Report on the Infinium 450k Methylation Array Analysis Workshop: April 20, 2012 UCL,London, UK

A new platform for DNA methylome analysis is Illumina''s Infinium HumanMethylation450. This technology is an extension of the previous HumanMethylation27 BeadChip and allows the methylation status of 12 samples per chip and 4 to 8 chips (total of 48 to 96 samples) to be assessed simultaneously for more than 480,000 cytosines across the genome. The platform incorporates two different probe types using different assay designs (InfiniumI and InfiniumII). Although this has allowed the assessment of more CpG sites, it has also introduced technical variation between the two probe types, which has complicated the analysis process. Many groups are working on normalization methods and analysis pipelines while many others are struggling to make sense of their new data sets. This motivated the organization of a meeting held at University College London that focused solely on the analysis methods and problems related to this new platform. The meeting was attended by 125 computational and bench scientists from 11 countries. There were 10 speakers, a small poster session and a discussion session.     

Report on the 2nd Annual Infinium Humanmethylation450 Array Workshop

The Illumina Infinium HumanMethylation450 BeadChip – the successor to their hugely popular HumanMethylation27 BeadChip – is arguably the most prevalent platform for large-scale studies of DNA methylome analysis. After the success of last year’s meeting¹ that discussed initial analysis strategies for this then-new platform, this year’s meeting (held at Queen Mary, University of London) included the presentation of now established pipelines and normalization methods for data analysis, as well as some exciting tools for down-stream analysis. The importance of defining cell composition was a new topic mentioned by most speakers. The epigenome varies between cell types and insuring that methylation differences are related to sample treatment and not a differing cell population is essential. The meeting was attended by 215 computational and bench scientists from 18 countries. There were 11 speakers, a small poster session, and a discussion session. Talks were recorded and are now freely available at http://www.illumina.com/applications/epigenetics/array-based_methylation_analysis/methylation-array-analysis-education.ilmn     

A systematic study of normalization methods for Infinium 450K methylation data using whole-genome bisulfite sequencing data

DNA methylation plays an important role in disease etiology. The Illumina Infinium HumanMethylation450 (450K) BeadChip is a widely used platform in large-scale epidemiologic studies. This platform can efficiently and simultaneously measure methylation levels at ∼480,000 CpG sites in the human genome in multiple study samples. Due to the intrinsic chip design of 2 types of chemistry probes, data normalization or preprocessing is a critical step to consider before data analysis. To date, numerous methods and pipelines have been developed for this purpose, and some studies have been conducted to evaluate different methods. However, validation studies have often been limited to a small number of CpG sites to reduce the variability in technical replicates. In this study, we measured methylation on a set of samples using both whole-genome bisulfite sequencing (WGBS) and 450K chips. We used WGBS data as a gold standard of true methylation states in cells to compare the performances of 8 normalization methods for 450K data on a genome-wide scale. Analyses on our dataset indicate that the most effective methods are peak-based correction (PBC) and quantile normalization plus β-mixture quantile normalization (QN.BMIQ). To our knowledge, this is the first study to systematically compare existing normalization methods for Illumina 450K data using novel WGBS data. Our results provide a benchmark reference for the analysis of DNA methylation chip data, particularly in white blood cells.     

Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array.     

Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array.     

Measuring the methylome in clinical samples: Improved processing of the Infinium Human Methylation450 BeadChip Array

The Infinium Human Methylation450 BeadChip Array^TM (Infinium 450K) is an important tool for studying epigenetic patterns associated with disease. This array offers a high-throughput, low cost alternative to more comprehensive sequencing-based methodologies. Here we compare data generated by interrogation of the same seven clinical samples by Infinium 450K and reduced representation bisulfite sequencing (RRBS). This is the largest data set comparing Infinium 450K array to the comprehensive RRBS methodology reported so far. We show good agreement between the two methodologies. A read depth of four or more reads in the RRBS data was sufficient to achieve good agreement with Infinium 450K. However, we observe that intermediate methylation values (20–80%) are more variable between technologies than values at the extremes of the bimodal methylation distribution. We describe careful processing of Infinium 450K data to correct for known limitations and batch effects. Using methodologies proposed by others and newly implemented and combined in this report, agreement of Infinium 450K data with independent techniques can be vastly improved.     

Genome-scale analysis of DNA methylation in colorectal cancer using Infinium HumanMethylation450 BeadChips

Illumina’s Infinium HumanMethylation450 BeadChip arrays were used to examine genome-wide DNA methylation profiles in 22 sample pairs from colorectal cancer (CRC) and adjacent tissues and 19 colon tissue samples from cancer-free donors. We show that the methylation profiles of tumors and healthy tissue samples can be clearly distinguished from one another and that the main source of methylation variability is associated with disease status. We used different statistical approaches to evaluate the methylation data. In general, at the CpG-site level, we found that common CRC-specific methylation patterns consist of at least 15,667 CpG sites that were significantly different from either adjacent healthy tissue or tissue from cancer-free subjects. Of these sites, 10,342 were hypermethylated in CRC, and 5,325 were hypomethylated. Hypermethylated sites were common in the maximum number of sample pairs and were mostly located in CpG islands, where they were significantly enriched for differentially methylated regions known to be cancer-specific. In contrast, hypomethylated sites were mostly located in CpG shores and were generally sample-specific. Despite the considerable variability in methylation data, we selected a panel of 14 highly robust candidates showing methylation marks in genes SND1, ADHFE1, OPLAH, TLX2, C1orf70, ZFP64, NR5A2, and COL4A. This set was successfully cross-validated using methylation data from 209 CRC samples and 38 healthy tissue samples from The Cancer Genome Atlas consortium (AUC = 0.981 [95% CI: 0.9677–0.9939], sensitivity = 100% and specificity = 82%). In summary, this study reports a large number of loci with novel differential methylation statuses, some of which may serve as candidate markers for diagnostic purposes.     

Measuring the methylome in clinical samples: Improved processing of the Infinium Human Methylation450 BeadChip Array

The Infinium Human Methylation450 BeadChip Array^TM (Infinium 450K) is an important tool for studying epigenetic patterns associated with disease. This array offers a high-throughput, low cost alternative to more comprehensive sequencing-based methodologies. Here we compare data generated by interrogation of the same seven clinical samples by Infinium 450K and reduced representation bisulfite sequencing (RRBS). This is the largest data set comparing Infinium 450K array to the comprehensive RRBS methodology reported so far. We show good agreement between the two methodologies. A read depth of four or more reads in the RRBS data was sufficient to achieve good agreement with Infinium 450K. However, we observe that intermediate methylation values (20–80%) are more variable between technologies than values at the extremes of the bimodal methylation distribution. We describe careful processing of Infinium 450K data to correct for known limitations and batch effects. Using methodologies proposed by others and newly implemented and combined in this report, agreement of Infinium 450K data with independent techniques can be vastly improved.     

An evaluation of analysis pipelines for DNA methylation profiling using the Illumina HumanMethylation450 BeadChip platform

The proper identification of differentially methylated CpGs is central in most epigenetic studies. The Illumina HumanMethylation450 BeadChip is widely used to quantify DNA methylation; nevertheless, the design of an appropriate analysis pipeline faces severe challenges due to the convolution of biological and technical variability and the presence of a signal bias between Infinium I and II probe design types. Despite recent attempts to investigate how to analyze DNA methylation data with such an array design, it has not been possible to perform a comprehensive comparison between different bioinformatics pipelines due to the lack of appropriate data sets having both large sample size and sufficient number of technical replicates. Here we perform such a comparative analysis, targeting the problems of reducing the technical variability, eliminating the probe design bias and reducing the batch effect by exploiting two unpublished data sets, which included technical replicates and were profiled for DNA methylation either on peripheral blood, monocytes or muscle biopsies. We evaluated the performance of different analysis pipelines and demonstrated that: (1) it is critical to correct for the probe design type, since the amplitude of the measured methylation change depends on the underlying chemistry; (2) the effect of different normalization schemes is mixed, and the most effective method in our hands were quantile normalization and Beta Mixture Quantile dilation (BMIQ); (3) it is beneficial to correct for batch effects. In conclusion, our comparative analysis using a comprehensive data set suggests an efficient pipeline for proper identification of differentially methylated CpGs using the Illumina 450K arrays.     

Novel imprinted single CpG sites found by global DNA methylation analysis in human parthenogenetic induced pluripotent stem cells

Genomic imprinting is the process of epigenetic modification whereby genes are expressed in a parent-of-origin dependent manner; it plays an important role in normal growth and development. Parthenogenetic embryos contain only the maternal genome. Parthenogenetic embryonic stem cells could be useful for studying imprinted genes. In humans, mature cystic ovarian teratomas originate from parthenogenetic activation of oocytes; they are composed of highly differentiated mature tissues containing all three germ layers. To establish human parthenogenetic induced pluripotent stem cell lines (PgHiPSCs), we generated parthenogenetic fibroblasts from ovarian teratoma tissues. We compared global DNA methylation status of PgHiPSCs with that of biparental human induced pluripotent stem cells by using Illumina Infinium HumanMethylation450 BeadChip array. This analysis identified novel single imprinted CpG sites. We further tested DNA methylation patterns of two of these sites using bisulfite sequencing and described novel candidate imprinted CpG sites. These results confirm that PgHiPSCs are a powerful tool for identifying imprinted genes and investigating their roles in human development and diseases.     

Discovery of cross-reactive probes and polymorphic CpGs in the Illumina Infinium HumanMethylation450 microarray

DNA methylation, an important type of epigenetic modification in humans, participates in crucial cellular processes, such as embryonic development, X-inactivation, genomic imprinting and chromosome stability. Several platforms have been developed to study genome-wide DNA methylation. Many investigators in the field have chosen the Illumina Infinium HumanMethylation microarray for its ability to reliably assess DNA methylation following sodium bisulfite conversion. Here, we analyzed methylation profiles of 489 adult males and 357 adult females generated by the Infinium HumanMethylation450 microarray. Among the autosomal CpG sites that displayed significant methylation differences between the two sexes, we observed a significant enrichment of cross-reactive probes co-hybridizing to the sex chromosomes with more than 94% sequence identity. This could lead investigators to mistakenly infer the existence of significant autosomal sex-associated methylation. Using sequence identity cutoffs derived from the sex methylation analysis, we concluded that 6% of the array probes can potentially generate spurious signals because of co-hybridization to alternate genomic sequences highly homologous to the intended targets. Additionally, we discovered probes targeting polymorphic CpGs that overlapped SNPs. The methylation levels detected by these probes are simply the reflection of underlying genetic polymorphisms but could be misinterpreted as true signals. The existence of probes that are cross-reactive or of target polymorphic CpGs in the Illumina HumanMethylation microarrays can confound data obtained from such microarrays. Therefore, investigators should exercise caution when significant biological associations are found using these array platforms. A list of all cross-reactive probes and polymorphic CpGs identified by us are annotated in this paper.     

Causes of blood methylomic variation for middle-aged women measured by the HumanMethylation450 array

To address the limitations in current classic twin/family research on the genetic and/or environmental causes of human methylomic variation, we measured blood DNA methylation for 479 women (mean age 56 years) including 66 monozygotic (MZ), 66 dizygotic (DZ) twin pairs and 215 sisters of twins, and 11 random technical duplicates using the HumanMethylation450 array. For each methylation site, we estimated the correlation for pairs of duplicates, MZ twins, DZ twins, and siblings, fitted variance component models by assuming the variation is explained by genetic factors, by shared and individual environmental factors, and by independent measurement error, and assessed the best fitting model. We found that the average (standard deviation) correlations for duplicate, MZ, DZ, and sibling pairs were 0.10 (0.35), 0.07 (0.21), -0.01 (0.14) and -0.04 (0.07). At the genome-wide significance level of 10^?7, 93.3% of sites had no familial correlation, and 5.6%, 0.1%, and 0.2% of sites were correlated for MZ, DZ, and sibling pairs. For 86.4%, 6.9%, and 7.1% of sites, the best fitting model included measurement error only, a genetic component, and at least one environmental component. For the 13.6% of sites influenced by genetic and/or environmental factors, the average proportion of variance explained by environmental factors was greater than that explained by genetic factors (0.41 vs. 0.37, P value <10^?15). Our results are consistent with, for middle-aged woman, blood methylomic variation measured by the HumanMethylation450 array being largely explained by measurement error, and more influenced by environmental factors than by genetic factors.     

Reducing the risk of false discovery enabling identification of biologically significant genome-wide methylation status using the HumanMethylation450 array

Background

The Illumina HumanMethylation450 BeadChip (HM450K) measures the DNA methylation of 485,512 CpGs in the human genome. The technology relies on hybridization of genomic fragments to probes on the chip. However, certain genomic factors may compromise the ability to measure methylation using the array such as single nucleotide polymorphisms (SNPs), small insertions and deletions (INDELs), repetitive DNA, and regions with reduced genomic complexity. Currently, there is no clear method or pipeline for determining which of the probes on the HM450K bead array should be retained for subsequent analysis in light of these issues.

Results

We comprehensively assessed the effects of SNPs, INDELs, repeats and bisulfite induced reduced genomic complexity by comparing HM450K bead array results with whole genome bisulfite sequencing. We determined which CpG probes provided accurate or noisy signals. From this, we derived a set of high-quality probes that provide unadulterated measurements of DNA methylation.

Conclusions

Our method significantly reduces the risk of false discoveries when using the HM450K bead array, while maximising the power of the array to detect methylation status genome-wide. Additionally, we demonstrate the utility of our method through extraction of biologically relevant epigenetic changes in prostate cancer.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-51) contains supplementary material, which is available to authorized users.     

Validation of a DNA methylation reference panel for the estimation of nucleated cells types in cord blood

Cord blood is widely used as surrogate tissue in epigenome-wide association studies of prenatal conditions. Cell type composition variation across samples can be an important confounder of epigenome-wide association studies in blood that constitute a mixture of cells. We evaluated a newly developed cord blood reference panel to impute cell type composition from DNA methylation levels, including nucleated red blood cells (nRBCs). We estimated cell type composition from 154 unique cord blood samples with available DNA methylation data as well as direct measurements of nucleated cell types. We observed high correlations between the estimated and measured composition for nRBCs (r = 0.92, R² = 0.85), lymphocytes (r = 0.77, R² = 0.58), and granulocytes (r = 0.72, R² = 0.52), and a moderate correlation for monocytes (r = 0.51, R² = 0.25) as well as relatively low root mean square errors from the residuals ranging from 1.4 to 5.4%. These results validate the use of the cord blood reference panel and highlight its utility and limitations for epidemiological studies.     

基于TCGA数据库结肠癌RBL1基因甲基化水平的诊断与预后分析

视网膜母细胞瘤样蛋白1(RBL1/p105)是视网膜母细胞瘤蛋白质家族的成员之一,RBL1通常被认为是抑癌基因,在细胞增殖、凋亡、分化中发挥重要作用。该文探讨RBL1基因在结肠癌诊断和预后中的作用和可能的机制。利用癌症基因组图谱(TCGA)数据库中的结肠癌病例资料和芯片数据,筛选RBL1基因差异甲基化位点,并通过Cox回归模型研究甲基化位点与结肠癌患者预后之间的关系。该研究发现,待研究的癌组织RBL1基因启动子甲基化水平高于癌旁组织(0.120±0.012 vs 0.113±0.008, P=0.000 04)。随后,研究者采用受试者工作特征(ROC)曲线来评价RBL1基因启动子甲基化水平的诊断价值,用曲线下面积(AUC)作为诊断价值的评判标准。研究发现,启动子区甲基化的AUC达到0.732,对应的灵敏度为80.5%、特异度为55.3%。Cox多因素分析结果发现, cg04086771高甲基化是结肠癌患者预后的独立危险因素(P=0.002)。该研究通过对TCGA数据库的挖掘,发现RBL1基因启动子区甲基化均值可能与结肠癌发病风险有关,同时RBL1基因的甲基化位点的甲基化水平对结肠癌的预后有影响。     

In Silico Analysis of Crop Science： Report on the First China-UK Workshop on Chips, Computers and Crops

A workshop on ＂Chips, Computers and Crops＂ was held in Hangzhou, China during September 26-27, 2008. The main objective of the workshop was to bring together China and UK scientists from mathematics, bioinformatics and plant molecular biology communities to exchange ideas, enhance awareness of each others＇ fields, explore synergisms and make recommendations on fruitful future directions in crop science. Here we describe the contributions to the workshop, and examine some conceptual issues that lie at the foundations and future of crop systems biology.