Plotting %Occupied by %PF to optimize loading for the NovaSeq X/X Plus, NovaSeq 6000, and iSeq 100

The NovaSeq X/X Plus, NovaSeq 6000, and iSeq 100 sequencing instruments use patterned flow cells that have a fixed number of nanowells where libraries cluster. Because of this fixed metric, the reported cluster density is identical when comparing runs from the same instrument type. To optimize loading concentrations on these platforms, the %Occupied and % Pass Filter (%PF) metrics can be plotted in Sequencing Analysis View (SAV) to determine if a run was underloaded, optimally loaded, or overloaded. Follow the instructions below for plotting these metrics and reviewing the resulting plot to optimize library loading. To view the %Occupied data, SAV v2.4.5 or higher is required for NovaSeq 6000 data and SAV v2.4.7 or higher is required for iSeq 100 data. SAV v3.0 or higher is required to view run metrics data for the NovaSeq X/X Plus, and can also be used to view NovaSeq 6000 and iSeq 100 data. SAV installers can be downloaded from the Sequencing Analysis Viewer Support page.

A video tutorial on how to create the %Occupied by %Pass Filter plot in SAV v2.4.x can be found here.

Note: The %Occupied metric is not available for data from other Illumina sequencing instruments.

Plotting %Occupied by %Pass Filter in SAV v2.4.x:

1. Load run data into SAV and select the Imaging tab.

2. Select the Scatter Plot tool from the toolbar to bring up a plotting window.

3. On the Data tab to the right of the window, scroll to select %Occupied for the "X Values" and %Pass Filter for the "Y Values”. A plot will automatically be generated.

Plotting %Occupied by %Pass Filter in SAV v3.0:

1. Load the run metrics data into SAV and then select the Images tab.

1. In the Images tab, select Custom Plots

1. In the Custom Plots window, ensure that the Scatter plot radio button is selected.

2. In the X Axis Value dropdown menu, select %Occupied

3. In the Y Axis Value dropdown menu, select % Pass Filter

4. Optional: The Color By dropdown menu can be used to color code the scatter plot points according to various criteria.

Reviewing Plots:

Underclustered:

• Points fall on a line with a positive slope from the bottom-left to the top-right of the plot.

  

Optimally Loaded:

• Points fall within a cloud of points with a positive slope in the body of the plot.

  

Overloaded:

• Points have a slightly negative, near-vertical slope and approach a % Occupied in the high 90’s.

  

If the resulting plot indicates the run is underloaded or overloaded, increase or decrease the loading concentration, respectively, to optimally load the library pool. If the plot indicates the run was optimally clustered, continue to use this loading concentration for future runs of this library type that are of similar size.

For more information, refer to the Cluster Optimization Overview Guide.

For any feedback or questions regarding this article (Illumina Knowledge Article #2308), contact Illumina Technical Support [e-mail].