HumMeth27QCReport: an R package for quality control and primary analysis of Illumina Infinium methylation data
© Mancuso et al; licensee BioMed Central Ltd. 2011
Received: 20 July 2011
Accepted: 19 December 2011
Published: 19 December 2011
The study of the human DNA methylome has gained particular interest in the last few years. Researchers can nowadays investigate the potential role of DNA methylation in common disorders by taking advantage of new high-throughput technologies. Among these, Illumina Infinium assays can interrogate the methylation levels of hundreds of thousands of CpG sites, offering an ideal solution for genome-wide methylation profiling. However, like for other high-throughput technologies, the main bottleneck remains at the stage of data analysis rather than data production.
We have developed HumMeth27QCReport, an R package devoted to researchers wanting to quickly analyse their Illumina Infinium methylation arrays. This package automates quality control steps by generating a report including sample-independent and sample-dependent quality plots, and performs primary analysis of raw methylation calls by computing data normalization, statistics, and sample similarities. This package is available at CRAN repository, and can be integrated in any Galaxy instance through the implementation of ad-hoc scripts accessible at Galaxy Tool Shed.
Our package provides users of the Illumina Infinium Methylation assays with a simplified, automated, open-source quality control and primary analysis of their methylation data. Moreover, to enhance its use by experimental researchers, the tool is being distributed along with the scripts necessary for its implementation in the Galaxy workbench. Finally, although it was originally developed for HumanMethylation27, we proved its compatibility with data generated with the HumanMethylation450 Bead Chip.
DNA methylation is an epigenetic mechanism that in vertebrates occurs most frequently at cytosines followed by guanines (CpG). This modification regulates gene expression and can be inherited through cell division, thus being essential for preserving tissue identities and guiding normal cellular development . Recent studies have also indicated that modulation of DNA methylation occurs during lineage-specific differentiation. For instance, methylation and transcriptional changes accompany myeloid versus lymphoid fate decisions . However, alterations in DNA methylation at specific regulatory sites have been linked to common diseases such as cancer, diabetes, multiple sclerosis, schizophrenia, and other neurodegenerative disorders [3–5].
As investigating the human DNA methylome has gained more and more interest in the last few years, several methods have been developed to detect cytosine methylation on a genomic scale. Among these, the Illumina Infinium Methylation Assay has proven reliable in genotyping labs worldwide. This hybridization-based technique offers quantitative methylation measurements at the single-CpG-site level and provides as accurate results as sequencing-based methylation assays (e.g. MethylCap-seq, MeDIP-seq, RRBS) .
More specifically, two Illumina platforms are available for high-throughput DNA methylation analysis so far: Golden Gate Veracode and Infinium. The Veracode Golden Gate Methylation represents an ideal platform for custom studies or biomarker validation, targeting 96 or 384 custom CpG sites per sample. For Infinium platform, two different Bead Chips are on the market: the HumanMethylation27 and the new HumanMethylation450 Bead Chips. The first enables scientists to interrogate 27,578 highly informative CpG sites located within the genomic regions upstream of 14,475 consensus coding sequence (CCDS) annotated at the NCBI database. Instead, the new HumanMethylation450 Bead Chip provides scientists with methylation levels of >450,000 CpG sites per sample, including coverage of all designable RefSeq genes (along with promoter, 5', and 3' regions, without bias against those lacking islands of CpG sites), CpG sites outside of CpG islands, non-CpG methylated sites identified in human stem cells, differentially methylated sites identified in tumor versus normal in multiple forms of cancer and across several tissue types, CpG islands outside of coding regions, microRNA promoter regions, and disease-associated regions identified through genome-wide association studies.
Like for other technologies, quality control is an important step in the analysis of methylation array data . Indeed, arrays with poor quality can significantly affect subsequent analysis and consequently invalidate their interpretation. But, while several R packages can assist scientists with quality control analysis of other data types (i.e. microarray [8–10], RNA-seq , target enrichment experiments ), only four are tailored to support DNA methylation research and, in particular, only two specifics for Illumina data. Of these, charm  implements analysis tools for DNA methylation data generated using Nimblegen microarrays and the McrBC protocol; it finds differentially methylated regions between samples, calculates percentage methylation estimates and includes array quality assessment tools. MassArray  is designed for the import, quality control, analysis, and visualization of methylation data generated using Sequenom's MassArray platform. Specific for Illumina, methylumi facilitates data manipulation with methods for quality control, normalization (only for Illumina Golden Gate), and plotting; while lumi handles Infinium data providing functions for quality and colour balance assessment, colour balance adjustment, background adjustment, normalization, and modelling of the methylation status. While these open source packages require knowledge and live interaction with the R programming environment, non-computational scientists could instead carry out the analysis with the Illumina Genome Studio software. However, since not all researchers may have access to this commercial software, we believe that a fully automated, open source and unique function-based tool would facilitate the analysis of methylation data even for wet-lab researchers.
DNA methylation samples and analysis
Samples used for the analysis.
JUR MET KIT2_BIO1
JUR MET KIT2_BIO2
JUR MET KIT1_BIO1
JUR MET KIT1_BIO2
Infinium Human Methylation450 Bead Chip was hybridized with the treated samples according to the standard protocol provided by Illumina. The fluorescently stained chip was imaged by the Illumina iScan Reader. Genome Studio Methylation Module was used to analyze intensities to assign site-specific DNA methylation β-values to each CpG site. HumMeth27QCReport was used to analyse raw files exported from Genome Studio software using default options: significance level p-value cutoff of 0.05, option to discard CpGs located on chromosome X set to false, and "Euclidean" distance for the clustering analysis.
HumMeth27QCReport is an extension package for the programming language and statistical environment R  (currently implemented for the version 2.12.1). The package has been developed to analyze the Illumina Infinium HumanMethylation27 Bead Chip, but it is already compatible with the new HumanMethylation450 panel. HumMeth27QCReport integrates functions from other packages like methylumi for data import and dye bias check, lumi for normalization, amap and Hmisc for PCA and clustering. It requires the R packages gplots, plotrix, WriteXLS, tcltk for graphics and data export steps, and the installation of Perl programming language to run the WriteXLS package.
An interface has also been designed for integrating HumMeth27QCReport into the Galaxy workbench and it is available through the Galaxy Tool Shed for anyone to add it to its own Galaxy instance.
The package workflow is summarized in Figure 1. HumMeth27QCReport requires as input three tab-delimited text files exported from the Genome Studio Methylation Module ("Sample table", "Control probe profile", and "Average Beta table") and reads into R using the basic function read. table(). After data import, the package first computes basic statistics, and then generates quality plots to monitor the Illumina Infinium sample-independent and sample-dependent internal quality controls. More in detail, sample-independent controls evaluate specific experimental steps and include:
● staining controls, which are used to examine the efficiency of the staining step;
● extension controls, which test the extension efficiency of A, T, C, and G nucleotides from a hairpin probe;
● target removal controls, which test the efficiency of the stripping step after the extension reaction. All target removal controls should result in low signal compared to the hybridization controls, indicating that the targets are removed efficiently after extension;
● hybridization controls, which test the overall performance of the entire assay using synthetic targets present at different concentrations.
Instead, sample-dependent controls evaluate performance across samples and include:
● bisulfite conversion controls, which assess the efficiency of bisulfite conversion of the genomic DNA;
● specificity controls, which monitor potential non-specific primer extension. Perfectly Matched (PM) controls correspond to A/T perfect match and should give high signal, instead Mismatched (MM) controls correspond to G/T mismatch and should give low signal;
● negative controls. The negative control probes are randomly permutated sequences that should not hybridize to the DNA template. The mean signal of these probes defines the system background;
● non-Polymorphic (NP) controls, which 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.
For each of the above-listed internal controls, HumMeth27QCReport generates plots to represent the percentage of background on signal and the percentage of CpGs not detectable at two different p-value cut-offs (0.01 and 0.05), as well as evaluates the average detection p-value of each sample. Low performance samples can be automatically removed from further analyses based on a user-defined cut-off (default p-value cut-off is 0.05).
Then, the package integrates methylumi and lumi functionalities to monitor and correct the dye-bias that is a common problem in two-colour microarray experiments [20, 21]. Firstly, it applies methylumi to plot intensities distribution at different cut-offs to easily monitor eventual dye bias present in the arrays; secondly, it performs quantile normalization provided by lumi to correct the eventual dye-bias. The assumption of quantile normalization is that the intensity distribution of the pooled methylated and unmethylated probes are similar for different samples. Because the total amount of CpG methylation can be significantly different from sample to sample in different conditions, a direct application of the normalization methods designed for expression microarray to the methylation data is inappropriate. To avoid this problem, our tool inherits the lumiMethyN function from the lumi package for probe level normalization (i.e., normalize the intensities of methylated and unmethylated probes instead of the summarized methylation levels).
Normalized M-values, defined in  and , are then used to compute Principal Component Analysis (PCA) and hierarchical clustering in order to assess sample similarities. We decided to use M-values instead of Beta-value because the first are more statistically valid for the differential analysis of methylation levels. As described in Du et al., M-values are calculated as the log2 ratio of the intensities of methylated probe versus unmethylated probe. An M-value close to 0 indicates a similar intensity between the methylated and unmethylated probes, which means that the CpG site is about half-methylated. Positive M-values is associated to a hyper-methylation while negative M-values mean the opposite.
The following basic statistics are exported into an excel file, to give a more accurate view of the quality of the dataset:
● summary values of the internal controls;
● summary of percentage of not-detected genes and average p-value for each sample;
● list of not-detected CpGs with p-value greater than 0.01 in more than 5% of the samples;
● list of not-detected CpGs with p-value greater than 0.05 in more than 5% of the samples.
Finally, the M-values are automatically exported into a text file so that they can be employed in following analysis such as identification of differentially methylated genes using the R Bioconductor package limma.
QC analysis of HumanMethylation450 Bead Chip arrays
In order to test the compatibility of our package with the new HumanMethylation450 Bead Chip, we performed the analysis of 12 control samples divided in 3 groups differently methylated: Jurkat (JUR), artificially methylated Jurkat (JUR MET), and unmethylated (UNMET). Samples in each group were treated with two different sodium bisulfite kits, QiaGen or EZ Gold. In addition, we included a sample with poor quality in the analysis (Table 1).
HumMeth27QCReport is an R package designed with wet lab researchers for automating the quality control and primary analysis of Illumina Infinium Methylation Assays. The novelty of our software is the automatic generation of ad-hoc quality plots that allow an easy monitor of sample-dependent and sample-independent QC parameters, as suggested by the Illumina Infinium guidelines. These plots represent a comprehensive tool for visual inspection of methylation datasets. Moreover, our software exports DNA methylation calls and basic statistics into text files that can be easily imported into other statistical software for further analysis, as well as computes sample similarities using unsupervised approaches, like PCA and hierarchical clustering. The software, compatible with the new HumanMethylation450 Bead Chip, is available at the CRAN repository. To enhance its use by non-computational researchers, we developed ad-hoc scripts for its implementation in the Galaxy workbench and made them available at the Galaxy Tool Shed.
Availability and requirements
Project name: HumMeth27QCReport
Operating system(s): Platform independent
Programming language: R
Dependecies: R (> = 2.13.0), methylumi, lumi, IlluminaHumanMethylation27k.db, IlluminaHumanMethylation450k.db, amap, Hmisc, gplots, plotrix, WriteXLS, tcltk
Other requirements: Perl
License: GNU GPLv2
Availability of supporting data
The data set supporting the results of this article and a further example data set are available at the tool homepage: http://biocore.crg.cat/wiki/HumMeth27QCReport
We thank Manuela Hummel for testing the package and providing us with very helpful comments, Toni Hermoso for his technical support, and Susana Iraola and Raquel Rabionet for useful discussions on DNA methylation research.
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