Spiking custom primers into the Illumina sequencing primers

Libraries prepared with custom sequencing primers require optimization to perform successfully on Illumina sequencing platforms. Combining (or “spiking”) custom primers into the Illumina primers is necessary when including PhiX or other standard Illumina libraries with the custom sample. Illumina cannot guarantee the performance or compatibility of custom primers, and it is up to the customer to validate primers for sequencing on a specific platform. When using custom sequencing primers, it is critical to know the recommended platform-dependent loading concentration, total volume, loading positions, and primer annealing temperatures. This bulletin provides information for spiking custom primers into the Illumina primers.

Spiking custom primers into Illumina sequencing primers

• The cartridge position, total volumes, and final concentration of custom primers for each platform are provided in the tables below.

• Calculate the volume of the custom primer to add to the Illumina primer cartridge position based on the final concentration of the custom primer in the cartridge*.

• After spiking in the custom primer, adjust with an appropriate pipette to half of the final volume, and at the cartridge position gently pipette up and down five times to mix thoroughly**.

• For MiSeq, MiniSeq, NextSeq 500/550 and NovaSeq 6000 platforms, do not check the “custom primer” box position in the sample sheet or during the run setup.

* To calculate how much custom primer to spike into the well, use the (C1)(V1) = (C2)(V2) equation where: C1 = the concentration of your custom primer (when using high concentration primer stock, such as 100 µM, the small additional volume to the final volume is negligible). V1 = solve for the volume of the custom primer to be spiked in C2 = the recommended custom primer final concentration from the chart below V2 = total volume of Illumina primer in the charts below

Example: (100 µM) (V1) = (0.5 µM) (680 µl) V1 = 3.4 µl

** MiSeq, NextSeq and MiniSeq cartridges: to increase successful measurement of the Illumina sequencing primers, the addition and mixing of custom primers, it is recommended to use slimmer tips or larger volumes of lower stock concentration custom primers.

Important Note: The guidelines below are based on the current primer volumes. To ensure accuracy, measure total primer volumes in the cartridge with a pipette before proceeding with setup.

iSeq 100

With the release of iSeq Control Software version 3.0, we are now able to sequence with custom primers on the iSeq 100. Due to the construction of the iSeq 100 cartridge, it is not possible to spike custom primers directly into the pre-loaded Illumina primers.

In lieu of spiking custom primers directly into reagent cartridge wells, the NextSeq 1000/2000 custom primer kit configurations are compatible with the iSeq 100 and should be used. Custom primers should be added to the appropriate Illumina primers, then loaded into the iSeq 100 reagent cartridge using the custom primer workflow. For available kit configurations and their part numbers, please refer to the NextSeq 1000/2000 Product Documentation.

Important Note: The NextSeq 1000/2000 Custom Primers Guide provides primer addition guidelines with volumes specific to the NextSeq 1000/2000. Please see below for iSeq 100-specific guidance for adding custom primers.

MiniSeq Because of the unique dual-indexing workflow on the MiniSeq, combinations of custom primers and Illumina primers are limited. Be aware of the custom primer dependencies outlined here.

MiSeq

* There is no option for a custom Index 2 (i5) primer since the template uses the grafted P5 primer on the surface of the flow cell.

NextSeq 500/550 Because of the unique dual-indexing workflow on the NextSeq 500/550, combinations of custom primers and Illumina primers are limited. Be aware of the custom primer dependencies outlined here.

NextSeq 1000/2000

Due to the construction of the NextSeq 1000/2000 cartridge, it is not possible to load custom primers into the Illumina primers.

If Illumina primers are needed to sequence PhiX during a run, a NextSeq 1000/2000 Custom Primer kit is required. Refer to the NextSeq 1000/2000 Product Documentation for instructions.

NovaSeq 6000 v1.5 Consumables Because of the primer design and dual-indexing workflow on the NovaSeq 6000, combinations of custom primers and Illumina primers are limited. Be aware of the custom primer dependencies outlined here.

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 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.

Using Custom Primers in Place of Illumina Primers

If only the custom primer is needed, custom primers can be used in place of the Illumina primers. For instructions on how to set up a run using only custom primers, see the following guides:

• MiniSeq System Custom Primers Guide

• MiSeq System Custom Primers Guide

• NextSeq 500/550 System Custom Primers Guide

• NextSeq 1000/2000 Product Documentation - Custom Primers

• HiSeq System Custom Primers Guide

• NovaSeq Series Custom Primers Guide

• iSeq 100 Sequencing System - Custom Primers

• NovaSeq X Plus Product Documentation - Custom Primers

For more information on the indexing strategies for each instrument, refer to the Indexed Sequencing Overview Guide.

For any feedback or questions regarding this article (Illumina Knowledge Article #1542), contact Illumina Technical Support [e-mail].