How to Determine the Sex of Samples in a GenomeStudio Methylation Project

This article describes two commonly used methods for determining the sex of samples in a GenomeStudio Methylation project and provides step-by-step instructions for each approach.

The methods described assume a working knowledge of the GenomeStudio Methylation Module and the analysis of data generated from Infinium Methylation Arrays, such as the MethylationEPIC v2 array, or custom methylation arrays that contains a significant number of Y chromosome markers.

These approaches also assume that the project contains a mixture of male and female samples.

Methods for Determining the Sex of Samples in a GenomeStudio Methylation Project 1. Filter the Sample Methylation Profile for Y-chromosome markers. 1. In the Sample Methylation Profile table, select the "Filter" button (Figure 1).

Figure 1. Sample Methylation Profile table in GenomeStudio Methylation Module.

1. 1. In the subsequent pop-up window, create a filter with the condition "CHR = Y" to display markers located on the Y chromosome (Figure 2).

   

Figure 2. Filter Settings window for the Sample Methylation Profile table in GenomeStudio.

Important Note: If the data was analyzed using the manifest EPIC-8v2-0_A1.bpm, the chromosome designations differ from those used in other MethylationEPIC manifests by including the prefix "chr" (Figure 3).

**Figure 3.**Example of the CHR column displayed in a GenomeStudio Methlylation project created using the manifest file EPIC-8v2-0_A1.bpm.

In this case, the value input for the chromosome must include this prefix. Therefore, the filter must be set to "Chr = chrY", otherwise, GenomeStudio will not correctly recognize the chromosome and the filter will not return the expected results (Figure 4).

Figure 4. Filter Settings for a GenomeStudio Methylation project using the EPIC-8v2-0_A1.bpm manifest.

1. 1. Select "OK". The markers displayed in the Sample Methylation Profile table will now be limited to Y-chromosome markers only (Figure 5).

   

Figure 5. Sample Methylation Profile table filtered for Y chromosome using the manifest EPIC-8v2-0_A1.bpm.

1. Inspect the Y-chromosome intensity values for the samples in the Sample Methylation Profile table.

   1. In the Sample Methylation Profile table, select the Box plot button (Figure 6).

   

Figure 6. Box plot button in the Sample Methylation Profile table.

1. 1. In the subsequent pop-up window, select the "Intensity" subcolumn. Female samples generally show near-background intensities, while male samples exhibit noticeably higher intensity values (Figure 7).

Figure 7. Y-chromosome Intensity Box plot of Y chromosomes.

Alternative method

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

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