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, 35 (5), e29

A New Method for Accurate Assessment of DNA Quality After Bisulfite Treatment

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A New Method for Accurate Assessment of DNA Quality After Bisulfite Treatment

Mathias Ehrich et al. Nucleic Acids Res.

Erratum in

  • Nucleic Acids Res. 2014 Oct 29;42(19):12331

Abstract

The covalent addition of methylgroups to cytosine has become the most intensively researched epigenetic DNA marker. The vast majority of technologies used for DNA methylation analysis rely on a chemical reaction, the so-called 'bisulfite treatment', which introduces methylation-dependent sequence changes through selective chemical conversion of non-methylated cytosine to uracil. After treatment, all non-methylated cytosine bases are converted to uracil but all methylated cytosine bases remain cytosine. These methylation dependent C-to-T changes can subsequently be studied using conventional DNA analysis technologies. The bisulfite conversion protocol is susceptible to processing errors, and small deviation from the protocol can result in failure of the treatment. Several attempts have been made to simplify the procedure and increase its robustness. Although significant achievements in this area have been made, bisulfite treatment remains the main source of process variability in the analysis of DNA methylation. This variability in particular impairs assays, which strive for the quantitative assessment of DNA methylation. Here we present basic mathematical considerations, which should be taken into account when analyzing DNA methylation. We also introduce a PCR-based assay, which allows ab initio assessment of the DNA quality after bisulfite treatment and can help to prevent inaccurate quantitative measurement resulting from poor bisulfite treatment.

Figures

Figure 1.
Figure 1.
Box plot graphic depicting the variability of repeated measurements for each step in the process (Step 1: bisulphite treatment; Step 2: PCR; Step 3: MassCLEAVE; Step 4: MALDI-TOF MS analysis). Boxes are centered on the median and range from the lower to the upper quartile. Whiskers indicate the interquartile range. Red whiskers indicate the standard deviation from the mean. Bisulfite treatment and PCR can be identified as the greatest source of process variability. The post-PCR processing (MassCLEAVE) and, in particular, the MALDI analysis show high precision in repeated measurements.
Figure 2.
Figure 2.
Panel (A) shows the probability distributions for observed methylation ratios based on the binomial distribution and different amounts of starting molecules. Shown are examples for 10, 25, 50 75 and 90% methylated molecules in the starting template. With a sample size of 3000 molecules, 95% of all randomly sampled probes will contain between 48 and 52% methylated DNA when the DNA sample contains 50% methylated DNA (red colored distribution). However, when the DNA sample contains only 300 molecules, this range is expanded from 43 to 57% (blue colored distribution). Panel (B) shows the 95% confidence intervals for sampling-means as a function of the number of the sampled molecules. Shown are results for 10 (blue), 25 (red) and 50% (black) methylated molecules in the starting template.
Figure 3.
Figure 3.
Gradient PAGE gel with CYBR Gold staining showing the DNA fragmentation of untreated genomic DNA (left) and after bisulfite treatment at varying temperatures (from left to right: 50, 70 and 80°C). The figure indicates that an increase of the incubation temperature during bisulfite treatment results in increased DNA fragmentation.
Figure 4.
Figure 4.
Panel (A) shows a schematic representation of the different PCR amplicons and their genomic context on chromosome 11. All PCR amplicons share a subset of CpG sites (indicated as red stripes), which were used for comparison of methylation ratios. Panel (B) shows an agarose gel for the six amplification products of the IGF2 region. Shown are PCR results for the six amplicons shown in panel (A) for four different bisulfite treatment incubation temperatures. The gel picture confirms that increasing incubation temperatures during bisulfite treatment lead to a decrease in the obtainable amplification length.
Figure 5.
Figure 5.
Bar graphs showing the number of high quality mass spectra for each amplicon length (two panels on the left). The panels on the right side show the corresponding standard deviations of the quantitative measurements. The bar graphs show results for different bisulfite incubation protocols. The results from 16 h incubation at constant temperature are shown in the upper two panels and results from a cycled incubation protocol are shown in the lower two panels. A total of 18 reactions were performed for each amplicon. Cycled incubation and lower incubation temperatures result in higher amplification success for longer amplicons and lower standard deviations on the determination of methylation ratios.
Figure 6.
Figure 6.
Correlation between the results obtained from the quality control assays and PCR success from additional genomic targets of varying length. The bar graphs in panel (A) and (B) show the results from the quality control assays similar to Figure 5. The QC assay indicates that incubation at 90°C limits amplification to only short amplicons (<300 bp), whereas incubation at 70°C results in decreased amplification success for amplicons around 500 bp in length. Panel (C) and (D) show results for further 39 PCR amplicons of different genomic regions ranging in length from 200 to 700 bp. Panel (c) shows the percentage of successful quantitative measurements in relationship to the amplicon length. Panel (D) shows a gel picture of the PCR results. Both confirm the results predicted from the use of the QC assay (panel a and b).

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