Initiating a Sequencing Run

This section is only applicable to protocols utilizing standard SBS chemistry. If your protocol utilizes XLEAP-SBS chemistry, refer to XLEAP-SBS Sequencing Protocol.

This step initiates a sequencing run in one of four modes:

• Cloud mode—Run is selected from a list of planned runs in NextSeq 1000/2000 Control Software. During sequencing, CBCL data is uploaded to BaseSpace Sequence Hub. After sequencing, DRAGEN in BaseSpace Sequence Hub starts automatically.
• Hybrid mode—Run is selected from a list of planned runs in NextSeq 1000/2000 Control Software. After sequencing, on-instrument analysis starts automatically. CBCL data and DRAGEN secondary analysis output files are stored in the selected output folder.
• Local mode—A sample sheet in v2 file format is manually imported into NextSeq 1000/2000 Control Software. After sequencing, on-instrument analysis starts automatically. CBCL data and DRAGEN secondary analysis output files are stored in the selected output folder. If Proactive, Run Monitoring and Storage is selected, analysis can also be initiated through BaseSpace Sequence Hub apps after sequencing completion.
• Standalone mode—Set up a run, following the instructions in NextSeq 1000/2000 Control Software to generate CBCL data.

Opening the visor during the pre-run check or the run can cause run failure.

Keep hands clear of instrument during visor opening and closing to avoid injury.

1. Configure the run mode, as described in Configure Run Mode.
2. Select Start.
3. Enter your BaseSpace Sequence Hub sign-in credentials, and then select Sign In.
4. If you selected Proactive, Run Monitoring and Storage, select the Workgroup containing your run created in Instrument Run Setup in BaseSpace Sequence Hub.

A workgroup selection is required to avoid errors. Make sure you have selected a workgroup before proceeding.

5. Select Next.
6. Select your run.
7. Confirm that the Analysis, Run Length, and Secondary Analysis version matches the correct run.

Analysis displays Cloud_ to indicate that analysis occurs in BaseSpace Sequence Hub.

8. Select Review.
9. [Optional] Enter custom read primer and custom index primer locations.

For information on preparing and adding custom primers, refer to Custom Primers. Make sure to visit the Compatible Products page for your library prep kit to check if Illumina custom primers are necessary.

10. [Optional] Select a custom recipe. For more information, refer to Dark Cycle Sequencing.

If using the NextSeq 1000/2000 Control Software v1.3 or later and the Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit or the Illumina Stranded mRNA Prep kit, the custom recipe is automatically selected.

11. [Optional] To manually denature and dilute libraries, deselect the Denature and Dilute On Board checkbox. Refer to Manual Denature and Dilute.

The default selection is configured in the NextSeq 1000/2000 Control Software settings.

12. [Optional] To change the output folder, select the Output Folder field and enter a new location.

The Output Folder field is autopopulated from your default settings and is required unless Proactive, Run Monitoring and Storage is selected.

If you selected Proactive, Run Monitoring and Storage, Save to BaseSpace Sequence Hub displays Enabled.

If you selected Proactive and Run Monitoring, Save to BaseSpace Sequence Hub displays Disabled.

13. Review your run information, and then select Prep.
1. Configure the run mode, as described in Configure Run Mode.
2. Select Start.
3. If you selected Proactive, Run Monitoring and Storage or Proactive and Run Monitoring, enter your BaseSpace Sequence Hub sign-in credentials, and then select Sign In.
4. If you selected Proactive, Run Monitoring and Storage, select the BaseSpace Sequence Hub Workgroup to save your run in, and then select Next.

A workgroup selection is required to avoid errors. Make sure you have selected a workgroup before proceeding.

5. Select Choose... under Start With Sample Sheet, and navigate to the sample sheet in v2 formatting on the NextSeq 1000/2000 instrument, portable drive, or mounted network drive. Sample sheet file names cannot contain special characters.

NextSeq 1000/2000 Control Software v1.3 or later automatically detects the DRAGEN version from the sample sheet and if needed, prompts you to switch versions. The DRAGEN version must be installed on the system. For installation information, refer to Software Updates.

• Instrument Run Setup Used—Select the .zip folder containing the sample sheet v2 and supporting files if applicable. Otherwise, select the sample sheet v2.
• Instrument Run Setup Not Used—Make sure that the secondary analysis supporting file is in the same directory as the sample sheet v2.

The selected sample sheet must be in v2 formatting. To create a sample sheet v2, download the generated sample sheet from Instrument Run Setup in BaseSpace Sequence Hub or edit a sample sheet v2 template provided on the NextSeq 1000/2000 support page. For more information on sample sheet v2 formatting and requirements, refer to the Sample Sheet v2 Resource. Make sure any files referenced in the sample sheet are in the same folder as the sample sheet.

6. Select Review.
7. [Optional] Enter custom read primer and custom index primer locations.

For information on preparing and adding custom primers, refer to Custom Primers. Make sure to visit the Compatible Products page for your library prep kit to check if Illumina custom primers are necessary.

8. [Optional] Select a custom recipe. For more information, refer to Dark Cycle Sequencing.

If using the NextSeq 1000/2000 Control Software v1.3 or later and the Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit or the Illumina Stranded mRNA Prep kit, the custom recipe is automatically selected.

9. [Optional] To manually denature and dilute libraries, deselect the Denature and Dilute On Board checkbox. Refer to Manual Denature and Dilute.

The default selection is configured in the NextSeq 1000/2000 Control Software settings.

10. [Optional] To change the output folder, select the Output Folder field and enter a new location.

The Output Folder field is autopopulated from your default settings and is required unless Proactive, Run Monitoring and Storage is selected.

If you selected Proactive, Run Monitoring and Storage, Save to BaseSpace Sequence Hub displays Enabled.

If you selected Proactive and Run Monitoring, Save to BaseSpace Sequence Hub displays Disabled.

11. Review your run information, and then select Prep.
1. Configure the run mode, as described in Configure Run Mode.
2. Select Start.
3. If you selected Proactive, Run Monitoring and Storage or Proactive and Run Monitoring, enter your BaseSpace Sequence Hub sign-in credentials, and then select Sign In.
4. If you selected Proactive, Run Monitoring and Storage, select the BaseSpace Sequence Hub Workgroup to save your run in, and then select Next.
5. Select Set Up New Run.
6. In the Run Name field, enter a unique name of your preference to identify the current run.

The run name can contain alphanumeric characters, dashes, hyphens, and underscores.

7. For Read Type, select how many sequencing reads to perform:
• Single Read—Perform one read, which is the simpler, faster option.
• Paired End—Perform two reads, the consensus of which generates higher-quality data and provides more accurate alignment.
8. Enter the number of cycles performed in each read:

There is no maximum number of index cycles, but the sum of the read cycles and index cycles must be less than the number of cycles indicated on the cartridge label plus 38 (or plus 27 if using the 300-cycle cartridge in the NextSeq 1000/2000 P3 Reagents kit). Refer to Supported Number of Cycles.

Read 1—Enter 1–301 cycles.

Index 1—Enter the number of cycles for the Index 1 (i7) primer. For a PhiX-only run, enter 0 in both index fields.

Index 2—Enter the number of cycles for the Index 2 (i5) primer.

Read 2—Enter up to 301 cycles. This value is typically the same as the Read 1 value.

9. If you selected Proactive, Run Monitoring and Storage, select Choose... to import a sample sheet.

NextSeq 1000/2000 Control Software v1.3 or later automatically detects the DRAGEN version from the sample sheet and if needed, prompts you to switch versions. The DRAGEN version must be installed on the system. For installation information, refer to Software Updates.

The selected sample sheet must be in v2 formatting. To create a sample sheet v2, download the generated sample sheet from Instrument Run Setup in BaseSpace Sequence Hub or edit a sample sheet v2 template provided on the NextSeq 1000/2000 support page. For more information on sample sheet v2 formatting and requirements, refer to the Sample Sheet v2 Resource. Make sure any files referenced in the sample sheet are in the same folder as the sample sheet.

10. [Optional] Enter custom read primer and custom index primer locations.

For information on preparing and adding custom primers, refer to the NextSeq 1000 and 2000 Custom Primers Guide (document # 1000000139569). Make sure to visit the Compatible Products page for your library prep kit to check if Illumina custom primers are necessary.

11. [Optional] Select a custom recipe. For more information, refer to Dark Cycle Sequencing.
12. [Optional] To manually denature and dilute libraries, deselect the Denature and Dilute On Board checkbox. Refer to Manual Denature and Dilute.

The default selection is configured in the NextSeq 1000/2000 Control Software settings.

13. [Optional] To change the output folder, select the Output Folder field and enter a new location.

The Output Folder field is autopopulated from your default settings and is required unless Proactive, Run Monitoring and Storage is selected.

14. Select Prep.
1. Make sure that the cartridge was previously thawed and inverted 10 times to mix before loading the flow cell (gray tab removed) and diluted library.
2. Select Load.

The NextSeq 1000/2000 Control Software opens the visor and ejects the tray.

3. Place the cartridge onto the tray with the label facing up and the flow cell inside the instrument. Push cartridge in until it locks into seat.

4. Select Close to retract the cartridge and close the visor.

The NextSeq 1000/2000 Control Software displays information from the scanned consumables after ~3 minutes.

5. [Optional] Select Eject Cartridge to remove the cartridge.

The visor opens after 1 minute and ejects the cartridge.

6. Select Sequence.

Pre-run checks include an instrument check followed by a fluidics check. The fluidics check pierces the cartridge seals, which causes 3-4 popping sounds to emit from the instrument. This sound is expected. The reagent is now passed through the flow cell.

Consumables cannot be reused after the fluidics check starts.

1. Wait about 15 minutes for pre-run checks to complete.

The run starts automatically after successful completion.

2. If an error occurs during the instrument check, select Retry to redo the check.

When a check is in progress, the circle for that check is animated.

3. To troubleshoot recurring errors, refer to Error Message Resolution.
1. Monitor run progress and metrics as they appear on the Sequencing screen.
• Estimated run completion—The approximate date and time of run completion. The estimated run completion metric requires 10 previous runs to calculate accurate run completion time.
• Average %Q30—The average percentage of base calls with a Q-score ≥ 30.
• Projected Yield—The expected number of bases called for the run.
• Total Reads PF—The number of paired end (if applicable) clusters passing filtering (in millions).
• Real Time Demux—Status of demultiplexing when initiated at the beginning of Read 2 following completion of Read 1, Index 1, and Index 2 cycles. Status displays Complete even if index cycles are not performed. Not available for Cloud mode runs.
• Real Time Alignment—Status of Read 1 alignment when initiated at the beginning of Read 2 following completion of Read 1, Index 1, and Index 2 cycles. Not available for Cloud mode runs.

Q30 and yield metrics appear after cycle 26.

2. To monitor run processes, select the control software menu, and then select Process Management.
3. To cancel a run, select End Run. Refer to Cancel a Run for more information on canceling runs.
4. Unload consumables from the instrument. Remove the cartridge from the instrument within 3 days.
1. When sequencing is complete, select Eject Cartridge.

The software ejects the used cartridge from the instrument.

2. Remove the cartridge from the tray.
3. Remove the flow cell from the cartridge.
4. Dispose of the flow cell, which has electronic components, in accordance with applicable standards for your region.
5. [Optional] Remove the drainage plug under the Illumina logo on the side of the cartridge over an appropriate area (ie, sink or hazardous liquid waste receptacle). Keep the plug facing horizontal or downward away from your face. Drain used reagents in accordance with applicable standards for your region. If automatic reagent purge is not enabled, drain time depends on the cartridge size.

This set of reagents contains potentially hazardous chemicals. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Ventilation should be appropriate for handling of hazardous materials in reagents. Wear protective equipment, including eye protection, gloves, and laboratory coat appropriate for risk of exposure. Handle used reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and regulations. For additional environmental, health, and safety information, refer to the SDS at support.illumina.com/sds.html.

6. Recycle the cartridge shell, and then dispose of the reagent cartridge. Refer to Recycle Cartridge Components for information.
7. Dispose of the reagent cartridge.

A post-run wash is not necessary because fluidics are discarded with the cartridge.

8. Select Close Door to reload the tray and return to the Home screen.

The software automatically reloads the tray and sensors confirm cartridge removal.

The cartridge plastic may be recyclable according to your local solid waste recycling guidelines. Check with your local solid waste recycler or your municipality’s specific instructions for recycling.

The following are the cartridge components. The reagent well plate and flow cell are not recyclable.

  1. Flow cell tray
  2. Flow cell shell
  3. Piercer
  4. Valve assembly
  5. Rotor valve cover
  6. Bottom shell
  7. Reagent well plate
  8. Top shell

Recycle the cartridge shell as follows.

1. Make sure that the cartridge is drained.
2. Remove the punch tool from the fixture.

  1. Punch tool
  2. Fixture
3. Position the wider edge of the fixture parallel to the benchtop.
4. Rest the cartridge on top of the fixture's prybars. It is not necessary to push the cartridge down.

  1. Prybars
  2. Rear prybars
5. Align the cartridge with the two rear prybars.
6. Align the punch tool with the label on top of the cartridge.

7. Using heavy force, press down quickly on the punch tool to separate the cartridge shell.

If not enough force is applied, the cartridge shell might not separate completely. If the cartridge shell is only partially separated, remove the cartridge from the fixture, close the shell, and then apply additional force with the punch tool.

8. Remove the piercer from the cartridge.

  1. Piercer
9. Remove the cover from the forward rotor valve assembly.

10. To remove the flow cell shell, perform one of the following actions:
• Insert a flat head screwdriver between the flow cell shell and lower assembly, and then lift.

• Pull the flow cell shell off the lower assembly.

11. To remove the reagent well plate from the lower assembly, disconnect one snap on one side of the lower assembly. Then remove the reagent well plate.

12. Disconnect both snaps on the flow cell tray, and then remove the flow cell tray.
13. Remove the metal spring from the flow cell tray.
14. Disconnect the four snaps on the valve assembly by pushing the snaps down and away from the reagent well plate edge.

15. Remove the valve assembly by pulling the underside of the assembly.
16. Dispose of the reagent well plate, which might contain unused reagents, in accordance with applicable standards for your region.

Cleaning the cartridge tray is only necessary if the reagent has leaked onto the cartridge tray.

1. Remove the cartridge from the instrument.
2. Put on a new pair of powder-free gloves and any additional protective wear.
3. Spray 10% bleach solution onto a cloth.
4. Wipe the cartridge tray using the cloth, and then immediately remove the bleach solution using a heavy duty wipe.

The bleach stains the cartridge tray if not removed immediately.

5. Spray 70% ethanol solution on the cartridge tray and immediately remove using a heavy duty wipe.
6. Set the cartridge tray back to load position.