How to use custom primers on the NovaSeq X/X Plus

The NovaSeq X/X Plus supports Custom Primers using three available Custom Primer (CP) wells and a Custom Primer Recipe XML file available on the NovaSeq X Plus Product Files page. The custom primers will be diluted in the NovaSeq X Series Custom-Primer buffer, available from the NovaSeq X Series Reagents Kits page on the Illumina website.

Note on PhiX: When custom primers are used for Read 1 or Read 2, the software directs the instrument to pull from the CP1 and CP2 wells. Therefore, Illumina primers pre-loaded into the reagent are not used for the sequencing run. If Illumina primers are not used for Read 1 or Read 2, the optional Illumina PhiX control will not be sequenced. To use the PhiX control with custom primers, see section 'How to spike in custom primers into Illumina Primer Wells on the NovaSeq X/X Plus' below or contact Illumina Technical Support for guidance.

Note on Lanes: When custom primers are used on the NovaSeq X / X Plus, the custom primer is applied to the entire flow cell and cannot be selected for individual lanes.

Prepare the Custom Primers

1. If the custom primers are stored frozen, thaw them at room temperature and then store on ice.

2. Dilute the primers to a final concentration of 0.3 µM using the NovaSeq X Series Custom-Primer buffer. For the primer volumes, see Table 1.

3. When combining primers to make the custom Index Read or any custom read primer mixture, the final concentration of each primer must be 0.3 µM. This will result in a higher total primer concentration and is expected.

   1. Example: For Index 1 and Index 2 primer mix, prepare each primer solution at 0.6 µM, then combine 3.5 ml of each primer solution for a final volume of 7.0 ml with each primer at 0.3 µM and a total concentration of 0.6 µM.

Table 1: Custom Primer well positions and volumes in milliliters (ml)

Custom Primer	Well Position	Volume (ml) for 10B and 25B Flow Cell	Volume (ml) for 1.5B Flow Cell	Conc. (µM)
Custom Read 1 Primer	CP1	5.0	3.0	0.3
Custom Read 2 Primer	CP2	5.0	3.0	0.3
Custom Index 1 and 2 Primer Mix	CP3	6.0	5.0	0.3

Add Custom Primers to the Reagent Cartridge

1. Use a low-lint tissue to wipe clean the foil seal covering each custom primer position listed in Table 1.

2. Using a clean pipette tip, pierce the foil seal covering each custom primer position.

3. Add the appropriate volumes of custom primer to the correct position on the reagent cartridge.

• Avoid touching the foil seal when dispensing the primer.

Setup sequencing run to use Custom Primers

Using custom primers is specified during run setup. After selecting the appropriate run, add a custom recipe file to the run.

1. Navigate to the NovaSeq X Plus Product Files Support Page and download the Custom Primer Recipe XML file.

2. Save the Custom Primer Recipe XML file to the instrument, typically to the Desktop or Downloads directory.

3. In the Review run screen, select Select file... under Custom Recipe.

4. Navigate to the directory where the XML file was saved on the instrument, then select the custom recipe XML file.

5. After reviewing the run setup information, select Load consumables to proceed.

How to spike in custom primers into Illumina Primer Wells on the NovaSeq X/X Plus

The NovaSeq X/X Plus uses two primer solutions that combine primers for Read 1 and 2, and Index 1 and 2. If custom primers are only required for specified Reads, spike-in the concentrated primers into the appropriate wells for the Reads that require custom primers. When spiking in custom primers, no changes to the run set up are needed and a custom recipe is not required.

Table 2. provides the well positions, Illumina primer volume, and the recommended custom primer (CP) volume to add in microliters (µl) assuming a starting concentration of 100 micromolar (µM) for the custom primer stock that will be used.

Table 2.

Kit Type	Primer Mix	Reads Primed	Cartridge Position	Primer Fill Vol. (ml)	Final Conc. (µM)	CP Conc. (µM)	CP Vol. (µl)
1.5B	TP21	Read 1/2	28	4.6	0.3	100	13.8
	TP14	Index 1/2	21	4.7	0.3	100	14.1
10B	TP21	Read 1/2	28	7.7	0.3	100	23.1
	TP14	Index 1/2	21	6.9	0.3	100	20.7
25B	TP21	Read 1/2	28	8.1	0.3	100	24.3
	TP14	Index 1/2	21	6.9	0.3	100	20.7

For any feedback or questions regarding this article (Illumina Knowledge Article #7779), contact Illumina Technical Support [e-mail].