DNA Methylation data analysis

Motivation

DNA methylation is a chemical tag on DNA that helps control whether genes are on or off. In cancer, methylation patterns often change early and in consistent ways, which can silence tumor-suppressor genes or activate cancer-related programs. Because these signals can be measured in tumor tissue and even in blood (cell-free DNA), methylation is a powerful source of biomarkers for early detection, tumor classification, prognosis, and treatment monitoring.^[1]

Data Analysis

Following the tutorial ^[2], we operate analysis on Galaxy:

1. Quality Control with Falco
   • Result: Look into 'Per base sequence content', we observe elevated T and reduced C, which is expected for bisulfite-treated libraries because unmethylated cytosines are converted and sequenced as thymine.
2. Aligment with bwameth (bwa caller with bisulfite-aware)
3. MethylDackel: A tool for processing bisulfite sequencing alignments
   • Potential:
     • Determine the position-dependent methylation bias in the dataset, producing diagnostic SVG images (mbias)
       • Result: Looking at the CPG methylation % plot, we identify where each curve becomes stable, and trim the unstable prefix/subfix.
     • Extract methylation metrics from an alignment file in BAM/CRAM for mat (extract)
       • Input: 1-Region of interest:CpGIslands.bed, 2-Score file: converted fraction CpG by Wig/BedGraph-to-bigWig
       • Tool: computeMatrix, plotProfile
       • Result: Mean methylation profile around CpG islands (±1 kb)
4. Metilene: calling differentially methylated regions from bisulfite sequencing data
   • Result: compare methylation between two groups and report contiguous genomic segments where the groups differ consistently (DMRs) over the regions of interest.

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1. Lakshminarasimhan, R., & Liang, G. (2016). The Role of DNA Methylation in Cancer. Advances in experimental medicine and biology, 945, 151–172. https://doi.org/10.1007/978-3-319-43624-1_7 ↩︎

2. https://training.galaxyproject.org/training-material/topics/epigenetics/tutorials/methylation-seq/tutorial.html ↩︎