> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/instrumentation/miniseq/instrumentation-miniseq-reference_material-list/000002029.md).

# 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 Primers**Custom 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 & 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.

**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 Kits**The 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](https://support.illumina.com/downloads/miniseq-custom-primers-guide-1000000002700.html).

\
\
\ <br>

|                                                                                                                                                                                                                                                                                                                                                                        |
| :--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: |
| *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)*.* |


---

# Agent Instructions
This documentation is published with GitBook. GitBook is the documentation platform designed so that both humans and AI agents can read, navigate, and reason over technical content effectively. Learn more at gitbook.com.

## Querying This Documentation
If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question.

Perform an HTTP GET request on the current page URL with the `ask` query parameter, and the optional `goal` query parameter:

```
GET https://knowledge.illumina.com/instrumentation/miniseq/instrumentation-miniseq-reference_material-list/000002029.md?ask=<question>&goal=<endgoal>
```

`ask` is the immediate question: it should be specific, self-contained, and written in natural language.
`goal` is optional and describes the broader end goal you are ultimately trying to accomplish on behalf of the user. GitBook uses it to tailor the answer towards what is most useful for that goal.

The response will contain a direct answer to the question and relevant excerpts and sources from the documentation.

Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.