How to use custom primers on the MiniSeq

Using Custom Primers on the MiniSeq System

When using custom primers with the MiniSeq, it is important to follow specific setup procedures to ensure successful sequencing. This article outlines how to specify and load custom primers, primer position assignments, and key contingencies related to the dual-indexed workflow.

Specifying Custom PrimersCustom primers must be designated during run setup if using the dedicated custom primer wells (13, 14, and 15) to ensure the system recognizes and uses the correct wells for custom primers:

• • Via MiniSeq Control Software: During run setup.

  • Via Local Run Manager (LRM): On the Create Run screen, select the checkboxes for each custom primer being used (Read 1, Index 1, Index 2, Read 2).

The system uses the Primers.xml file to record which primer positions were used. This file is located in the Reagents folder of the run directory.

Primer Position Assignments

Primer Type	Position	Description
Custom Primers	15	Custom Read 1 Primer
	14	Custom Read 2 Primer
	13	Custom Index 1 and Index 2 Primers
Illumina Primers	24	Illumina Read 1 Primer
	25	Illumina Read 2 Primer
	28	Illumina Index 1 and Index 2 Primer Mix

• Note: If Illumina primers are not used for insert Read 1 and insert Read 2, the optional Illumina PhiX control library will not be sequenced.

Primer Contingencies & CompatibilityDue to the dual-indexed sequencing workflow on the MiniSeq, users must load complementary primers when using custom primers for any read. This ensures proper sequencing chemistry and data quality.

MiniSeq Standard Reagent Kits

• • If using a custom Read 1 primer, users must also use an Index 2 primer.

  • If using a custom Read 2 primer, users must also use an Index 1 primer.

In most cases, failure to load the appropriate complementary primer will result in sequencing failure or incomplete data. There may be specific use cases where this guidance is not appropriate, though this is relevant to the vast majority of library designs.

Combination	Read 1 Primer	Index 1 Primer	Index 2 Primer	Read 2 Primer
All Illumina	Illumina Read 1	Illumina Index 1	Illumina Index 2	Illumina Read 2
Custom Read 1	Custom Read 1	Illumina Index 1	Custom Index 2	Illumina Read 2
Custom Read 2	Illumina Read 1	Custom Index 1	Illumina Index 2	Custom Read 2
All Custom	Custom Read 1	Custom Index 1	Custom Index 2	Custom Read 2

MiniSeq Rapid Reagent KitsThe MiniSeq Rapid recipe allows for dual-indexed single-end runs. The Index 2 read is primed off the grafted P5 primer on the flow cell, rather than using an Illumina Index 2 sequencing primer. It is not possible to use custom Index 2 or custom Read 2 primers with MiniSeq Rapid reagents.

Combination	Read 1 Primer	Index 1 Primer
All Illumina	Illumina Read 1	Illumina Index 1
Custom Read 1	Custom Read 1	Illumina Index 1
Custom Index 1	Illumina Read 1	Custom Index 1
All Custom	Custom Read 1	Custom Index 1

Primer Preparation and Loading

Dilution: Use HT1 buffer to dilute primers to 0.3 µM in 2 ml total volume.

Loading:

• • Load 1 ml of each primer into the designated wells.

  • If using custom primers for both index reads, load 0.5 ml of each into position 13.

Custom Primer Loading Volumes by Position

Position	Volume	Primer Description
15	1 ml	Custom Read 1 Primer
14	1 ml	Custom Read 2 Primer
13	1 ml	Custom Index 1 Primer (in combination with the Custom Index 2 primer, if using)
13	1 ml	Custom Index 2 Primer (in combination with the Custom Index 1 primer, if using)

For more information, see MiniSeq System Custom Primer Guide.

For any feedback or questions regarding this article (Illumina Knowledge Article #2029), contact Illumina Technical Support [e-mail].