> 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/general/instrumentation-general-reference_material-list/000001891.md).

# Why sequencing 26 or more cycles in Read 1 is recommended

The following guidance maximizes data yield and quality for customers sequencing custom libraries. All Illumina libraries are designed to be sequenced with a minimum first read length of 26 cycles. Illumina also recommends designing custom libraries to be sequenced with a minimum of 26 cycles to ensure reliable base calling and quality scoring.

Illumina primary analysis software, Real-Time Analysis (RTA), is optimized and validated to use the first 25 cycles of Read 1 for quality metrics calculations. Illumina recommends running a minimum of 26 cycles for Read 1 to make sure that all RTA calculations are complete. If specified during run setup, Illumina sequencing systems can perform fewer than 25 cycles for Read 1 though this is not recommended. For runs with fewer than 25 cycles in Read 1, RTA reports pass filter percentages and calculated quality scores from the available Read 1 cycles. However, as RTA performance has not been tested under these conditions, the selection of reads passing filter and the quality scoring metrics reported for these runs is not equivalent or directly comparable to those runs with a Read 1 length of 25 or more cycles. For customers sequencing fewer than 25 cycles in Read 1, independent validation of the quality metrics is required.

For custom libraries with regions of low-diversity sequences such as internal barcodes, primer annealing sites, or homopolymers, consider increasing base diversity with the addition of a well-balanced library such as PhiX instead of sequencing a first read with fewer than 25 cycles. The percentage of PhiX to include with a custom library is instrument- and library-dependent and may require optimization. When sequencing libraries that use custom sequencing primers, use PhiX by spiking custom primers into Illumina primers. The following support bulletin provides additional information: [Spiking custom primers into the Illumina sequencing primers](https://support.illumina.com/bulletins/2016/04/spiking-custom-primers-into-the-illumina-sequencing-primers-.html).

\
\
\ <br>

|                                                                                                                                                                                                                                                                                                                                                                        |
| :--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: |
| *For any feedback or questions regarding this article (Illumina Knowledge Article #1891), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000001891%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 by asking a question.

Perform an HTTP GET request on the following URL with the `ask` and `goal` query parameters:

```
GET https://knowledge.illumina.com/instrumentation/general/instrumentation-general-reference_material-list/000001891.md?ask=<question>&goal=<user_goal>
```

`ask` is the immediate question: it should be specific, self-contained, and written in natural language.
`goal` is what the user is ultimately trying to achieve, the reason they need the answer. Sharing it helps GitBook give you a better, more relevant answer. A goal is most helpful when it describes the outcome the user wants rather than restating the question. For example, with `ask=how do I create an API token`, a goal like `automate deployments from our CI pipeline` lets GitBook tailor the answer to that use case.

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.