DNAm in baseline blood samples was determined using the Infinium HumanMethylation450K BeadChip Assay (Illumina.Inc, San Diego, CA, USA). Methodological details have been reported previously [31 (link)]. Data were normalized by pre-processing in GenomeStudio. In addition, probes with detection p-value>0.01, with missing values>10%, and targeting the X and Y chromosomes were excluded in data pre-processing. Methylation beta values of 58 mortality-related CpGs were extracted. The epigenetic clock, estimated by Hovarth’s DNAm age [8 (link)], was calculated using the online tool available at https://dnamage.genetics.ucla.edu/ .
Infinium humanmethylation 450k beadchip assay
Manufactured by Illumina
Sourced in United States
The Infinium HumanMethylation 450K BeadChip assay is a DNA methylation microarray platform developed by Illumina. It enables the interrogation of over 450,000 methylation sites across the human genome, providing a comprehensive analysis of DNA methylation patterns.
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Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Qiaamp dna mini protocol, by Qiagen (2 mentions)
Methylation module v1, by Illumina (2 mentions)
Rnase free dnase set, by Qiagen (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Agilent bioanalyzer, by Agilent Technologies (2 mentions)
Ez dna methylation kit, by Zymo Research (2 mentions)
Genomestudio, by Illumina (2 mentions)
Ez 96 dna methylation kit, by Zymo Research (1 mentions)
Infinium methylationepic beadchip kit, by Illumina (1 mentions)
450k array, by Illumina (1 mentions)
Ez dna methylationtm kit, by Zymo Research (1 mentions)
Qiaamp dna micro kit, by Qiagen (1 mentions)
Zymo ez dna methylationtm kit, by Zymo Research (1 mentions)
Ez dna methylation gold, by Zymo Research (1 mentions)
16 protocols using infinium humanmethylation 450k beadchip assay
1
Epigenetic Aging Biomarkers Analysis
2
DNA Methylation Analysis from Cord Blood
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DNA from cord blood underwent bisulfite conversion using the EZ-96 DNA methylation kit (Zymo Research Corporation, CA, USA). DNA methylation was measured using the Illumina Infinium HumanMethylation450k BeadChip assay at Illumina or in cohort-specific laboratories. Each cohort conducted its own quality control and normalization of methylation data, as detailed in the Supplementary Material (Supplementary File 1 ). In all analyzes, cohorts used normalized, untransformed β-values. As a consortium, we have found that extreme outliers in methylation data, likely caused by technical error or rare genetic variants, can have a large influence on results. Therefore, potential outliers were removed. Such outliers were defined using the Tukey method [45 ], in which an outlier is any value less than the lower quartile minus three-times the interquartile range, or more than the upper quartile plus three-times the interquartile range. This method is appropriate as it is not dependent on distributional assumptions of the data.
3
Comprehensive Bioinformatic Analysis of TCGA Colorectal Cancer
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Detailed procedures for data processing and reanalysis of DNA Methylation and RNA-seq data from TCGA data sets of stage II CC from TCGA are given in the Supplementary Materials . Briefly, DNA methylation reanalysis was performed in a group of 134 samples with available data [obtained by the Infinium Human Methylation 450K BeadChip assay (Illumina)] by using a set of 62 CpG sites that closely mapped to or nearby the 40 CpG sites of our initial classifier (see Supplementary Table S4 ). A gene-set enrichment analysis (GSEA) pathway analysis [17 (link)] was performed by comparing quartile 4 cases (tumours displaying the 25% higher beta values for DNA methylation) to quartile 1, 2, and 3 cases (tumours displaying the 75% lowest beta values) for two CpG sites of interest identified in this study (see above, CDH17 and LRP2). Significantly enriched pathways were retained considering a p-value inferior to 0.05 and a false discovery rate (FDR) inferior to 0.25. Immune cell subtype analysis was conducted using the ImmuCellAI web application (http://bioinfo.life.hust.edu.cn/ImmuCellAI#!/ (accessed on 1 January 2022)) [18 (link)]. Immune contexture analysis was performed by iAtlas [19 (link)] on the (https://isb-cgc.shinyapps.io/iatlas/ (accessed on 1 January 2022)) web application.
4
DNA Methylation Profiling Using Infinium Assays
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DNAm levels of subset I and II were assessed with the Infinium Methylation EPIC BeadChip kit (EPIC, Illumina.Inc, San Diego, CA, USA), and DNAm profiles of subset III and IV were determined with the Infinium Human Methylation450K BeadChip Assay (450K, Illumina.Inc, San Diego, CA, USA). As previously described,14 (link),25 (link),27 the assays were conducted following the manufacturer's instruction by the Genomics and Proteomics Core Facility at the German Cancer Research Center, Heidelberg, Germany (DKFZ). In data pre-processing, signals of probes with detection P-value >0·01, >10% missing values, and probes targeting the X and Y chromosomes were excluded.14 (link),25 (link),27 ,28 (link)
5
Epigenome-wide DNA Methylation Analysis in ALSPAC
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ALSPAC collected (peripheral) blood at ages 7 and 17 and DNA was extracted. Epigenome-wide DNAm at specific CpG sites was measured for ~ 1000 individuals using the Infinium® HumanMethylation450K BeadChip assay (Illumina, Inc., CA, USA). DNAm data were pre-processed, including background correction and subset quantile normalization using the pipeline described by Touleimat and Tost [27 (link)] (further details in the ARIES cohort profile [21 (link)]). Estimation of white blood cell counts (CD8T cells, CD4T cells, Natural Killer, B cells, Monocytes and Granulocytes) was done using the Houseman algorithm [28 (link)]. Cross-reactive and polymorphic probes identified by Chen et al. [29 (link)] and probes on sex chromosomes were removed prior to downstream analysis (n = 453,723 probes). Due to few participants with non-Caucasian or missing ethnicity, and few non-singleton births, these were removed.
6
Transcriptome and Methylome Profiling of DLPFC
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Details on RNAseq and methylation data are published (De Jager et al. 2014 ; Ng et al. 2017 ). Briefly, RNA from 168 individuals was extracted from DLPFC with the miRNeasy mini kit (Qiagen, Venlo, Netherlands) and the RNase free DNase Set (Qiagen, Vento, Netherlands). RNA concentration was quantified using Nanodrop (Thermo Fisher Scientific, Waltham, MA), and RNA quality was assessed using an Agilent Bioanalyzer. RNAseq was performed using Illumina HiSeq with 101 bp paired-end reads with an average depth of 90 m reads. The trimmed reads were aligned to the reference genome using Bowtie and the expression FPKM values were estimated using RSEM; see supplement for normalization details.
DNA from 222 individuals was extracted from DLPFC using the Qiagen QIAamp DNA mini protocol. DNA methylation data were generated using Illumina Infinium HumanMethylation450k Bead Chip assay. Raw data were further processed using Methylation Module v1.8 from the Illumina Genome Studio software suite to generate a beta value for each cytosine guanine dinucleotide (CpG); see supplement for normalization details.
DNA from 222 individuals was extracted from DLPFC using the Qiagen QIAamp DNA mini protocol. DNA methylation data were generated using Illumina Infinium HumanMethylation450k Bead Chip assay. Raw data were further processed using Methylation Module v1.8 from the Illumina Genome Studio software suite to generate a beta value for each cytosine guanine dinucleotide (CpG); see supplement for normalization details.
7
TCGA Methylation Data Analysis Pipeline
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TCGA methylation data was analysed on the Illumina Infinium HumanMethylation 450K BeadChip assay. We accessed TCGA methylation “level 2” data for 263 HNSC, 223 LUSC and 125 LUAD individuals. We estimated the methylated level of each CpG site by calculating the M-value (log2(ratio of methylated and unmethylated probes)) using TCGA level 2 data [19 (link)]. Methylation level 2 data is already background-corrected.
8
DNA Methylation Analysis of Cord Blood Samples
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DNA samples at birth were obtained from cord blood, drawn from the umbilical cord upon delivery in accordance with standard procedures. DNA methylation analysis of these samples was performed using the Illumina Infinium HumanMethylation450K BeadChip assay [19 (link)]. All DNA methylation wet-lab and pre-processing analyses were performed at the University of Bristol as part of the ARIES project. Following extraction, DNA was bisulfite-converted using the Zymo EZ DNA MethylationTM kit (Zymo, Irvine, CA). The Illumina 450 K array was used to quantify DNA methylation at over 485,000 CpG sites across the genome. The arrays were then scanned using an Illumina iScan and initial quality review was assessed using GenomeStudio. Samples then underwent a number of further quality control processes. For each sample, the estimated level of DNA methylation at each CpG site was reported as a beta value (β), ranging from 0 (no cytosine methylation) to 1 (complete cytosine methylation) [18 (link)].
9
DNA Methylation Profiling of Liver Cell Lines
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DNA was extracted from the two cell lines (Huh7 and L02) using a QIAamp DNA Micro kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. Bisulfite modification of 1 µg DNA was conducted using an EZ DNA Methylation kit (Zymo Research Corp., Irvine, CA, USA) according to the manufacturer's protocol. The Illumina Infinium HumanMethylation 450K BeadChip assay was performed according to Illumina's standard protocol (Illumina, Inc., San Diego, CA, USA). Experiments with Huh7 and L02 cells were performed in triplicate to avoid false positive and false negative results.
10
DNA Methylation Profiling of Longitudinal Samples
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DNA samples were extracted from cord blood on delivery, and from peripheral blood samples in childhood (age 7) and in adolescence (age 15–17) according to established procedures27 (link). DNA was bisulfite-converted using the Zymo EZ DNA MethylationTM kit (Zymo, Irvine, CA) and then DNA methylation of over 485,000 CpG sites was quantified using the Illumina Infinium HumanMethylation450K BeadChip assay (HM450; Illumina Inc., CA). Arrays were scanned using the Illumina iScan, and GenomeStudio (version 2011.1; Illumina Inc.) was used to extract signal intensities and assess initial quality review.
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