# How to properly format and use a custom reference genome in MiSeq Reporter

MiSeq instruments with MiSeq Control Software (MCS 2.6) or earlier include several pre-installed reference genomes that are used during the alignment and variant calling steps of many [MiSeq Reporter analysis workflows](http://support.illumina.com/downloads/miseq-reporter-workflow-reference-guides.html), which can also utilize user-added custom reference genomes. A custom reference genome file must be properly formatted and added to the MiSeq Reporter and [Illumina Experiment Manager](http://support.illumina.com/sequencing/sequencing_software/experiment_manager.html) (IEM) Genome repositories so that it can be used for analysis in MiSeq Reporter. Custom reference genomes cannot be used for analysis in BaseSpace.

**General tips:**

* The file must be in FASTA format using the \*.fa extension, not the \*.fasta extension.
* Use a text editor such as Notepad or TextEdit, or a source code editor like [Notepad++](https://notepad-plus-plus.org/), to edit your file.
* Sequence identifiers are limited to 24 characters and can only contain letters, numbers, hyphens, and underscores. All other characters, including blank spaces, are not allowed in the sequence identifiers.
  * Examples of good and bad sequence identifiers:\
    ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-2c81895bc6c2f932949e3e7db04bea8a09a6cc93%2Fimage1.png?alt=media\&token=ca796e21-f847-4128-b983-4cc32c5037c6)
* For the sequences, each line in the file is limited to 80 characters in length. The sequences must contain only the characters A, T, C and G and may use only upper-case characters. Ns, blank spaces, and lower case characters are not allowed.
* Each line in the file must end with a line feed character (LF), not a carriage return (CR), nor both a CR and LF. Some source code editors such as Notepad++ include an option to view all characters, which displays invisible characters such as end of line characters. In Notepad++, to change all end of line characters to LF:
  * Go to **Edit** > **EOL Conversion** > **Unix Format**.

**Add the reference file to the MiSeq Reporter Genome repository:**

1. On the MiSeq PC, navigate to the Genome repository. The default path is C:\Illumina\MiSeq Reporter\Genomes.
2. In the Genomes folder (repository), create a new folder and give the new folder a genome name. Avoid using any spaces in the name.
3. Place the custom reference genome FASTA file inside the new folder.

**Add the reference file to the Illumina Experiment Manager (IEM) Genome repository:**

1. Open IEM and select the **Folder Settings** button. Take note of the Genome Repository path.
2. Navigate to the **Genomes** folder shown in the Genome Repository path.

* Note: The default Genomes directory is often hidden by default. Either copy and paste the path from the folder settings to a new Computer window or configure Windows to view hidden files in order to follow the full path. To view hidden files, open the Windows **Control Panel**, select **Folder Options**, and click on the **View** tab. Select **Show hidden files, folders and drives** and uncheck the **Hide extensions for known file types** setting.

3. In the **Genomes** folder, create a new folder and give the folder a genome name (do not use spaces in the name), then place your custom reference genome FASTA file inside the new folder.
4. Close and re-open IEM.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #2212), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000002212%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |