# About

The MethylAid web application provides different tab-panels for displaying filter control plots, sample-dependent and sample-independent diagnostic plots of the various quality control probes present on the 450k Human Methylation array.

On a filter control plot you can highlight a single sample by using a mouse click (clicking in `empty space` will undo the highlighting). The highlighted sample will be visible on all plots.

On the left of the plot area there is a conditional-control panel with widgets providing additional interaction specific to each plot. When provided, samples can be colored by metadata using a drop down menu to select a particular column.

A checkbox is present to allow highlighting of the outliers which will also be visible on all plots. An overview of the determined outliers is given in a separated tabpanel. Furthermore, each plot can be downloaded as a png image.

# Contact

m.van_iterson@lumc.nl

Department of Molecular Epidemiology, Leiden University Medical Center,

Leiden, The Netherlands

# Description of the filter plots

Five filter plots are proposed to determine bad-quality samples. A plot based on the median Methylated and Unmethylated log2 intensity, non-polymorphic sample-dependent quality control probes, Type I bisufite conversion quality control probes, sample-dependent hybridization quality control probes and a plot representing per sample the fraction of probes above background using the detection p-value based on the negative controls. To make optimal use of the plotting area and filtering out bad quality samples easier by using just one threshold all plots except based on the the detection p-value are 45 degrees rotated like the tranditional microarray MA-plots. The default thresholds are based on our experience with 450k data that we have seen so far. We have obtained good results with excluding samples with:

1. a median Methylated and Unmethylated log2 intensity smaller than 10.5; Rotated MU plot
2. an average log2 intensity of the expected signals in green and red channel of non-polymorphic controls smaller than 11.75; Overall sample-dependent control plot
3. an average log2 intensity of converted Bisulfite Type I controls in green and red channel smaller than 12.75; Bisulfite converion control plot
4. an average log2 intensity of High and Low hybridization controls (green channel) smaller than 13.25; Overall sample-dependent control plot
5. less 95% of their probes above the background signal;Detection p-value plot

# Description of the quality control probes

Below you find a detailed description of the quality control probes taken from “Infinium HD Assay Methylation Protocol Guide” (Part # 15019519 Rev. B ).

Bisulphite conversion controls (Type I): These controls use the Infinium I probe design and allele- pecific single base extension to monitor efficiency of bisulfite conversion. If the bisulfite conversion reaction was successful, the 'C' (Converted) probes will match the converted sequence and be extended. If the sample has unconverted DNA, the 'U' (Unconverted) probes will be extended. There are no underlying C bases in the primer landing sites, except for the query site itself. Performance of bisulfite conversion controls C1, C2, and C3 should be monitored in the Green channel, and controls C4, C5, and C6 should be monitored in

Red channel.

Bisulphite conversion controls (TypeII): These controls use Infinium II probe design and single base extension to monitor the efficiency of bisulfite conversion. If the bisulfite conversion reaction was successful, the 'A' base will be incorporated and the probe will have intensity in the Red channel. If the sample has unconverted DNA, the 'G' base will be incorporated across the unconverted cytosine, and the probe will have elevated signal in the Green channel.

Specificity controls I: These controls are designed to monitor allele-specific extension for Infinium I probes. The methylation status of a particular cytosine is carried out following bisulfite treatment of DNA through the use of query probes for unmethylated and methylated state of each CpG locus. In assay oligo design, the A/T match corresponds to the unmethylated status of the interrogated C, and the G/C match corresponds to the methylated status of C. G/T mismatch controls check for non-specific detection of methylation signal over unmethylated background. PM controls correspond to A/T perfect match and should give high signal. MM controls correspond to G/T mismatch and should give low signal. Performance of GT Mismatch controls should be monitored in both green and red channels. The Controls dashboard table lists expected outcome for controls probes.

Specificity controls II: These controls are designed to monitor extension specificity for Infinium II probes and check for potential non-specific detection of methylation signal over unmethylated background. Specificity II probes should incorporate the 'A' base across the non-polymorphic T and have intensity in the Red channel. In case of non-specific incorporation of the 'G' base, the probe will have elevated signal in the Green channel.

Non-polymorphic controls: Non-polymorphic controls test the overall performance of the assay, from amplification to detection, by querying a particular base in a non-polymorphic region of the bisulfite genome. They let you compare assay performance across different samples. One non-polymorphic control has been designed to query each of the four nucleotides (A,T, C,and G). Controls Table on page 236 lists the bead type IDs. The target with the C base results from querying the opposite whole genome amplified strand generated from the converted strand.

Negative controls: Negative control probes are randomly permutated sequences that should not hybridize to the DNA template. Negative controls are particularly important for methylation studies because of a decrease in sequence complexity after bisulfite treatment. The mean signal of these probes defines the system background. This is a comprehensive measurement of background, including signal resulting from cross-hybridization, as well as non-specific extension and imaging system background. The GenomeStudio platform uses the Average signal and standard deviation of 600 negative controls to establish detection limits for the methylation probes. Performance of negative controls should be monitored in both green and red channels.

Staining controls: Staining controls are used to examine the efficiency of the staining step in both the red and green channels. Staining controls have dinitrophenyl (DNP) or biotin attached to the beads. Controls Table on page 236 lists the control names, relevant color channel, and expected intensity of biotin and DNP labeling controls. These controls are independent of the hybridization and extension step. Both red and green channels can be evaluated using the Staining Controls.

Extension controls: Extension controls test the extension efficiency of A, T, C, and G nucleotides from a hairpin probe, and are therefore sample-independent. Both red (A,T) and green (C,G) channels are monitored. Controls Table on page 236 lists the control names, the relevant color channel, and the expected intensity of the extension controls.

Target removal: Target removal controls test the efficiency of the stripping step after the extension reaction. In contrast to allele-specific extension, the control oligos are extended using the probe sequence as template. This process generates labeled targets. The probe sequences are designed such that extension from the probe does not occur. All target removal controls should result in low signal compared to the hybridization controls, indicating that the targets were removed efficiently after extension. The target removal controls are present in the hybridization buffer RA1. Performance of target removal controls should only be monitored in the green channel. Controls Table on page 236 lists control names and expected signal.

Hybridization controls: The hybridization controls test the overall performance of the entire assay using synthetic targets instead of amplified DNA. These synthetic targets complement the sequence on the array perfectly, allowing the probe to extend on the synthetic target as template. The synthetic targets are present in the hybridization buffer (RA1) at three levels, monitoring the response from high-concentration (5 pM), medium-concentration (1 pM), and low-concentration (0.2 pM) targets. All bead type IDs should result in signal with various intensities, corresponding to the concentrations of the initial synthetic targets. Performance of hybridization controls should only be monitored in the green channel. Controls Table on page 236 lists control names for the hybridization controls and expected intensities.