# 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   | 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          | Custom           | Custom           | Custom          |

See the [MiniSeq System Custom Primer Guide](https://support.illumina.com/downloads/miniseq-custom-primers-guide-1000000002700.html) 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](https://support.illumina.com/downloads/miniseq-custom-primers-guide-1000000002700.html).

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #2029), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000002029%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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