TITLE: Automated procedure for estimation of methylation levels in MS-HRM analysis
AUTHORS: Sally Samsø Mathiasen, Jan Bińkowski, Tina Kjeldsen, Tomasz K Wojdacz, Lise Lotte Hansen

[DESCRIPTION]
Testing for disease-related DNA methylation changes provides clinically relevant information in personalized patient care. Methylation-Sensitive High-Resolution Melting (MS-HRM) is a method used for measuring methylation changes and has already been used in diagnostic settings. This method utilizes one set of primers that initiate the amplification of both methylated and non-methylated templates. Therefore, the quantification of the methylation levels using MS-HRM is hampered by the PCR bias phenomenon. Some approaches have been proposed to calculate the methylation level of samples using the high-resolution melting (HRM) curves. However, limitations of the methylation calculation using MS-HRM have not been evaluated systematically and comprehensively. We used the Area Under the Curve (AUC), a derivative of the HRM curves, and least square approximation (LSA) to establish a procedure that allowed us to infer methylation levels in an MS-HRM experiment and assess the limitations of that procedure for the assays’ specific methylation level measurement. The developed procedure allowed, with certain limitations, estimation of the methylation levels using HRM curves. 

[STEPS]
SECTION: DATA IMPORT
1. MS-HRM data preparation for the analyses (when using Light Cycler system – other PCR systems may require adjusting of the data format) 
SECTION: DATA IMPORT
1.1. Normalize the HRM curves using the Gene Scanning software (we recommend default settings for the normalization). 

SECTION: DATA IMPORT
1.2. Generate difference plots for each normalized melting curve with the 100% methylation melting curve as the baseline/reference.

SECTION: DATA IMPORT
1.3. If data for any of the samples contain obvious outliers, you may consider removing the outliers (at this point only note the name of the outlier). 
SECTION: DATA IMPORT
1.4. Export the difference plot as a text file. 
An example of layout of the file obtained from LightCycler480 instrument (Roche) see supplementary material S8-S11 File.
 

SECTION: CALCULATION OF EXPERIMENT SPECIFIC STANDARD CURVE
2. Methylation levels estimation procedure 
SECTION: CALCULATION OF EXPERIMENT SPECIFIC STANDARD CURVE
2.1. Open the Methylation Levels Calculator (MLC) (S2 File Methylation Levels Calculator (MLC)). 
 



SECTION: CALCULATION OF EXPERIMENT SPECIFIC STANDARD CURVE
2.2. Open the exported text file exported from LC480 instrument (see for an example file: S10-S11 MGMT assay text_without outliers).

SECTION: CALCULATION OF EXPERIMENT SPECIFIC STANDARD CURVE
2.3. Select and copy all the data in the text file and paste it into the sheet “imported data” starting in cell B3. Make sure that the names in rows 2 and 3 agree (For the purpose of this example, the outliers are removed in the text file)
 

 

SECTION: CALCULATION OF EXPERIMENT SPECIFIC STANDARD CURVE
2.4. The MLC will now automatically calculate AUC and display the AUC value for each control and sample. That value is shown in raw 1 of the “Imported data” sheet of the MLC.
SECTION: CALCULATION OF EXPERIMENT SPECIFIC STANDARD CURVE
2.5. Check if the value of AUC for each replicate (replicates of each control are marked with the same colours) in row 1 agree within acceptable range (if not consider excluding outliers by replacing the values with 0. Compare the outlier with the findings of 1.4).

 


SECTION: CALCULATION OF EXPERIMENT SPECIFIC STANDARD CURVE
2.6. Go to the sheet: “0 variable”. 
 


SECTION: CALCULATION OF EXPERIMENT SPECIFIC STANDARD CURVE
2.7. Go to the sheet: “1 variable”. 
 

SECTION: CALCULATION OF EXPERIMENT SPECIFIC STANDARD CURVE
2.8. Go to the sheet: “1 variable after LSA”. 
 

SECTION: CALCULATION OF EXPERIMENT SPECIFIC STANDARD CURVE
2.9. Go to: Data > Solver > Solve on the pop-up window > OK in the next pop-up window. Now the M value has been calculated by LSA and the standard curve was recalculated accordingly - the blue curve in fig1 in panel 3 sheet “1 variable”.

 

SECTION: CALCULATION OF EXPERIMENT SPECIFIC STANDARD CURVE
2.10. Go to the sheet: “2 variables” 

 

SECTION: CALCULATION OF EXPERIMENT SPECIFIC STANDARD CURVE
2.11. Go to the sheet: “2 variables after LSA”. 
 

SECTION: CALCULATION OF EXPERIMENT SPECIFIC STANDARD CURVE
2.12. Go to: Data > Solver > Solve on the pop-up window and click OK in the next pop-up window. Now the M and N value have been calculated by LSA and standard curve was recalculated accordingly - the blue curve in fig 1 in panel 3 sheet “2 variable”. 

 

SECTION: ESTIMATION OF THE METHYLATION LEVEL IN UNKNOWN SAMLES
3. ESTIMATION OF THE METHYLATION LEVEL IN UNKNOWN SAMLES
 

SECTION: ESTIMATION OF THE METHYLATION LEVEL IN UNKNOWN SAMLES
3.1. Go to the sheet: “Polynomial trend function (PTF)”

 

SECTION: ESTIMATION OF THE METHYLATION LEVEL IN UNKNOWN SAMLES
3.2. Go to the sheet: “Unknown sample calculation (USC)”

 

SECTION: CALCULATION OF THE EXPRIMENT SPECIFIC DETECTION WINDOW
4. CALCULATION OF THE EXPRIMENT SPECIFIC DETECTION WINDOW
SECTION: CALCULATION OF THE EXPRIMENT SPECIFIC DETECTION WINDOW
4.1. Go to the sheet: “Cut off (CO)”

 

SECTION: CALCULATION OF THE EXPRIMENT SPECIFIC DETECTION WINDOW
4.2. Go to sheet: “Detection window” 

 

SECTION: CALCULATION OF METHYLATION LEVELS IN THE ASSAY SPECIFIC DETECTION WINDOW
5. CALCULATION OF METHYLATION LEVELS IN THE ASSAY SPECIFIC DETECTION WINDOW
SECTION: CALCULATION OF METHYLATION LEVELS IN THE ASSAY SPECIFIC DETECTION WINDOW
5.1. Go to the sheet: “2 variables within DW”.

 

SECTION: CALCULATION OF METHYLATION LEVELS IN THE ASSAY SPECIFIC DETECTION WINDOW
5.2. Go to: Data > Solver > Solve on the pop-up window and click OK in the next pop-up window. Now the M and N value have been calculated by LSA and standard curve was recalculated accordingly.

 

SECTION: CALCULATION OF METHYLATION LEVELS IN THE ASSAY SPECIFIC DETECTION WINDOW
5.3. Go to the sheet “PTF within DW”. PTF (Polynomial Trend Function).

 

SECTION: CALCULATION OF METHYLATION LEVELS IN THE ASSAY SPECIFIC DETECTION WINDOW
5.4. Go to the sheet “USC within DW” USC (Unknown Sample Calculations)