A PhiX validation run confirms proper hardware and software
performance of the instrument. The Illumina PhiX control library is a
well-balanced genome with relatively equal representation of A, T, G,
and C nucleotides. PhiX lacks an index and is not an appropriate tool
for assessing Index Read performance. Below are the instructions for
setting up a PhiX validation run on NovaSeq sequencing platforms.
The optimal loading concentration for a PhiX validation run on the
NovaSeq system is 250 pM. Refer to the NovaSeq
Denature and Dilute Guide for the denaturation and dilution
procedure. Load denatured PhiX into the library tube just prior to run
setup. The uncapped library tube is loaded into position 8 of the
NovaSeq flowcell (SP, S1, S2, or S4) can be used for a PhiX
validation run; to test all four lanes, a NovaSeq S4 300 cycle kit
- For S4 runs, 4 tubes of PhiX will be
required per flowcell.
- Open the NovaSeq Control Software (NVCS)
Sequence on the Home screen and select one or both
- Follow the prompts on the screen to load the flow
- Note: Do not place
anything on top of the flow cell door.
- Follow the prompts on the screen to load the cluster, SBS, and
buffer cartridges, and to empty the large and small waste
- (Optional) If uploading the run to BaseSpace
Sequence Hub, select Run Monitoring Only. If Run
Monitoring and Storage is selected, a sample sheet will be
- On the Run Setup screen:
- Enter run parameters –
typically 2 x 151.
- Set number of cycles for Index 1
and Index 2 to zero.
- Specify the desired
network output location.
- Review the run parameters, then
proceed to the pre-run checks.
- After the checks have
successfully completed, your sequencing run will begin.