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. The following instructions can be used for setting up a PhiX validation run for the NextSeq 1000/2000 with either Hybrid mode or Local mode.

If the instrument is connected to the Internet, run set up using Hybrid mode, Proactive or Run Monitoring using BaseSpace Sequencing Hub is highly encouraged 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 mode will need to be used.

Hybrid Mode:

1. Log into BaseSpace Sequence Hub.
2. Navigate to the Runs tab.
3. Select New Run on the top-right side.
4. Then select NextSeq 1000/2000.




 

5. Provide a Run name.
6. Select Local Analysis.
7. Select DRAGEN FASTQ Generation ensuring that the version chosen matches the version on your instrument.




 

8. Select Illumina DNA Prep for Library Prep Kit.
9. Select IDT For Illumina Nextera DNA Unique Dual Indexes Set A B C D for Index Adapter Kit.
10. For Index Reads, select No Index. PhiX does not contain index adapters so it will not successfully sequence through index reads.
11. For Read Type, Read 1 length, and Read 2 length, select the best option for the PhiX test run being performed. This information will likely be provided to you by Illumina support.




 

12. Enter PhiX into the SampleID section under Sample Data.
13. Select Next.




 

14. Keep all default analysis settings.
15. Select Submit Run.




 

16. 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.
17. In the NextSeq 1000/2000 Control Software, select the hamburger menu on the top-right side.
18. Select Settings.
19. Make sure that Online Run Setup is selected.
20. Make sure that Proactive and Run Monitoring is selected for BaseSpace Option.
21. Select Save.




 

22. Start run setup.
23. Sign into BaseSpace and choose appropriate Workgroup if applicable.
24. Select PhiX run that was created under the Select Run screen.
25. Select Review.
26. Confirm all run configurations and output location.
27. Proceed with loading the cartridge and the rest of run setup.
28. 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.

1. Log into BaseSpace Sequence Hub.
2. Navigate to the Runs tab.
3. Select New Run on the top-right side.
4. Then select NextSeq 1000/2000.




 

5. Provide a Run name.
6. Select Local Analysis.
7. Select Choose DRAGEN FASTQ Generation ensuring that the version chosen matches the version on your instrument.




 

8. Select Illumina DNA Prep for Library Prep Kit.
9. Select IDT For Illumina Nextera DNA Unique Dual Indexes Set A B C D for Index Adapter Kit.
10. For Index Reads select No Index. PhiX does not contain index adapters so it will not successfully sequence through index reads.
11. For Read Type, Read 1 length, and Read 2 length, select the best option for the PhiX test run being performed. This information will likely be provided to you by Illumina support.




 

12. Enter PhiX into the SampleID section under Sample Data.
13. Select Next.




 

14. Keep all default analysis settings.
15. Select the down chevron next to submit run.
16. Select Export Sample Sheet.




 

17. 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’s important to keep the Sample Sheet saved on the network drive or thumb drive as saving it locally on the instrument can cause errors.
18. In the NextSeq 1000/2000 Control Software select the hamburger menu on the top-left corner.
19. Select settings.
20. Make sure that Local Run Setup is selected.
21. Make sure that None is selected for BaseSpace Option.
22. Select Save.




 

23. Start run set-up.
24. On the SELECT RUN screen, select Start with Sample Sheet and navigate to the sample sheet, then select Open.
25. Select Setup.




 

26. On the run review screen, confirm run configuration and output folder location.
27. Proceed with loading the cartridge and the rest of run set up.