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 Primers

Custom primers must be designated during run setup:

• • Via MiniSeq Control Software: During run setup, if using the dedicated custom primer wells.

  • 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). This ensures the system recognizes and uses the correct wells for custom primers.

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 Read 1 or Read 2, the optional Illumina PhiX control library will not be sequenced.

Primer Contingencies & Compatibility

Due 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.

• • If using a custom Read 1 primer, you must also load the reverse complement of the Index 2 primer.

  • If using a custom Read 2 primer, you must also load the reverse complement of the Index 1 primer.

In most cases, failure to load the appropriate complementary primer will result in sequencing failure or incomplete data.

MiniSeq Standard Reagent Kits

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	Custom Index 1	Custom Index 2	Illumina Read 2
Custom Read 2	Illumina Read 1	Custom Index 1	Custom Index 1	Custom Read 2
All Custom	Custom	Custom	Custom	Custom

See the MiniSeq System Custom Primer Guide for MiniSeq Rapid Reagent Kit Compatible Primer Combinations.

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 well.

  • 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
13	0.5 ml each	Custom Index 1 Primer and Custom Index 2 Primer
13	1 ml	Custom Index 2 Primer

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 [email protected].