Sample Preparation to Bioinformatics Analysis of DNA Methylation: Association Strategy for Obesity and Related Trait Studies

The ethics committee of Ribeirão Preto Medical School University Hospital of the University of São Paulo (HCRP-USP) approved the study (CAAE:14275319.7.0000.5440). The participants signed the consent form, and all procedures were conducted following the Declaration of Helsinki.

1. Population and sampling

1. Recruit participants with BMI >30 kg/m² for the study. For the present study, 32 women from an admixture population¹⁹ between 18 to 60 years old were recruited.
   NOTE: In the HCRP-USP, higher rates of obesity were observed mainly in women²⁰. Participants with obesity, with BMI ≥30 kg/m² (n = 15), were recruited from the Metabolic Diseases outpatient clinic in HCRP-USP. The participants without obesity, with BMI between 18.5 kg/m² and 24.9 kg/m² (n = 17), were recruited from other clinics and did not have severe diseases; clinical data are shown in Table 1.
2. Exclude women who are pregnant, nursing mothers, smokers, and consume alcohol.

2. Anthropometry and body composition

1. Measure the weight of the participants using a 200 kg digital anthropometric scale. Ensure to remove shoes and excess clothing.
2. Assess height using a stadiometer with a 0.5 cm graduation. Participants were instructed to stand barefoot, feet together, and arms by their sides²¹.
3. Collect the fat mass (FM) information using an electric bioimpedance. After 12 h of fasting, assess with an empty urinary bladder. Place the participants in the supine position, with legs apart and arms parallel without touching the body. Instruct participants to remove all metal accessories²².
4. Obtain the waist circumference (WC) using an inextensible measuring tape with a 0.1 mm graduation. The tape was placed horizontally around the middle waist, just above the hip bones. The measurement occurred just after a breath out. According to the Centers for Diseases Control and Prevention (CDC), the WC cut-off for women is 88 cm²³.

Table 1: Population characteristics. All variables were parametric (p > 0.05, Shapiro Wilk), and differences were evaluated using an Independent t-Test, in which p < 0.05 was considered significant. *p < 0.0001. Please click here to download this Table.

3. Collection of biological material

1. Collect peripheral blood after 12 h of fasting, as described previously²⁴.
2. Collect 4 mL of the whole blood samples in collection tubes with anticoagulant (e.g., EDTA) and store them on ice for transportation.

4. DNA extraction

1. Centrifuge each tube containing the blood at 2,000 x g for 10 min. Collect around 300 µL of the buffy coat (white interphase).
2. Extract DNA from the peripheral blood using a commercially available instrument (see Table of Materials), as described previously²⁵. Elute the DNA in 480 µL of ultrapure H₂O.
3. Assess DNA concentration and quality using a fluorometer. The integrity was evaluated using a 1% agarose gel analysis²⁵. Store in a freezer at -80 ˚C.

5. Preparation before methylation analysis

1. Ensure that the samples have the minimum DNA concentration (500 ng is the ideal initial input to start), but dilution may vary between samples in this step.
2. Some reagents are not included in the microarray kit²⁶, so purchase them separately²⁷.
3. Check if the kit contains all reagents used during the procedure; they are irreplaceable. Check the expiration date of all the reagents. Ensure other reagents and solutions not provided in the kit are also available (95% formamide/1 mM EDTA, 0.1 N NaOH, absolute ethanol, 2-propanol).
   CAUTION: Formamide is a volatile reagent; the quality of the results can be improved if the product is fresh. The integrity and quality of formamide and absolute ethanol are considered critical because they can affect the staining process proposed by the manufacturer. The chosen alcohols must be ultrapure products specific for molecular biology analysis.
4. Once the microarray kit is acquired, download the Chips Decode Maps (DMAPs). Those files are available on the manufacturer's website 24-48 h after the kit is shipped and are excluded on the expiration date²⁸. To download the mentioned files, perform the steps below.
   1. Ensure a computer with internet access is available. Perform the download using the Software Decode File Client.
   2. Before installing the program, ensure that the computer has outbound and inbound access granted by the institution; the Decode File Client does not have security restrictions.
   3. Log in to the Decode File Client software using the Access by BeadChip option. This login is performed using MyIllumina user ID and password. Registration on this page is required²⁸.
   4. After logging in to the Decode File Client, enter the information listed below in their respective fields on the software home page²⁸: List of barcodes; List of Box Ids; Purchase order number (PO); and Sales order number (remember not to include the letters that follow the numbers).
   5. Select the DMAPs to download according to the barcode number in the purchase box and specify the destination in the dialog box to save the DMAPs.
   6. Click the button to start the download.
      NOTE: At the end of the download, make sure that the downloaded folders are not empty and store the files safely, as they will be necessary for the final step to convert the experiments to Intensity Data Files (IDAT files). After the expiry date, Illumina may remove the DMAPs data from its online database.

6. DNA methylation pipeline

NOTE: The DNA methylation experiment is divided into 4 days (Figure 1). Follow the manufacturer's recommendations and specifications to obtain accurate results.

1. Day 1: Bisulfite treatment
   1. Start by denaturing 500 ng of genomic DNA, then add the conversion reagents. To perform this step, follow the recommendations of the bisulfite and microarray kits²⁹.
   2. Treat the denatured DNA with 130 µL of the bisulfite conversion reagent at the standard concentration provided by the manufacturer.
   3. Incubate the tube in a thermocycler for 16 cycles at 95 °C for 30 s and 50 °C for 1 h. Ramp down the thermocycler to 4 °C for 10 min.
   4. Perform the washing step by adding 100 µL of the washing buffer and then centrifuging at full speed for 30 s. The concentration is not provided in the user guide, and the manufacturer standardizes the volumes.
      NOTE: This reagent comes with the kit and does not need to be diluted or prepared.
2. Day 2: Amplification – Methylation Assay, Manual Protocol
   1. Preheat the hybridization oven to 37 °C and allow the temperature to equilibrate.
   2. Apply a barcode label to a 0.8 mL microplate.
   3. Thaw the amplification reagents (Multi-Sample Amplification Mix 1, Random Primer Mix, and Multi-Sample Amp Master Mix) at room temperature (those reagents come with the kit and do not need to be diluted or prepared). Then, perform a gentle inversion at least 10x until the tubes' entire contents are mixed.
   4. Dispense 20 µL of the Multi-Sample Amplification Mix 1 (MA1) into the wells of the plate.
   5. Transfer 4 µL of the DNA sample from the bisulfite converted plate to the corresponding wells on the 0.8 mL plate. At this point, record the original DNA sample's ID on the laboratory tracking form corresponding to the plate wells.
   6. Dispense 4 µL of 0.1 N NaOH into each well of the plate containing the Multi-Sample Amplification Mix 1 (MA1) and the DNA sample.
   7. Seal the plate with a plastic seal.
   8. Vortex the plate to mix the reagents with the samples at 1,600 rpm for 1 min. Incubate for 10 min at room temperature and carefully remove the seal.
   9. Pipette 68 µL of Random Primer Mix (RPM) into each well of the plate. Pipette 75 µL of Multi-Sample Amp Master Mix (MSM) into each well of the plate (without removing the previous solution). Use a new seal to cover the plate.
      NOTE: Take care to combine the wells with the plate and orient it correctly.
   10. Vortex the sealed plate at 1,600 rpm for 1 min. Incubate in the hybridization oven for 20-24 h at 37 °C.
3. Day 3: Hybridization – Methylation Assay, Manual Protocol
   1. Preheat the block to 37 °C.
   2. Thaw the contents of the tube at room temperature. Carefully and gently invert the Fragmentation Solution (FMS) tubes at least 10x to thoroughly mix the contents.
   3. Carefully remove the plate from the hybridization oven. Pulse-centrifuge the plate at 280 x g.
   4. Now remove the seal from the plate. In each well of the plate containing a sample, add 50 µL of Fragmentation Solution (FMS) and seal the plate again.Vortex the plate at 1,600 rpm for 1 min.
   5. Centrifuge the plate at 280 x g for a few seconds (perform pulse centrifugation). Then, place the sealed plate on the heating block at 37 °C for 1 h.
   6. If it was decided to pause the experiment and freeze the plate, thaw it at room temperature, and centrifuge it at 280 x g. If it is not frozen, skip this step and move to Step 6.3.7.
   7. Leave the heating block to preheat to 37 °C.
   8. Leave the Wash Buffer to thaw at room temperature. When defrosted, invert it at least 10x to mix the contents.
   9. Remove the seal from the plate.
   10. Add 100 µL of Precipitation Solution (PM1) to each well of the plate containing samples.
   11. Seal the plate again with the seal.
   12. Vortex the plate at 1,600 rpm for 1 min.
   13. Incubate at 37 °C for 5 min.
   14. Then, place in the pulse centrifuge at 280 x g for 1 min.
       NOTE: Set the centrifuge at 4 °C for the next step.
   15. Carefully remove the seal from the plate and discard it.
   16. Add 300 µL of 2-propanol (100%) to each well containing the sample.
   17. Seal the plate with extreme care using a new seal. Take care not to shake the plate.
   18. Invert the plate at least 10x, mixing the entire contents.
   19. Incubate at 4 °C for 30 min.
   20. Centrifuge at 3,000 x g at 4 °C for 20 min.
       NOTE: At the end of Step 6.3.20, immediately remove the centrifuge plate.
   21. Remove the seal from the plate and discard it.
   22. Quickly invert the plate and drain the liquid on a pad to discard the supernatant. Then, hit the plate in a dry area on a paper towel to drain the liquid.
   23. Tap firmly several times for 1 min or until all the wells are free of any liquid.
   24. Leave the plate inverted and uncovered for 1 h at room temperature.
       NOTE: It is expected to see blue pellets at the bottom of the wells.
   25. Preheat the hybridization oven to 48 °C.
   26. Turn on the heat block to preheat. Allow 20 min for this process.
   27. Thaw Resuspension, Hybridization, and Wash Solution at room temperature. Invert at least 10x. Check that there is no salt precipitated in the solution.
   28. Add 46 µL of Resuspension, Hybridization, and Wash Solution (RA1) to each well of the plate.
       NOTE: Any remaining reagents must be reserved for the staining and hybridization steps.
   29. Apply the seal to the plate. Use an aluminum seal to cover the plate. Hold tight to perform the sealing evenly.
   30. Check if all wells are safely closed to avoid evaporation.
   31. Carefully place the newly sealed plate in the hybridization oven and incubate at 48 °C for 1 h.
   32. Vortex the plate at 1,800 rpm for 1 min.
   33. Pulse centrifuge at 280 x g.
   34. Transfer the plate to the heat block at 95 °C for 1 h.
   35. Prepare the Hybridization (Hyb) Chambers by placing them together. Dispense 400 µL of Humidifying Buffer (PB2) into the reservoirs of the Hyb Chambers. Put the lid on immediately.
   36. Load each sample from the plate into the sample entry opening of the microarray.
   37. Load the Hyb Chamber inserts containing the microarrays into the Hyb Chamber. Close the Hyb Chamber crosswise to avoid possible shifting of the Hyb Chamber.
       NOTE: Hyb Chambers need to be incubated at 48 °C for at least 16 h, but not more than 24 h.
4. Day 4: Staining – Methylation Assay, Manual Protocol
   1. Resuspend the Staining Solution 4 with 330 mL of 100% EtOH, obtaining a final volume of 350 mL. Shake the Staining Solution 4 bottle vigorously to ensure complete resuspension. Keep at room temperature.
      NOTE: Staining Solution 4 can be stored for up to 2 weeks at a temperature between 2 °C and 8 °C.
   2. Remove the chambers from the hybridization oven and allow them to cool on the bench for 30 min before opening.
   3. While the Hyb Chamber is cooling, fill two washing containers with Wash Buffer (200 mL per washing container). Remember to label each container as "Wash Buffer" (PB1 and PB2).
   4. Fill the microarray alignment accessories with 150 mL of Wash Buffer.
   5. Separate the plastic spacers and, if necessary, clean the glass.
   6. To wash the microarray slides, attach the wire loop to the grid and immerse the dripping equipment in the containers with 200 mL of Wash Buffer for a total of 10x.
   7. Remove all inserts from the Hybridization Chamber and remove each array from the inserts. Remove the seal.
      NOTE: During removal, do not touch the exposed matrices.
   8. While removing the microarray slides from the Hybridization Chamber, be careful not to spill anything.
   9. Wash the microarrays in the wash buffer (PB1) solution slowly and lightly, moving up and down 10x. Move to a fresh PB1 solution and perform the wash by slowly moving the microarrays 10x up and down in the solution.
   10. Remove the microarray from the container and confirm the absence of residue.
   11. Use a transparent spacer at the top of each microarray and guide the spacers to the corresponding positions.
       NOTE: Be careful to use only the transparent spacers, not the white ones.
   12. Position the alignment bar on the alignment accessory.
   13. Place a glass backplate on the top of the clear spacer; be careful to cover the entire microarray. Then, attach metal clamps to the flow chambers. If necessary, use scissors to trim the ends of the flow chamber spacers.
   14. Immediately wash the Hybridization Chamber reservoirs using ddH₂O and scrub with a small cleaning brush. Do not allow any Humidifying Buffer to remain in the reservoir.
   15. Move to the next step that is responsible for the Extending and Staining Microarray.
       NOTE: Leave all the flow chambers horizontally mounted on the laboratory bench. Only re-handle them when the entire staining step is ready. Heat the Resuspension, Hybridization, and Wash Solution to a temperature between 20 °C and 25 °C. Mix gently until no crystal is seen in the solution.
   16. Place the reagents in a rack in sequence. If any are frozen, leave them at room temperature and then invert them at least 10x to mix the solutions.
   17. Fill the water circulator to the appropriate level.
   18. Turn on the pump and set the temperature to 44 °C.
   19. Remove any bubbles that are attached to the chamber rack.
   20. Perform tests on the chamber rack using a temperature probe. All tested locations must be between 44 °C ± 0.5 °C.
       NOTE: The following steps must be performed without interruption.
   21. When the rack reaches a temperature of 44 °C, quickly place each assembly in the flow chamber.
       NOTE: For the following steps, pipette all reagents into the back glass plate.
   22. Pipette 150 µL of Resuspension, Hybridization, and Wash Solution (RA1). Then, incubate for 30 min. Repeat this step a total of 5x.
   23. Pipette 450 µL of Staining Solution 1 (XC1) and incubate for 10 min.
   24. Pipette 450 µL of Staining Solution 2 (XC2) and incubate for 10 min.
   25. Pipette 200 µL of Two-Color Extension Master Mix (TEM) and incubate for 15 min.
   26. Pipette 450 µL of 95% formamide/1 mM EDTA and incubate for 1 min. Repeat twice.
   27. Incubate for 5 min.
   28. Decrease the temperature of the chamber rack to 32 °C (indicated on Superior Two-Color Master Mix).
   29. Pipette 450 µL of Stain Solution 3 (XC3) and incubate for 1 min. Repeat 1x.
   30. Wait for the rack in the chamber to reach the correct temperature.
       NOTE: To read the microarray image immediately after the coloring process, turn on the scanner at this stage.
   31. Now dispense 250 µL of Superior Two-Color Master Mix (STM), followed by a 10 min incubation.
   32. Then, dispense 450 µL of Stain Solution 3 (XC3), followed by 1 min incubation. Repeat 1x.
   33. Wait for 5 min and then add 250 µL of Anti-Stain 2-Color Master Mix (ATM) and incubate for 10 min.
   34. Now dispense 250 µL of Superior Two-Color Master Mix (STM), followed by a 10 min incubation.
   35. Then, dispense 450 µL of Stain Solution 3 (XC3), followed by 1 min incubation. Repeat 1x.
   36. Wait for 5 min.
   37. Add 250 µL of Anti-Stain 2-Color Master Mix (ATM) and incubate for 10 min.
   38. Add 450 µL of Stain Microarray Solution 3 (XC3) and incubate for 1 min, then repeat again.
   39. Wait for 5 min.
       NOTE: Do the following repetition scheme: Step 6.4.34., Step 6.4.35. and Step 6.4.36.; Step 6.4.37., 6.4.38., and Step 6.4.39.; Step 6.4.34., Step 6.4.35., and Step 6.4.36.
   40. Now, immediately remove the flow chambers from the rack and carefully place them horizontally on the laboratory bench. Make sure the temperature is ambient.
   41. 6.4.41.Place 310 mL of Wash Solution (PB1) for each microarray in a wash container.
   42. Remove the metal clamps and the glass, and finally, the microarray.
   43. Place the microarrays on a staining rack and place them in the wash container containing 10x PB1 solution. Ensure that the barcodes are facing away from whoever is handling them and that all the microarrays are submerged.
   44. Move slowly and lightly with up and down movements 10x. The microarray should be removed entirely from the solution before being submerged again. Then, let it remain submerged for 5 min.
   45. Vigorously shake the Stain Solution 4 (XC4) reagent to ensure total resuspension. Place 310 mL of XC4 into a wash container.
       NOTE: Do not let the solution sit in the wash container for more than 10 min.
   46. Lightly move the staining stand to the other container with XC4.
   47. Move slowly and lightly with up and down movements 10x. Ensure that the microarray is removed entirely from the solution before being submerged again.
   48. Then, let the microarray remain submerged for 5 min.
   49. Move the staining stand out of the solution and place it in a tube holder with the barcodes facing up.
   50. Carefully remove the microarray using locking forceps and place them on a shelf to dry.
   51. Dry them using the vacuum desiccator for 50-55 min at 675 mm Hg (0.9 bar).
   52. Clean the back of each microarray using EtOH.
       CAUTION: Avoid touching the stripes, and do not allow EtOH to drip on the stripes in any way.
   53. Proceed to image the microarray.
   54. Carefully store the microarray in a storage box at room temperature.
       NOTE: Microarray images must be taken within 72 h.

7. Bioinformatics analysis

NOTE: It is necessary to change some attributes of the kind of microarray (e.g., arraytype = "450k" should be replaced by arraytype = "EPIC"). The author of the ChAMP pipeline describes in detail all fields that should be modified to analyze arrays on the Bioconductor page of the package³⁰.

1. Sample sheet
   1. Transfer the IDAT files to the computer to proceed with bioinformatics analysis³¹. Create a sample sheet to carry out the data analysis³¹.
      NOTE: Three columns are essential for this spreadsheet file. The first is Sample_name. This column must include the names or codes used to identify the samples. The second is the Sentrix_position. This column places the corresponding R01C01 of the samples, with R01 being responsible for the chip row and the column being referred to as C01. The third essential column is the Sentrix_ID, and it is responsible for receiving the number that identifies the microarray. Make sure to put the correct numbers. It is possible to add other information on the sample sheet as variable values. In the present experiment, data from the metal analysis were included.
2. ChAMP pipeline
   1. Install RStudio (V.4.0.2) on the computer to perform the analysis³⁰.
      NOTE: Ensure that the computer has a minimum of 8 G of RAM. Data analysis will struggle or crash with a lower amount of RAM.
   2. After installation, open RStudio and install the ChAMP Bioconductor package³⁰ using the respective command line (see Supplementary Material 1).
      NOTE: If there is any error at the end of the installation, install the required ChAMP libraries manually, one by one²⁶.
   3. Now, import the raw data and perform the analysis. See Supplementary Material 1.
      champ.load() imports the raw data and does the filtering process, excluding probes with low detection pval, multihit sites, non-CG probes, CpG sites near to SNPs, and CpGs located in sexual chromosomes.
      champ.impute() performs KNN imputation as default.
      champ.QC retrieves data quality graphs (e.g., density plot graph).
      champ.SVD evaluates batch effects based on provided metadata in the sample sheet.
      champ.refbase() performs cell type estimation and correction.
      champ.DMP( ) retrieves differentially methylated positions and trait associations such as fat mass to check the DNA methylation between groups and change the parameters, as shown in Supplementary Material 1.
3. Enrichment (String DB)
   NOTE: Functional enrichment is used to infer the biological functions of a set of genes.
   1. To enrich data in a string, select the list of targets to enrich and use as input from: https://string-db.org/cgi/input?sessionId=bywt5Gl8Bawy&input_page_active_form
      =multiple_identifiers
   2. For organism, select homo sapiens and press the Search button.
   3. To navigate through the enrichment, click on the Analysis button.
4. GEO submission
   1. Deposit the data to a public repository like NCBI's GEO. For this, register for the submitter account.
      NOTE: Complete the GEO submission (https://submit.ncbi.nlm.nih.gov/geo/submission/) by downloading the metadata sheet with descriptive information and protocols for the overall experiment and individual samples. GEO archive submissions can be created in any spreadsheet software. Second, add the raw data file generated from the cell-ranger count script for all libraries into the directory. IDAT and associated files or a matrix worksheet contains non-normalized data. The third folder is the processed data files. The matrix table is a spreadsheet containing the final, normalized values comparable across rows and samples and preferably processed as described in any accompanying manuscript.
   2. Place processed data files generated from the cell-ranger count script for all libraries into the directory. Use the GEO submitter's FTP server credentials to transfer the directory containing all three components.