Report on the Infinium 450k Methylation Array Analysis Workshop

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.     

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.     

Agreement in DNA methylation levels from the Illumina 450K array across batches,tissues, and time

Epigenome-wide association studies (EWAS) have focused primarily on DNA methylation as a chemically stable and functional epigenetic modification. However, the stability and accuracy of the measurement of methylation in different tissues and extraction types is still being actively studied, and the longitudinal stability of DNA methylation in commonly studied peripheral tissues is of great interest. Here, we used data from two studies, three tissue types, and multiple time points to assess the stability of DNA methylation measured with the Illumina Infinium HumanMethylation450 BeadChip array. Redundancy analysis enabled visual assessment of agreement of replicate samples overall and showed good agreement after removing effects of tissue type, age, and sex. At the probe level, analysis of variance contrasts separating technical and biological replicates clearly showed better agreement between technical replicates versus longitudinal samples, and suggested increased stability for buccal cells versus blood or blood spots. Intraclass correlations (ICCs) demonstrated that inter-individual variability is of similar magnitude to within-sample variability at many probes; however, as inter-individual variability increased, so did ICC. Furthermore, we were able to demonstrate decreasing agreement in methylation levels with time, despite a maximal sampling interval of only 576 days. Finally, at 6 popular candidate genes, there was a large range of stability across probes. Our findings highlight important sources of technical and biological variation in DNA methylation across different tissues over time. These data will help to inform longitudinal sampling strategies of future EWAS.     

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.     

Monitoring the phase space of ecosystems: Concept and examples from the Quillow catchment,Uckermark

Ecosystem research benefits enormously from the fact that comprehensive data sets of high quality, and covering long time periods are now increasingly more available. However, facing apparently complex interdependencies between numerous ecosystem components, there is urgent need rethinking our approaches in ecosystem research and applying new tools of data analysis.The concept presented in this paper is based on two pillars. Firstly, it postulates that ecosystems are multiple feedback systems and thus are highly constrained. Consequently, the effective dimensionality of multivariate ecosystem data sets is expected to be rather low compared to the number of observables. Secondly, it assumes that ecosystems are characterized by continuity in time and space as well as between entities which are often treated as distinct units.Implementing this concept in ecosystem research requires new tools for analysing large multivariate data sets. This study presents some of them, which were applied to a comprehensive water quality data set from a long-term monitoring program in Northeast Germany in the Uckermark region, one of the LTER-D (Long Term Ecological Research network, Germany) sites.The effective dimensionality was assessed by the Correlation Dimension approach as well as by a Principal Component Analysis and was in fact substantially lower than the number of observables. Continuity in time, space and between different types of water bodies was studied by combining Self-Organizing Maps with Sammon's Mapping. Groundwater, kettle hole and stream water samples exhibited some overlap, confirming continuity between different types of water bodies. Clear long-term shifts were found at the stream sampling sites. There was strong evidence that the intensity of single processes had changed at these sites rather than that new processes developed. Thus the more recent data did not occupy new subregions of the phase space of observations.Short-term variability of the kettle hole water samples differed substantially from that of the stream water samples, suggesting different processes generating the dynamics in these two types of water bodies. However, again, this seemed to be due to differing intensities of single processes rather than to completely different processes.We feel that research aiming at elucidating apparently complex interactions in ecosystems could make much more efficient use from now available large monitoring data sets by implementing the suggested concept and using corresponding innovative tools of system analysis.     

Meeting report on membrane transport and metabolism 4th International Congress on Amino Acids Vienna,Austria, August 7–11, 1995

Summary The session on Membrane Transport was a lively, interactive one with substantial audience participation in the discussion periods. Progress in this area of amino acid science is rapid as the genes encoding the transporter proteins are being cloned and characterized. The contributors to the platform session represented institutions from several countries giving the meeting a truly international flavor. Most of the participants contributed articles to this volume. The platform speakers were Dr. John McGivan (United Kingdom), Dr. Marçal Pastor-Anglada (Spain), Dr. Bruce Stephens (USA), Dr. G. Gazzola and Dr. V. Dall'Asta (Italy), Dr. Carol MacLeod (USA), Miss Maria Rivera-Correa (Puerto Rico), Dr. Ellen Closs (Germany), Dr. Manuel Palacín (Spain), Dr. Ovidio Bussolati (Italy), Dr. Suresh Tate (USA), Dr. S. Nakamura (Japan).     

Sequence-matched probes produce increased cross-platform consistency and more reproducible biological results in microarray-based gene expression measurements

Cancer derived microarray data sets are routinely produced by various platforms that are either commercially available or manufactured by academic groups. The fundamental difference in their probe selection strategies holds the promise that identical observations produced by more than one platform prove to be more robust when validated by biology. However, cross-platform comparison requires matching corresponding probe sets. We are introducing here sequence-based matching of probes instead of gene identifier-based matching. We analyzed breast cancer cell line derived RNA aliquots using Agilent cDNA and Affymetrix oligonucleotide microarray platforms to assess the advantage of this method. We show, that at different levels of the analysis, including gene expression ratios and difference calls, cross-platform consistency is significantly improved by sequence- based matching. We also present evidence that sequence-based probe matching produces more consistent results when comparing similar biological data sets obtained by different microarray platforms. This strategy allowed a more efficient transfer of classification of breast cancer samples between data sets produced by cDNA microarray and Affymetrix gene-chip platforms.     

基于加权Ripley's K-function的多尺度景观格局分析——以江苏盐城滨海湿地为例

以1987,1992,1997,2002,2007年的遥感影像为例,首次尝试使用加权Ripley's K-function的多尺度格局分析方法,计算了20年来景观异质性在江苏盐城滨海湿地的时间变化和空间分布趋势。通过对研究区的样带划分以及景观类型的点状化处理,建立滨海湿地样带图层和1987—2007年间各类型景观的点格局数据库,从而分析滨海湿地不同类型景观的空间聚集特征变化。基于加权Ripley's K-function的计算表明,在各级空间尺度和时间变化上,各类型湿地的斑块都呈现出空间聚集分布状态,且1987年以来,不同湿地类型的聚集空间特征尺度和空间分布强度均出现了大幅的增减变化,除互花米草滩之外的自然湿地的聚集空间特征尺度和强度都有明显下降甚至少到无法被检测到,而人工湿地却呈现聚集特征尺度和强度的双增长,且该聚集程度有逐渐增强的趋势。分析表明,既考虑样点的空间位置信息又考虑样点分布范围的加权Ripley's K-function方法能很好地表征湿地景观在多尺度上的变异,且与传统空间景观指数等分析方法的结论在一定程度上保持一致。     

Genome-wide DNA methylation profiling of recurrent and non-recurrent chordomas

Chordomas are an aggressive rare type of malignant bone tumors arising from the remnant of the notochord. Chordomas occur mainly in vertebral bones and account for 1–4% of malignant bone tumors. Management and treatment of chordomas are difficult as they are resistant to conventional chemotherapy; therefore, they are mainly treated with surgery and radiation therapy. In this study, we performed DNA methylation profiling of 26 chordomas and normal nucleus pulposus samples plus UCH-1 chordoma cell line using the Illumina Infinium HumanMethylation450 BeadChips. Combined bisulfite restriction analysis and bisulfite sequencing was used to confirm the methylation data. Gene expression was analyzed using RT-PCR before and after 5-aza-2’-deoxycytidine (5-azaDC) treatment of chordoma cell lines. Analysis of the HumanMethylation450 BeadChip data led to the identification of 8,819 loci (2.9%) that were significantly differentially methylated (>0.2 average β-value difference) between chordomas and nucleus pulposus samples (adjusted P < 0.05). Among these, 5,868 probes (66.5%) were hypomethylated, compared to 2,951 (33.5%) loci that were hypermethylated in chordomas compared to controls. From the 2,951 differentially hypermethylated probes, 33.3% were localized in the promoter region (982 probes) and, among these, 104 probes showed cancer-specific hypermethylation. Ingenuity Pathway Analysis indicates that the cancer-specific differentially methylated loci are involved in various networks including cancer disease, nervous system development and function, cell death and survival, cellular growth, cellular development, and proliferation. Furthermore, we identified a subset of probes that were differentially methylated between recurrent and non-recurrent chordomas. BeadChip methylation data was confirmed for these genes and gene expression was shown to be upregulated in methylated chordoma cell lines after treatment with 5-azaDC. Understanding epigenetic changes in chordomas may provide insights into chordoma tumorigenesis and development of epigenetic biomarkers.     

Report of the 13(th) Genomic Standards Consortium Meeting, Shenzhen, China, March 4-7, 2012

This report details the outcome of the 13(th) Meeting of the Genomic Standards Consortium. The three-day conference was held at the Kingkey Palace Hotel, Shenzhen, China, on March 5-7, 2012, and was hosted by the Beijing Genomics Institute. The meeting, titled From Genomes to Interactions to Communities to Models, highlighted the role of data standards associated with genomic, metagenomic, and amplicon sequence data and the contextual information associated with the sample. To this end the meeting focused on genomic projects for animals, plants, fungi, and viruses; metagenomic studies in host-microbe interactions; and the dynamics of microbial communities. In addition, the meeting hosted a Genomic Observatories Network session, a Genomic Standards Consortium biodiversity working group session, and a Microbiology of the Built Environment session sponsored by the Alfred P. Sloan Foundation.     

A novel in situ probe for oxygen uptake rate measurement in mammalian cell cultures

The newly developed in situ oxygen uptake rate (in situ OUR) probe presented in this article is based on the in situ microscope technology platform. It is designed to measure the oxygen uptake rate (OUR) of mammalian cells, an important parameter for metabolic flux analysis, inside a reactor (in situ) and in real-time. The system isolates a known volume of cell culture from the bulk inside the bioreactor, monitors the oxygen consumption over time, and releases the sample again. The sample is mixed during the measurement with a new agitation system to keep the cells in suspension and prevent oxygen concentration gradients. The OUR measurement system also doubles as a standard dissolved oxygen (DO) probe for process monitoring when it is not performing OUR measurements. It can be equipped with two different types of optical sensors (i.e., DO, pH) simultaneously or a conventional polarographic DO-probe (Clark type). This new probe was successfully tested in baby hamster kidney perfusion cell cultures.     

'Single molecule': theory and experiments,an introduction

At scales below micrometers, Brownian motion dictates most of the behaviors. The simple observation of a colloid is striking: a permanent and random motion is seen, whereas inertial forces play a negligible role. This Physics, where velocity is proportional to force, has opened new horizons in biology. The random feature is challenged in living systems where some proteins - molecular motors - have a directed motion whereas their passive behaviors of colloid should lead to a Brownian motion. Individual proteins, polymers of living matter such as DNA, RNA, actin or microtubules, molecular motors, all these objects can be viewed as chains of colloids. They are submitted to shocks from molecules of the solvent. Shapes taken by these biopolymers or dynamics imposed by motors can be measured and modeled from single molecules to their collective effects. Thanks to the development of experimental methods such as optical tweezers, Atomic Force Microscope (AFM), micropipettes, and quantitative fluorescence (such as Förster Resonance Energy Transfer, FRET), it is possible to manipulate these individual biomolecules in an unprecedented manner: experiments allow to probe the validity of models; and a new Physics has thereby emerged with original biological insights. Theories based on statistical mechanics are needed to explain behaviors of these systems. When force-extension curves of these molecules are extracted, the curves need to be fitted with models that predict the deformation of free objects or submitted to a force. When velocity of motors is altered, a quantitative analysis is required to explain the motions of individual molecules under external forces. This lecture will give some elements of introduction to the lectures of the session 'Nanophysics for Molecular Biology'.     

Assessment of methylation level prediction accuracy in methyl-DNA immunoprecipitation and sodium bisulfite based microarray platforms

In this study, we verified the accuracy of two array methods—methylated DNA immunoprecipitation coupled with CpG island microarrays (MeDIP-CGI-arrays) and sodium bisulfite conversion based microarrays (BC-arrays)—in predicting regional methylation levels as measured by pyrosequencing of bisulfite converted DNA (BC-pyrosequencing). To test the accuracy of these methods we used the Agilent Human CpG island and the Illumina HumanMethylation27 microarrays respectively, and compared microarray outputs to the data from targeted BC-pyrosequencing assays from several genomic regions of corresponding samples. We observed relatively high correlation with BC-pyrosequencing data for both array platforms, R = 0.87 for BC-Array and R = 0.79 for MeDIP-CGI array. However, MeDIP-CGI array were less reliable in predicting intermediate levels of DNA methylation. Several bioinformatics strategies, to ameliorate the performance of the MeDIP-CGI-Arrays did not improve the correlation with BC-pyrosequencing data. The high scalability, low cost and simpler analysis of BC-arrays, together with the recent extended coverage may make them a more versatile methylation analysis tool.     

Analysis of activity and inhibition of oxygen-dependent enzymes by optical respirometry on the LightCycler system

There is currently a need for a method capable of measuring the activity and inhibition of biologically relevant oxygenases in a format that enables the convenient, fast, and cost-efficient generation of dose-response information. Here we describe a low-volume luminescence-based assay for the measurement of such oxygen-dependent enzymes. The assay employs a photoluminescent oxygen-sensitive probe and glass capillary microcuvettes measured on the Roche LightCycler detection platform. Three discrete types of oxygen probe were evaluated for this application: (i) solid-state coatings, (ii) soluble macromolecular MitoXpress probe, both phosphorescent porphyrin-based, and (iii) a luminescent Ir(III)-based nanoparticle probe. Measurement parameters were optimised and subsequently applied to the analysis of three biologically relevant oxygenases, namely cytochrome P450 (CYP), monoamine oxygenase (MAO), and cyclooxygenase (COX). CYP enzymes are central players in drug detoxification while specific inhibitors of MAO and COX are important for therapeutic intervention and treatment of neurological and inflammatory diseases, respectively. To determine assay utility, oxygen consumption catalysed by all three enzyme types was measured and the effect of specific inhibitors determined. The panel included the MAO-A/B inhibitors clorgyline, toloxatone, deprenyl, and the COX-1/2 inhibitors niflumic acid, nimesulide, SC-560, ketoprofen, and phenylbutazone. IC₅₀ values were then compared with literature values. The measurement methodology described allows the low-volume analysis of biologically relevant oxygenases and displays the requisite sensitivity and throughput to facilitate routine analysis. It is also applicable to other O₂-dependent enzymes and enzymatic systems.     

A systematic assessment of normalization approaches for the Infinium 450K methylation platform

The Illumina Infinium HumanMethylation450 BeadChip has emerged as one of the most popular platforms for genome wide profiling of DNA methylation. While the technology is wide-spread, systematic technical biases are believed to be present in the data. For example, this array incorporates two different chemical assays, i.e., Type I and Type II probes, which exhibit different technical characteristics and potentially complicate the computational and statistical analysis. Several normalization methods have been introduced recently to adjust for possible biases. However, there is considerable debate within the field on which normalization procedure should be used and indeed whether normalization is even necessary. Yet despite the importance of the question, there has been little comprehensive comparison of normalization methods. We sought to systematically compare several popular normalization approaches using the Norwegian Mother and Child Cohort Study (MoBa) methylation data set and the technical replicates analyzed with it as a case study. We assessed both the reproducibility between technical replicates following normalization and the effect of normalization on association analysis. Results indicate that the raw data are already highly reproducible, some normalization approaches can slightly improve reproducibility, but other normalization approaches may introduce more variability into the data. Results also suggest that differences in association analysis after applying different normalizations are not large when the signal is strong, but when the signal is more modest, different normalizations can yield very different numbers of findings that meet a weaker statistical significance threshold. Overall, our work provides useful, objective assessment of the effectiveness of key normalization methods.