# A quick visualization method to help determine the sex of given samples in a GenomeStudio Methylation project

The following method assumes a working knowledge of the GenomeStudio Methylation Module, and the analysis of data from an Infinium Methylation Array, such as Methylation450K, MethylationEPIC, or MethylationEPIC v2, or a custom methylation array that contains a significant number of markers being assayed that are on the Y chromosome. It also assumes that there is a mixture of samples with regard to sex.

Step 1: filter the sample methylation profile for Y chromosome markers, then compare the intensities for the samples across the project.

* In the sample Methylation Profile table, click on the "Filter" button\
  ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-3f2cd65c94b4261573b445bbae98d3864c9637ca%2Fimage1.png?alt=media)
* In the subsequent pop-up window, create a filter: "CHR = Y"\
  ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-d28ea097e091d827a5759a443d17d6e68b14c406%2Fimage2.png?alt=media)
  * IMPORTANT NOTE - If your data was analyzed using the manifest "EPIC-8v2-0\_A1.bpm", the chromosome designations are unlike all other MethylationEPIC manifests, in that they include a prefix "chr".\
    ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-a0c2edc113585deaf3925712605e2e41477a08a6%2Fimage3.png?alt=media)

    In this case, the Value input for the chromosome must include this prefix, i.e. the filter must read "Chr = chrY", otherwise the filter will not recognize the chromosome designation correctly.\
    ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-82025efdbd43409ae995d929382421e6c5d7bdeb%2Fimage4.png?alt=media)
* Click on "OK". The displayed markers in the Sample Methylation Profile table will now consist only of Y-chromosome markers\
  ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-7458b2e2f38d33d37c8ed7d3ec1af069d0b852a9%2Fimage5.png?alt=media)

Step 2 - inspect the Y-chromosome intensity values for the samples in the Sample Methylation Profile table.

* In the Sample Methylation Profile, click on the Box plot button\
  ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-cbf58f7399f648e38b39b813313564bd5dfe74d6%2Fimage6.png?alt=media)
* In the subsequent pop-up window, select the "Intensity" sub column. Females will have near background intensities, while males will show elevated intensity boxes.\
  ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-1e67851511c9a6b1114882d7cb3105625053f80f%2Fimage7.png?alt=media)

**Alternative method**

The following publication describes an alternate method utilizing the beta values from the X chromosome to determine sex from Infinium Methylation data:

Wang Y, Hannon E, Grant OA, Gorrie-Stone TJ, Kumari M, Mill J, Zhai X, McDonald-Maier KD, Schalkwyk LC. DNA methylation-based sex classifier to predict sex and identify sex chromosome aneuploidy. BMC Genomics. 2021 Jun 28;22(1):484. doi: 10.1186/s12864-021-07675-2. PMID: 34182928; PMCID: PMC8240370.

<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8240370/>

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