How to use BeadArray Controls Reporter (BACR) to quickly analyze Infinium Methylation Array Control Data

The BeadArray Controls Reporter (BACR) software can be used to quickly analyze the built-in controls on Infinium Methylation Arrays, including Infinium MethylationEPIC v2.

Why use BeadArray Controls Reporter?

• Computationally faster than making a GenomeStudio 2011.1 project to analyze data.

• Helpful for large datasets that may be analyzed with third-party software.

• Automatically compares expected positive signal with expected negative signal to remove background and flags controls that deviate from the expected relative intensity patterns with yellow highlighting.

• Populates a spreadsheet with results, which is convenient for keeping records/documenting run performance data.

How to run an analysis of Methylation Controls with BACR

1. Ensure that data output files from scanner are labeled with 12-digit barcode of BeadChip, and that these folders contain .idat files. Be sure not to use the DMAP folders that are also named by BeadChip barcode; the contents of the folder should contain .idat files and not .gz DMAP files.

2. Make a folder on the computer that contains the following:

• all IDAT folders to be included in analysis

• .bpm manifest file

• .csv Sample sheet. For instructions on how to prepare sample sheet, refer to the Sample Sheet Creation section in the BeadArray Controls Reporter Software Guide. Figure 1. Example of folder containing IDAT Files for all samples on a BeadChip

1. Open Bead Array Controls Reporter and select the 'Browse' button to assign the 'IDAT File Location'. The IDAT File Location is the parent folder containing all the required files listed above-- in this example, 'Demo Data EPIC.'

Figure 2. Assigning the IDAT File Location

1. Select 'Options' > 'OutputDirectory' to configure where the output file will be saved. If this step is skipped, the output file will be saved under Documents by default. Figure 3. Modifying the Output Directory

2. Click 'Start Analysis' to run the application.

3. Open the output file using Microsoft Excel or a similar spreadsheet program. The output file will be saved to the selected location and will have the name of the parent folder with the word 'controls' appended to the end of the file name.

4. Interpret the results

• The products of the calculations performed by BACR will be flagged with yellow highlighting in the spreadsheet, if the thresholds set in the BACR application are exceeded. It is recommended to conduct a more thorough evaluation of the controls using GenomeStudio 2011.1 Controls Dashboard visualization tool if values are flagged as yellow.

• In some cases, the thresholds (including the Background Correction Offset) may need to be adjusted slightly depending on the scanner that was used.

BACR Options - Customizing Threshold Settings

To adjust the thresholds used by BACR to flag calculation products as yellow, select 'Set Thresholds' from the Options menu.

Figure 4. Modifying threshold values

Figure 5. Default Output file thresholds

** If the FFPE restore kit was used, set the Restoration Threshold to 1. Otherwise, leave at default setting (0). ** Only adjust Thresholds that have a value of 1 by default. Do not adjust Thresholds with default value of 5.

Refer to the BeadArray Controls Reporter Software Guide for more information on the calculations conducted to arrive at the output file results. This is an excellent documentation on calculations and interpretation of Methylation Controls.

How to prepare a Sample Sheet

The Sample Sheet template can be downloaded from the Illumina website. Reformat the barcode/Sentrix ID as a 'Number' with 0 decimal places in Excel before saving. Excel will default back to scientific notation, but the values should be in Number format before saving the .csv file.

Figure 6. Example of Sample Sheet

Resources:

• BeadArray Controls Reporter Installer

• Illumina Knowledge: Sample Sheet Guidelines for GenomeStudio

For any feedback or questions regarding this article (Illumina Knowledge Article #3398), contact Illumina Technical Support [e-mail].