8, Maintain the Instrument.

The Yoke is not seated properly on the Buffer-Pin Valve.

Check the position of the Buffer-Pin Valve Lever (Yoke) to see that the Yoke is seated properly on the Buffer-Pin Valve.

If the lever does not move up and down freely, close the door and restart the instrument. After the instrument has restarted, check the lever movement. If the lever does not move up and down freely, contact Applied Biosystems.

If the lever moves up and down freely, push the PDP block all the way back against the wall. There should be a 0.5mm gap between the edges of block and back wall.


The Check Valve is clogged.

Filaments, or particles, are clogging the PDP.


Run the Wash Pump and Channels wizard.


See “Wash the pump chamber and channels” and “flush the water trap (pump trap)” in Chapter 8, Maintain the Instrument.





The Bubble Detect error message displays on the screen.

Unable to remove bubbles.

Occurs when the bubble remove/fill array wizard fails to complete.

Air bubbles are in the PDP or PDP channels.


Run the Remove Bubble wizard. See:

“Start the instrument” and “Start the computer” in Chapter 2, Start the System.

“Remove bubbles from the polymer pump” in Chapter 8, Maintain the Instrument.

Install desired polymer type on instrument.

1. Power off the instrument.

2. Power off the computer.

3. Power on the instrument.

4. Power on the computer.

5. Perform Fill Array Wizard or Replenish Polymer Wizard.

6. Run the Remove Bubble wizard.

7. Perform Spatial and Spectral calibrations.

See:


• “Remove bubbles from the polymer pump” in Chapter 8, Maintain the Instrument.





Poor signal and resolution after polymer type change.

The Anode Buffer Container (ABC) level is low.

Do not use if the buffer level is too low or the seal has been compromised. A fill tolerance of ± 1 mm is acceptable.

See “Change the anode buffer container (ABC) and Change the cathode buffer container (CBC)” in Chapter 8, Maintain the Instrument.

The Cathode Buffer Container (CBC) level is low.

Do not use if the buffer level is too low or the seal has been compromised. A fill tolerance of ± 0.5mm is acceptable.

The Check Valve is clogged.

Filaments, or particles, clogging the Polymer Delivery Pump (PDP).

See the figure above.



See:

• “Wash the pump chamber and channels and the Flush the water trap (pump trap)” in Chapter 8, Maintain the Instrument.

• The Check Valve is clogged symptom in this table.

The Buffer-Pin Valve does not move.

Polymer crystallizations have formed around the Buffer-Pin Valve.

If you see any crystals, leaks, and dried residue around the Buffer-Pin Valve, clean the valve and the array locking lever immediately.

Add DI water to the buffer solution to dissolve crystals.

Note: Use the lint-free swabs, included in the PDP Cleaning kit (PN 4359572).

If leaks persist, contact Applied Biosystems.

See “Daily instrument maintenance tasks” in Chapter 8, Maintain the Instrument.

Ensure that the maintenance schedule is followed per 3500 Series Data Collection Software notifications.

If leaks persist, contact Applied Biosystems.

The vent hole behind the Buffer-Pin Valve is clogged.

Clean the vent hole behind the Buffer-Pin Valve with DI water.


The PDP block is not in the correct position.

See the “Polymer Delivery Pump (PDP) is extremely noisy while running maintenance wizard” symptom included in this table.


See “Polymer Delivery Pump (PDP) is extremely noisy while running maintenance wizard” symptom included in this table.





Any of the following visual or audible conditions:

• Unstable current• Arc-detect errors• A crackling noise

at the beginning of electrophoresis

• A blue lightning symbol below the oven

• An error message regarding electrical current

• Electric discharge

The buffer level is below the fill line.

Verify that buffer level is at or above the fill line.

See “Instrument operational procedures”in Chapter 8, Maintain the Instrument.

The buffer spilled on top of the CBC.

IMPORTANT! Ensure that the environment (humidity) is non-condensing.

Wipe away spills, moisture, and condensation with a lint-free lab cloth.


See:

• Chapter 8, Maintain the Instrument.

• Troubleshoot appendix.

The buffer spilled on top of the Autosampler.

The condensation on the CBC.

The condensation around the septa.

The condensation on the lower part of the oven door, near the array header.

The condensation inside the oven.

There is not enough fluid in larger chamber of ABC, or the anode spilled into smaller overflow chamber.

Pipette the buffer from the smaller overflow chamber to the larger chamber. Ensure that the buffer is filled to within ± 1 mm of the fill line.

See “Change the anode buffer container (ABC)” in Chapter 8, Maintain the Instrument.

For more details see the product insert included in the product package.





The run times out 20 minutes after filling.

The situation is most likely to occur during a spatial calibration run.

The conditions are as follows:

• POP-6™ polymer• 8-capillary instrument • Oven and Detection cell

heater off • Pump pressure near low end

of tolerance

Pre-heat the oven and detection cell while you prepare for a run (detection cell temperature is set by the software). Preheating helps mitigate subtle first-run migration rate effects. The preheat function automatically turns off after 2 hours of instrument inactivity. Applied Biosystems recommends that you pre-heat the oven for at least 30 minutes before you start a run if the instrument is cold (room temperature <20 °C).

It is not necessary to wait for the oven, and the detection cell, to reach the final temperature before running a spatial calibration

If the run is started when the indicators (on the dash board) are red, the run will not start until the oven and detection cell heater reach operating temperature (all indicators are green).

See:

• “Check instrument status” in Chapter 3, Set Up and Run.

• “Prepare the instrument” in Chapter 3, Set Up and Run.

• “Prepare for the spectral calibration” in Chapter 5, Calibrate and Check Performance.





Table 2 shows the consumable issues.

Table 2 Consumables issues


Residual seal on top of the pouch fitment.


The top seal of the pouch has become delaminated and left the polyethylene behind on the pouch fitment.

Using a pipette tip, remove the entire seal from the pouch fitment before use on the instrument.

See “Routine instrument cleaning” in Chapter 8, Maintain the Instrument.



Software and data issues Table 3 Software and data issues


Insufficient Polymer in Polymer Delivery Pump error message.

Insufficient polymer in polymer delivery pump.

If the pump wash completes and error occurs after the wash:

1. Power off the instrument.

2. Power off the computer.

3. Power on the instrument.

4. Power on the computer.

Follow the prompts in the Fill Array Wizard window and complete all the steps.

See:


• “Fill capillary array with fresh polymer” in Chapter 8, Maintain the Instrument.

No plate is detected in position A.

The plate is in position B.

Plate Setup Error when linking plate layout to a plate not in Plate Position A.


1. Move plate from position B to position A.

2. Create a plate layout, and click Link Plate.

3. Click OK in the Plate Setup Error message dialog box.

4. On the left pane, click Load Plates for run.

5. Manually link the plate to position B.

See “Link the plate” and “Load the plate in the instrument” in Chapter 3, Set up and Run.

Do not use Quick Start, instead open plate and link via the main workflow.

Note: When using the Quick Start, the system expects the plate to be on position A.

The Autosampler has not completed initialization.

Click OK to accept the error. Manually load the plate to the correct position in the “load plates for run” view.

It takes approximately 10 seconds for the instrument to initialize after the instrument door is closed.

Malfunctioning plate sensor(s). Contact Applied Biosystems. Contact Applied Biosystems.


Software and data issues

The system is unable to read RFID.

Malfunctioning RFID label. Try an old CBC (or ABC, pouch or array). If the RFID is able to read, then use a different (new) CBC (or ABC, pouch or array).

If the RFID is still unable to read, contact Applied Biosystems.


The Load plate for run message does not display correctly.

See the figures below.

The window is not refreshing properly.

Click OK in the error message and follow the prompts.

See “Load plate troubleshooting” in the Troubleshoot appendix.

Table 3 Software and data issues (continued)




The Change Polymer Type Wizard does not complete.

This condition indicates a problem with the Delivery Pump (PDP) or with the Check Valve (CV).

Visually check the ABC. It must be at least half full after the Change Polymer Type Wizard completes the pump wash cycle.

IMPORTANT! Use the Conditioning Reagent pouch and an empty ABC to conduct the wash.

If the fluid inside the ABC is not at least half full after the pump wash cycle is completed using the Change Polymer Type Wizard, then contact Applied Biosystems.


Contact Applied Biosystems.

The system status in the Dashboard is not updated.

The 3500 Series Data Collection Software did not refresh the dashboard information automatically.

Click Refresh. See “Check system status in the Dashboard” in Chapter 2, Start the System.

If the problem persists, restart the computer and the instrument on a weekly basis as part of maintenance.

See “Weekly instrument maintenance tasks” in Chapter 8, Maintain the Instrument.

Dashboard shows more consumables than the actual amount, after installing new CBC or ABC.

The 3500 Series Data Collection Software did not refresh the dashboard information automatically.

Click Refresh after changing or installing consumables.

See ‘Check system status in the Dashboard” in Chapter 2, Start the System.

If the problem persists:

• Replenish the polymer installed on the instrument from the Maintenance Wizards, or

• Quick Start a run

See:

• “Use the Maintenance Wizards to perform operations” in Chapter 8, Maintain the Instrument

• “Quick Start a run” in Chapter 3, Set Up and Run.





The operating system's Java update scheduler error message displays.

The Java updater is unable to complete the update.

Close the Java update scheduler.

Note: The Java update scheduler does not affect the performance of the 3500 Series Data Collection Software or the quality and accuracy of the data collected.


A Column fill error displays in the plate grid view.

Assays are being assigned to un-selected wells.

• Select the wells that should not include the assay and deselect the assay only to those wells, or

• Use the table view to add the assay to the samples

See “Assign plate contents” in Chapter 3, Set Up and Run.

The Estimated Time Remaining appears longer than expected.

The Estimated Time Remaining in the top panel of the Monitor Run screen is based on the time remaining in the instrument run. This value is calculated based on a unique sequence of instructions. This estimate is adjusted after the completion of every step in an injection.

To view time remaining per injection, scroll to the the Time Remaining column in the Injection List Details.

See “Monitor the run” in Chapter 3, Set Up and Run.





In the Sizing Overlay Report, the 3500 Series Data Collection Software plots the sizing overlay for all capillaries including those that fail sizing.

See the picture below.

Plots displayed in Sizing Overlay Plot are based on the samples selected.

Select only the capillaries that pass sizing to be included in the report.

Avoid selecting the capillaries that fail sizing in the report.

See:

• “Report options” in Chapter 4, Review Results.

• “Normalization size standards provided” in Chapter 6, Manage Library Resources.

• “QC protocols library (primary analysis – HID)” in Chapter 6, Manage Library Resources.

• “Review results troubleshooting” in the Troubleshoot appendix.





Zoom errors in electropherogram graphical displays (Monitor Run, Review Results, Spectral and Performance Check):

• The zoom feature does not re-baseline the sample data view, or

• The X axis of the sample plot does not stay at the bottom of the screen. It moves up toward the region the user has zoomed in on, making data difficult to review

The zoom feature does not re-baseline the sample data view.

No action. See Chapter 4, Review Results.

During the first run of any dye set for a new array, mirror spectral peaks can be seen in the raw data electropherogram display.

See the picture below.

The first Spectral run for any Matrix standard uses a default Spectral stored in the 3500 Series Data Collection Software. Mirror Peaks are only observed while the run is in progress and not when the Spectral is completed.

No action. See “First Spectral Run with a New Array” in Chapter 5, Calibrate and Check Performance.





Peak heights in the Spectral report are different from the values seen when viewing the spectral data in the electropherogram display of the 3500 Series Data Collection Software.

The raw data electropherogram display in the software does not have the Run Scale Divisor applied to the data as is done in the final peak height values displayed in the Spectral report.

No action. No additional information is required.





The Spectral peaks in the raw data view appear to be in the wrong order or there are extraneous peaks that may adversely affect the spectral profile.

Septa contamination. Replace the CBC septa. See “Change the cathode buffer container (CBC)” in Chapter 8, Maintain the Instrument.

Breakdown products in Matrix Standard.

If the spectral calibration has passed, verify that the peaks in the spectral profile do not contain gross overlaps, dips, or other irregularities. If the peaks in the spectral profile are separate and distinct, the spectral data for this capillary is acceptable.

Note: Extra, and small, peaks do not affect the data.

The software searches for the highest peak for each dye, uses those peaks for calibration, and then it determines pass/fail.

See “Review normalized data” in Chapter 4, Review Results.

If the peaks in the spectral profile are not separate and distinct, the calibration run is not acceptable.

Verify that:

• The correct chemistry, dye set, and run module were selected during setup. If not, correct and repeat the run.

• The reagents are not expired. If expired, change reagents, and repeat the run.

Note: Check the expiration dates and storing condition of the Matrix standard. If the Matrix standard has expired or has been compromised, use a new calibration standard and repeat the run.

Extra peaks in the raw data or “Bad dye order detected” error message.

See picture below.

Bubbles in the polymer system. Select the Bubble Remove Wizard to clear the bubbles.

See Chapter 8, Maintain the Instrument.

Possible contaminant or precipitate.

Properly bring the polymer to room temperature. Do not heat to thaw rapidly. Swirl to dissolve any solids.

Replace the polymer if it has expired.

Expired polymer. Replace the polymer.





AnyDye Set Spectral Calibration fails.

Lack of peaks.

By default, the 3500 Series Data Collection Software is looking for 6 peaks if AnyDye Set is selected.

AnyDye spectral can fail due to the lack of peaks or incorrect peak order (the order not matching the specified order in dye-set file).

• Select AnyDye set• De-select the dyes that will

not be present.

See “Spectral calibration” in Chapter 5, Calibrate and Check Performance.

The Performance check and/or spectral calibration run fails.

You cannot accept a failed run. No history is stored for a failed run.

• The Accept button is dimmed.

• The .ab1 or .fsa files are not generated; therefore, no history is saved.

Note: Select the History tab to see a list of previous calibrations.

The number of failed capillaries exceeds the threshold (defaults: 1 for 8-cap, 3 for 24-cap).

To generate a report for the failed calibration, click View Summary Report or View Detail Report before you click Reject Results. To save the report electronically, select CutePDF as the printer.

See:

“Sequencing install standard troubleshooting” in the Troubleshoot appendix.

See Chapter 5, Calibrate and Check Performance (Section 2: Performance check in).





The software does not create a plate from a plate template in the library.

The “Save As” option is used for saving plate templates.

Note: The “Save” option in the Library screen is not active.

Go to Define Plate Properties screen New Plate select Create new plate from Template.

IMPORTANT! Do not create a new plate from a template in the Library.

See “Plate libraries” and “Create a new plate” in Chapter 6, Manage Library Resources and Plate setup.

Specimen name and Amplicon name are specified in File Name Convention but not included in sample name.

The attributes of the Specimen name and Amplicon name are specified in the File Name Convention.

The Specimen name and Amplicon name are not included in the file name unless specified in the Customize Sample information section.

To view Specimen Name and Amplicon Name in the Customize Sample Information section, a File Name Convention, with corresponding attributes, must be assigned to a well.

Note: The Specimen Name and Amplicon Name fields can only be viewed in the Plate View and not the Table View of the Assign Plate Contests step of plate Setup.

Enter the Specimen name and Amplicon name in the Sample Name field in the Assign Plate Contents screen.

See Chapter 6, Manage Library Resources.

A basecalling error occurs when using MicroSEQ® ID (MSID) POP-7™ Assay.

The wrong mobility file is present in the MicroSEQ® ID POP-7™ Assay.

1. Duplicate the MicroSEQ® ID POP-7™ Assay (MSIDPOP7) template in the 3500 Series Data Collection Software.

2. Edit the basecalling protocol to use a POP-7™ assay mobility file.

3. Save the Template.

See the Secondary Analysis: Sequencing appendix.




Section 2 Amendments to the information presented in the user guide

Section 2 Amendments to the information presented in the user guide

Amendment to Chapter 1 Instrument and Software Description . . . . . . . . . . . . . . .24

Amendment to Chapter 3 Set Up and Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25

Amendment to Chapter 5 Calibrate and Check Performance . . . . . . . . . . . . . . . . . .33

Amendment to Chapter 6 Manage Library Resources . . . . . . . . . . . . . . . . . . . . . . .34

Amendment to Appendix B Secondary Analysis: Sequencing . . . . . . . . . . . . . . . . .38

Amendment to Appendix C Secondary Analysis: Fragment . . . . . . . . . . . . . . . . . .39



Amendment to Chapter 1 Instrument and Software Description

New concepts in the 3500 Series Data Collection Software

The 3500 Series Data Collection Software uses new elements to specify settings for data collection. Note that you no longer need to create or select an instrument protocol; it is part of an assay (described below).

New concept Specifies settings for ...

Primary Analysis (basecalling) protocol and templates

Basecalling

Secondary Analysis (MicroSEQ® ID) protocol

Location of the MicroSEQ ID Analysis Software for autoanalysis

File Name Convention and templates File naming

Results Group and templates Naming, sorting, and customizing the folders in which sample data files are stored

Assay and assay templates Data collection and processing. It contains:

• Instrument protocol (dye set and run configuration)

• Primary analysis (basecalling) protocol • Secondary analysis (MicroSEQ® ID)

protocol update for all types

Plate template Plate parameters and autoanalysis. Can also contain:

• Assays• File name conventions• Results groups


Amendment to Chapter 3 Set Up and Run


Name samples and assign sample types in the plate view

This section provides one way to name samples and assign sample types. For other ways to name samples, see the user guide.

1. Click a well, then type a sample name directly into the well, then press Enter.

2. Click-drag multiple wells.

3. Right-click and select Fill or Fill Series to populate the selected fields.

To use Fill Series, type a number as the last character of the named well).

You can copy and paste sample names instead of using fill commands.

4. At the bottom right of the Assign Plate Contents screen, expand the Customize Sample Info pane.

5. In the plate view, click-drag to select wells of interest.

6. Specify the Sample Type for the selected wells, then press Enter.

7. (Optional) Specify User Defined Fields and Comments. User Defined Fields contain additional attributes you can assign to a plate and are displayed only in Table View.

Note: For MicroSEQ® ID auto-analysis applications, User Defined Field 4 is used to specify MicroSEQ® ID Project Name and User Defined Field 3 is used to specify MicroSEQ® ID Project Name.



8. (Optional) For sequencing applications, specify amplicon and specimen name.

IMPORTANT! Amplicon and specimen names under Analysis are not used for MicroSeq® ID applications.

9. Repeat to assign the Sample Type for all named wells.

10. Go to “Assign assay, file name convention, and results group in the Plate View” in the user guide.

Note: For HID applications, include the well position in the allelic ladder sample names. Well position is needed to identify the position of allelic ladder samples during re-injection.

Prepare the plate assembly

1. Define the number of wells in the Define Plate Properties screen.• 96 = 96 standard plates and 8 strip standard tubes• 96-FastTube = 96 fast plates & 8 strip fast tubes• 384 = 384 well plates only

2. Load the plate in the instrument.

a. 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.

b. Close the instrument door to re-initialize the instrument.

Note: It takes, approximately, 10 seconds for the instrument to initialize after the instrument door is closed.



Plate assembly part numbers

The following table lists the names and part numbers for the 8-Strip tube plates.

For 8-Strip tube plates

Prepare the 8-Strip tube plate assembly on a clean, level surface. Do not heat plates that are sealed with septa.

IMPORTANT! Using the wrong plate base may affect performance.

8-Strip Tube Standard description

IMPORTANT! The array tips will be damaged if the plate retainer and septa strip holes do not align correctly.

Table 4 Applied Biosystems 3500/3500xL Genetic Analyzer consumables

Part number Description

4410228 96-Well retainer & base set (Standard)

4409530 96-Well retainer & base set (Fast)

4410235 384-Well retainer & base set (Standard) for 3500xL Genetic Analyzer

4410231 8-Tube retainer & base set (Standard)

4410233 8-Tube retainer & base set (Fast)

4410701 8-Strip Septa

4412614 96-Well Septa

4412520 384-Well Septa for 3500xL Genetic Analyzer

4410715 Septa Cathode Buffer Container

4306737 MicroAmp® Optical 96-Well Reaction Plate with Barcode

403081 MicroAmp® 96-Well Tray/Retainer Set

Note: The two adapters (Retainer and 96-well Tray), inside the set, are sold separately. See the description in the figure below.

4309849 MicroAmp® Optical 384-Well Reaction Plate with Barcode

4346906 MicroAmp® Fast Optical 96-Well Reaction Plate with Barcode, 0.1 ml

4358293 MicroAmp® Fast 8-Tube Strip, 0.1 ml

N8010580 MicroAmp® 8-Tube Strip, 0.2 ml



The following figure shows the components of the 8-Strip Tube Standard.

Note: The two adapters (Retainer and 96-well Tray), inside the set, are sold separately. See the description below.

Plate retainer

Septa

Well

96-Well Tray

Plate base

Retainer



8-Strip Tube Standard assembly

To assemble the 8-Strip Tube Standard, follow the numbered steps presented in the figure below.

If the reagents 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.

6) Plate retainer

5) Septa

3) Well

2) 96-Well tray

1) Plate base

4) Retainer



8-Strip Tube Fast description

IMPORTANT! The array tips will be damaged if the plate retainer and septa strip holes do not align correctly.

The following figure shows the components of the 8-Strip Tube Fast.

Plate retainer

Septa

Well

96-Well tray

Plate base



8-Strip Tube Fast assembly

To assemble the 8-Strip Tube Fast, follow the numbered steps presented in the figure below.

If the reagents 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.

5) Plate retainer

4) Septa

3) Well

2) 96-Well tray

1) Plate base



Start and stop a run

Define Pause, Resume, Abort

Pause

• When to use: Can be used when an instrument run is in progress. • Definition: Instrument run pauses after current injection is complete.

Resume Run

• When to use: Can be used when an instrument run has been paused.• Definition: Resume paused instrument run.

Abort Injection

• When to use: Can be used anytime during a run.• Definition: Immediately aborts the current injection and pauses the instrument

run. You can resume the run or terminate the injection list.

Terminate Injection List

• When to use: Can be used only after abort injection or pause run.

• Definition: Terminate the entire injection list.


Amendment to Chapter 5 Calibrate and Check Performance

Amendment to Chapter 5 Calibrate and Check Performance

When to perform a spectral calibration

Perform a spectral calibration for each dye set/polymer type combination you will use:

• Sequencing dye set/polymer type• Fragment dye set/polymer type• HID dye set/polymer type

Perform a spectral calibration when you:

• Use a dye set that you have not previously calibrated• Change the capillary array

IMPORTANT! When a new array is installed all spectral calibrations need to be performed.

• Change the polymer type• Have a service engineer perform an optical service procedure, such as

realigning or replacing the laser or CCD camera or mirrors on the instrument• See a decrease in spectral separation (pull-up/pull-down in peaks) in the raw or

analyzed data



Amendment to Chapter 6 Manage Library Resources

File name conventions library

File name conventions

The Specimen Name attribute is not functional. Even when selected, specimen name is not included in the file name.

Dye sets library

To calibrate a custom dye set using AnyDye, first create the dye set, then select the name of 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.

A spectral calibration creates a de-convolution matrix that compensates for dye overlap (reduces raw data from the instrument) in the 4-dye, 5-dye, 6-dye, or AnyDye data stored in each sample file.

AnyDye functionality limitations

Specify the following size standard dye colors when creating an AnyDye dye set:

• Any4Dye: Red• Any5Dye: Orange• Any6Dye: Orange and Purple

The dye order mapping option is highlighted in the following figure.

Analysis will fail for Any4dye if an Orange dye size standard is selected. By default, the size standard is defined with a Red dye in the Sizing Protocol.

If using an Orange dye size standard, create a new Size Standard definition with Orange as your dye color and a new Sizing Protocol.



MicroSeq® ID protocols library (secondary analysis)

MicroSeq® ID analysis protocol overview

A MicroSeq® ID protocol is the optional secondary analysis (auto-analysis) protocol for MicroSeq® ID Analysis Software v2.2 or later sequencing applications.

A MicroSeq® ID analysis protocol defines the secondary analysis software (MicroSeq® ID Analysis Software) location.

When you create a sequencing assay, you can optionally add a MicroSeq® ID analysis protocol to the assay. If you add this item from the library, a copy of the item is added to the 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 210.

Create a new MicroSEQ® ID analysis protocol

1. Access the MicroSEQ® ID Protocols library.

2. Click Create.

3. In the Create New MicroSEQ® ID Protocol dialog box (Figure 1 on page 36), specify settings (see Table 5 on page 36).

4. Select the remaining secondary analysis items, then click Save.

IMPORTANT! The auto-analysis settings you specify for the plate to run with this protocol must contain the same secondary software and location settings.



Figure 1 Create New MicroSeq® ID Protocol field l

Select any values. These fields are not used. See below for information.

Table 5 MicroSeq® ID Analysis protocol settings

Setting Description

Protocol Name Name of the protocol. Names must be unique.

Description Optional text entry.

Lock 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. Useful when your system includes the SAE module.

Application Type Automatically set to Sequencing.

Secondary Analysis Software IMPORTANT! The secondary analysis software must be installed and properly configured with the 3500 Series Data Collection Software before it is listed as a selection in this screen. For information on setting up the MicroSEQ® ID Analysis Software for auto-analysis, see the MicroSEQ® ID Analysis Software Getting Started Guide.

Secondary Analysis Software Instance

Computer on which the secondary analysis software is running.

Project Field not used.

Specify the MicroSEQ® ID software project in User Defined Field 4 in the Assign Plate Contents screen (described below).

Specimen Field not used.

Specify the MicroSEQ® ID software project in User Defined Field 3 in the Assign Plate Contents screen (described below).



Specify user-defined fields for MicroSEQ® ID project and specimen names

1. At the bottom right of the Assign Plate Contents screen, expand the Customize Sample Info pane.

2. In User Defined Field 4, enter the name of the MicroSEQ® ID project in which to store the data. The name you enter must exactly match the name of an existing project in the MicroSEQ® ID Analysis software.

3. In User Defined Field 3, enter the name of the MicroSEQ® ID specimen to create. The name you enter must follow the specimen naming conventions in MicroSEQ® ID Analysis software. For information, see the Creating a New Project topic in the MicroSEQ® ID Analysis software help.

Assign sample types and user-defined fields



Amendment to Appendix B Secondary Analysis: Sequencing

Auto-analyze projects in the sequencing analysis software

Auto-analysis can only be performed on the same computer that collects the sample files, therefore SeqScape® or MicroSEQ® ID Software must be co-installed and configured with the 3500 Series Data Collection Software on a Windows Vista® operating system. Automated basecalling occurs with KB™ Basecaller v.1.4.1.8 (calls pure or mixed bases with quality values) and secondary analysis occurs with SeqScape® or MicroSEQ® ID Software.

This procedure initially describes how to set up panels and bin sets in SeqScape® and then describes how to auto-analyze samples using the 3500 Series Data Collection Software. Once a run is complete, your data is seamlessly transferred into SeqScape® for analyzing, processing and reporting.

Auto-analysis with MicroSEQ® ID

Note: For information on setting up the MicroSEQ® ID Software to work with the 3500 Series Data Collection Software, refer to the MicroSEQ® ID Analysis Software v2.2 Getting Started Guide (4445126).


Amendment to Appendix C Secondary Analysis: Fragment

Amendment to Appendix C Secondary Analysis: FragmentFor detailed information on setting up a GeneMapper® ID-X analysis to autoanalyze in the 3500 Series Data Collection Software, refer to the GeneMapper® ID-X v 1.2 Installation Guide.

Part Number 4445098 Rev. A 12/2009 Stock Number 133UB13-01

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The purchase price of this Instrument includes a grant of a limited, non-transferable license under U.S. Patent No. 5,567,292 and method claims of its foreign counterparts, and under U.S. Patent No. 6,358,385 and element clams of its foreign counterparts, to use this particular instrument for electrophoresis methods employing fluorescence as a means of detection. No other licenses or rights are hereby conveyed either expressly, by implication, or estoppel including, but not limited to, any claims to a composition.

Label License Statement 30 / 31:

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