Overview
We have characterized the methylation landscape of the human genome by global analysis of patterns of CpG depletion and by direct sequencing of 3073 unmethylated domains and 2565 methylated domains from human brain DNA. Our results are reported in: Large-scale structure of genomic methylation patterns,Genome Research 16:157-163, 2006.
Methods
Sequence Generation
Genomic DNA from human tissues was digested into heavily methylated and completely unmethylated sequence fragments. Unmethylated regions were isolated by digestion with the restriction enzyme, McrBC, cloned into plasmid vectors and sequenced. Methylated regions were isolated and cloned based upon their resistance to a collection of methylation-sensitive enzymes (i.e., HpaII, HhaI, MaeII, BstUI, and AciI).
Pairs of sequence fragment ends (sequence tags) were derived by sequencing from the M13 forward and M13 reverse priming sites in the pZEro-1 vector. High throughput sequencing of very large numbers of these clones gave ample coverage in delineating the genome-wide methylation state.
Sequence Analysis
The analysis pipeline which identifies the inferred methylated and unmethylated regions within the human genome consists of the following steps:
- Locate the paired sequence fragment ends on the genome, restricting the distance between the ends to the size selection performed during library construction.
- Extract the intervening sequence for the library fragment from the genome assembly database and store sequences in FASTA format.
- Determine the methylation status of each library fragment by searching the sequence for library-specific restriction enzyme recognition sites, tabulate number of methylated, unmethylated CpGs per fragment.
- Visualize results on the UCSC Human Genome Browser. Process fragment scores into genome broswer custom track files, one track per chromosome.
For details, see: Sequence Analysis Pipeline
Results
Visualization
Results can be viewed as custom tracks on the
UCSC Human Genome Browser.
Here is an example screenshot showing the methylated and unmethylated fragments on the first part of chromosome 5:
The default display contains the following:
- The first track shows the status of the unmethylated fragments, (dark orange). The position of the fragment spans the start to the end coordinates. The height of the bar for each fragment corresponds to the number of CpG sites found during sequence analysis.
- The second track shows the status of methylated fragments, (blue). The height of the bar for each fragment corresponds to the number of binding sites for the restriction enzymes found during sequence analysis.
- Two Ucsc reference tracks are included: the Known Genestrack, (dark blue); and the CpG Islands track, (green).
Note: In order to minimize upload time, each link uploads custom track information on a single chromosome. Jumping to other chromosomes using the genome browser controls won't work; you must return to this page and click on the link for chromosome you wish to jump to.
| Human March 2006 assembly (hg18, NCBI build 36) | |||||
|---|---|---|---|---|---|
| chr1 | chr2 | chr3 | chr4 | chr5 | chr6 |
| chr7 | chr8 | chr9 | chr10 | chr11 | chr12 |
| chr13 | chr14 | chr15 | chr16 | chr17 | chr18 |
| chr19 | chr20 | chr21 | chr22 | chrX | chrY |
| Human May 2004 assembly (hg17, NCBI build 35) | |||||
|---|---|---|---|---|---|
| chr1 | chr2 | chr3 | chr4 | chr5 | chr6 |
| chr7 | chr8 | chr9 | chr10 | chr11 | chr12 |
| chr13 | chr14 | chr15 | chr16 | chr17 | chr18 |
| chr19 | chr20 | chr21 | chr22 | chrX | chrY |
| Human July 2003 assembly (hg16, NCBI build 34) | |||||
|---|---|---|---|---|---|
| chr1 | chr2 | chr3 | chr4 | chr5 | chr6 |
| chr7 | chr8 | chr9 | chr10 | chr11 | chr12 |
| chr13 | chr14 | chr15 | chr16 | chr17 | chr18 |
| chr19 | chr20 | chr21 | chr22 | chrX | chrY |
Downloads
The FASTA files containing sequence data for the fragments which were successfully aligned to the genome are available for download, organized by library and genome assembly.
| Assembly | Unmethylated | Methylated |
|---|---|---|
| Human March 2006 (hg18, NCBI build 36) | Brain McrBC.fa | Brain RE.fa |
| Human May 2004 (hg17, NCBI build 35) | Brain McrBC.fa | Brain RE.fa |
| Human July 2003 (hg16, NCBI build 34) | Brain McrBC.fa | Brain RE.fa |