Navigating Illumina Methylation Data: Biology vs. Technical Artefacts
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PROVIDER: PRJNA1121897 | ENA |
REPOSITORIES: ENA
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Project description:Illumina-based BeadChip arrays have revolutionized genome-wide DNA methylation profiling, pushing it into diagnostics. However, comprehensive quality assessment remains a challenge within a wide range of available tissue materials and sample preparation methods. This study tackles two critical issues: differentiating between biological effects and technical artefacts in suboptimal quality samples and the impact of the first sample on the Illumina-like normalization algorithm. We introduce three quality control scores based on global DNA methylation distribution (DB-Score), bin distance from copy number variation analysis (BIN-Score), and consistently methylated CpGs (CM-Score), that rely on biological features rather than internal array controls. These scores were explored and benchmarked across independent study cohorts. Additionally, we reveal deviations in beta values caused by different sample rankings with the Illumina-like normalization algorithm, verified these with whole-genome methylation sequencing data and showed effects on differential DNA methylation analysis. Our findings underscore the necessity of consistently utilizing a pre-defined normalization sample within the ranking process to boost the reproducibility of the Illumina-like normalization algorithm. In conclusion, our study delivers valuable insights, practical recommendations, and R functions designed to enhance the reproducibility and quality assurance of DNA methylation analysis, particularly for challenging sample types.
2024-12-19 | GSE269421 | GEO
Project description:We profiled DNA from liver and placenta technical replicate samples on the Illumina HumanMethylation450 BeadChip array. There technical replicates represent a single liver and a single placenta sample.
2014-02-19 | GSE52731 | GEO
Project description:Background: RNA-seq is revolutionizing the way we study transcriptomes. mRNA can be surveyed without prior knowledge of gene transcripts. Alternative splicing of transcript isoforms and the identification of previously unknown exons are being reported. Initial reports of differences in exon usage, and splicing between samples as well as quantitative differences among samples are beginning to surface. Biological variation has been reported to be larger than technical variation. In addition, technical variation has been reported to be in line with expectations due to random sampling. However, strategies for dealing with technical variation will differ depending on the magnitude. The size of technical variance, and the role of sampling are examined in this manuscript. Results: Independent Solexa/Illumina experiments containing technical replicates are analyzed. When coverage is low, large disagreements between technical replicates are apparent. Exon detection between technical replicates is highly variable when the coverage is less than 5 reads per nucleotide and estimates of gene expression are more likely to disagree when coverage is low. Although large disagreements in the estimates of expression are observed at all levels of coverage. Conclusions: Technical variability is too high to ignore. Technical variability results in inconsistent detection of exons at low levels of coverage. Further, the estimate of the relative abundance of a transcript can substantially disagree, even when coverage levels are high. This may be due to the low sampling fraction and if so, it will persist as an issue needing to be addressed in experimental design even as the next wave of technology produces larger numbers of reads. We provide practical recommendations for dealing with the technical variability, without dramatic cost increases.
2014-01-15 | GSE54068 | GEO
Project description:Background: RNA-seq is revolutionizing the way we study transcriptomes. mRNA can be surveyed without prior knowledge of gene transcripts. Alternative splicing of transcript isoforms and the identification of previously unknown exons are being reported. Initial reports of differences in exon usage, and splicing between samples as well as quantitative differences among samples are beginning to surface. Biological variation has been reported to be larger than technical variation. In addition, technical variation has been reported to be in line with expectations due to random sampling. However, strategies for dealing with technical variation will differ depending on the magnitude. The size of technical variance, and the role of sampling are examined in this manuscript. Results: Independent Solexa/Illumina experiments containing technical replicates are analyzed. When coverage is low, large disagreements between technical replicates are apparent. Exon detection between technical replicates is highly variable when the coverage is less than 5 reads per nucleotide and estimates of gene expression are more likely to disagree when coverage is low. Although large disagreements in the estimates of expression are observed at all levels of coverage. Conclusions: Technical variability is too high to ignore. Technical variability results in inconsistent detection of exons at low levels of coverage. Further, the estimate of the relative abundance of a transcript can substantially disagree, even when coverage levels are high. This may be due to the low sampling fraction and if so, it will persist as an issue needing to be addressed in experimental design even as the next wave of technology produces larger numbers of reads. We provide practical recommendations for dealing with the technical variability, without dramatic cost increases.
2014-01-15 | GSE54067 | GEO
Project description:Technical replicates reveal a sequencing batch effect among eight Illumina MiSeq runs on soil fungal communities
| PRJEB28018 | ENA
Project description:In CRC, 1) to identify epigenetic changes at inter-tumor and intra-tumor level, and 2) to relate intra-tumor clonality to clinical, molecular and histopathologic parameters. From 79 FFPE tumors, 3 different regions were macrodissected: invasive front (IF), digestive tract surface (DTS) and central bulk (CB). Clinical, molecular, and histopathologic parameters were stablished. Epigenetic analysis was performed using Infinium 450K beadchip (Illumina) and R statistics. Intra-tumor regions clustered together by patient. The biggest epigenetic changes were in IF vs DTS/CB. By patient, the most often divergent region was IF (49.4%) comparing with DTS and CB (25.3% in both). It did not correlate with histopathologic, molecular and clinical parameters.Epigenetic clonality is higher at intra-tumor level. The highest changes are observed in IF vs DTS/CB. No association with histopathologic, molecular, and clinical characteristics was found. Technical replicates of 12 samples previously hybridized on the Infinium HumanMethylation450 to demonstrate technique robustness.
2016-08-16 | GSE81359 | GEO
Project description:2-4-8 cell porcine embryo technical repeat dye-swap for Agilent EMPV1 (4x44k Format)
2013-02-25 | GSE34157 | GEO
Project description:RNA-seq: technical variability and sampling
| PRJNA234515 | ENA
Project description:We profiled DNA from liver and placenta technical replicate samples on the Illumina HumanMethylation450 BeadChip array. There technical replicates represent a single liver and a single placenta sample. Bisulphite converted DNA was hybridized to Illumina HumanMethylation450 BeadChip arrays
2014-02-19 | E-GEOD-52731 | biostudies-arrayexpress