How to set up a PhiX validation run on the NextSeq 1000/2000 sequencing platform

A PhiX validation run confirms proper hardware and software performance of the instrument. The Illumina PhiX control v3 library is a well-balanced genome with relatively equal representation of A, C, G, and T nucleotides. PhiX lacks an index and is not an appropriate tool for assessing Index Read performance.

The optimal loading concentration for a PhiX validation run on the NextSeq 1000/2000 system is 650 pM for Standard chemistry and P1/P2 XLEAP Chemistry. For all P3/P4 XLEAP chemistry kits and P1/P2 600 cycle kits the optimal loading concentration is 450 pM.

Use the following instructions to set up a PhiX validation run for the NextSeq 1000/2000 in either Hybrid mode or Local mode. If the instrument is connected to the Internet, set up the run in the Cloud of Hybrid mode with "Proactive and Run Monitoring" or "Proactive, Run Monitoring and Storage" (recommended for easy sharing with Illumina Support). If the instrument is not connected to the Internet or the use of BaseSpace Sequence Hub is blocked, Local or Standalone run setup mode is required.

Hybrid Mode

1. Log into BaseSpace Sequence Hub.

2. Navigate to the Runs tab.

3. Select New Run on the top-right side.

4. Select Run Planning.

1. Enter a Run name.

2. Select NextSeq 1000/2000 for Instrument Platform.

3. Select Local Analysis.

4. Select Next.

9.Select DRAGEN BCL Convert, ensuring that the version chosen matches the version on the NextSeq 1000/2000 instrument.

1. Select Not Specified for Library Prep Kit and Index Adapter Kit.

2. Select Next.

3. For Index Reads, select No Index. PhiX does not contain index adapters so it will not successfully sequence through index reads.

4. For Read Type, Read 1 length, and Read 2 length, select the best option for the PhiX test run being performed.

1. Enter PhiX into the SampleID section under Sample Data.

2. Keep all default analysis settings.

3. Select Next.

1. Select Save as planned.

Note: If Local Mode is using for run setup, use the Export option instead to generate a sample sheet for manual run setup.

1. When run set up is complete, and the run shows in the planned runs list in BaseSpace Sequence Hub, complete run setup on the instrument.

2. In the NextSeq 1000/2000 Control Software, select the hamburger menu in the top-left corner.

3. Select Settings.

4. Ensure that Online Run Setup is selected.

5. Verify Proactive and Run Monitoring is selected for BaseSpace Option.

6. Select Save.

1. Start run setup.

2. Sign into BaseSpace and choose appropriate Workgroup, if applicable.

3. Select the PhiX run created under the Select Run screen.

4. Select Review.

5. Confirm all run configurations and output location.

6. Proceed with loading the cartridge and the rest of run setup.

7. When the run has started, proceed to BaseSpace and generate a share link to provide to Illumina Support for run monitoring.

Local Mode

When using local mode, it is recommended to use BaseSpace Sequence Hub to generate the sample sheet. Use the Hybrid Mode instructions above (until step 17) to generate a sample sheet before starting run setup on the NextSeq 1000/2000.

1. After the Sample Sheet is exported, save it to a network drive connected to the instrument or a thumb drive connected to the instrument. Note: It is important to keep the Sample Sheet saved on the network drive or thumb drive as saving it locally on the instrument can cause errors.

2. In the NextSeq 1000/2000 Control Software select the hamburger menu on the top-left corner.

3. Select Settings.

4. Ensure that Local Run Setup is selected.

5. Verify that None is selected for BaseSpace Option.

6. Select Save.

1. Start run set-up.

2. On the SELECT RUN screen, select Start with Sample Sheet and navigate to the sample sheet, then select Open.

3. Select Setup.

1. On the run review screen, confirm run configuration and output folder location.

2. Proceed with loading the cartridge and the rest of run set up.