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. 2013 Jan;8(1):113-27.
doi: 10.4161/epi.23330. Epub 2012 Dec 20.

Identification of Differentially Methylated Regions Using Streptavidin Bisulfite Ligand Methylation Enrichment (SuBLiME), a New Method to Enrich for Methylated DNA Prior to Deep Bisulfite Genomic Sequencing

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Identification of Differentially Methylated Regions Using Streptavidin Bisulfite Ligand Methylation Enrichment (SuBLiME), a New Method to Enrich for Methylated DNA Prior to Deep Bisulfite Genomic Sequencing

Jason P Ross et al. Epigenetics. .
Free PMC article


We have developed a method that enriches for methylated cytosines by capturing the fraction of bisulfite-treated DNA with unconverted cytosines. The method, called streptavidin bisulfite ligand methylation enrichment (SuBLiME), involves the specific labeling (using a biotin-labeled nucleotide ligand) of methylated cytosines in bisulfite-converted DNA. This step is then followed by affinity capture, using streptavidin-coupled magnetic beads. SuBLiME is highly adaptable and can be combined with deep sequencing library generation and/or genomic complexity-reduction. In this pilot study, we enriched methylated DNA from Csp6I-cut complexity-reduced genomes of colorectal cancer cell lines (HCT-116, HT-29 and SW-480) and normal blood leukocytes with the aim of discovering colorectal cancer biomarkers. Enriched libraries were sequenced with SOLiD-3 technology. In pairwise comparisons, we scored a total of 1,769 gene loci and 33 miRNA loci as differentially methylated between the cell lines and leukocytes. Of these, 516 loci were differently methylated in at least two promoter-proximal CpG sites over two discrete Csp6I fragments. Identified methylated gene loci were associated with anatomical development, differentiation and cell signaling. The data correlated with good agreement to a number of published colorectal cancer DNA methylation biomarkers and genomic data sets. SuBLiME is effective in the enrichment of methylated nucleic acid and in the detection of known and novel biomarkers.

Keywords: biotin; bisulfite; enrichment; labeling; methylation; methylome; sequencing; streptavidin.


Figure 1. (A) Modified oligonucleotide sequences of adaptors and (B) a schematic showing the particular implementation of SuBLiME biotin labeling used in this study with minor steps emitted for clarity. First, sheared and repaired genomic DNA (gDNA) has annealed adaptor 2 (P2-BtnA and P2-BtnB) ligated to the repaired and A-tailed ends of the gDNA (shown in green). Next, the DNA is cut with Csp6I before ligation with the annealed hemi-methylated adaptor 1 (P1-BtnAM and P1-BtnB), which contains an overhanging 5′-TA-3′ (shown in red). The gDNA is then bisulfite-treated and the denatured DNA primed opposite the ligated P2-BtnA oligomer (shown in blue) and strand extension allowed to complete. Unconverted cytosines in the original bisulfite-treated material are now guanines in the newly copied DNA, while the polymerase adds adenines opposite converted uracils. Next, the original bisulfite-converted strand is degraded before priming in the other direction complementary to the P1 adaptor end. To label the DNA, the primer P1-BtnA (blue) was extended by Taq polymerase in the presence of 100 µM biotin-14-dCTP and the unlabeled deoxynucleotide triphosphates dTTP, dATP and dGTP. Finally, labeled material was enriched using streptavidin-coupled magnetic beads. Note that by design P2-BtnB contains no guanosines so no biotinylated-CTP can be added in the linker region. Therefore biotin labeled dCTP should only be added at sites of bisulfite non-conversion of cytosines.
Figure 2. A flow diagram of bisulfite conversion of DNA, SuBLiME biotin enrichment and library creation steps followed by the common primary analysis of the sequencing data and finally the directions and relationships between the multi-directional final analyses.
Figure 3. Methylation summary across the genome in 1 Mbp bins. Genome-wide data by chromosome are presented in a circle. The outermost track is a chromosome cytogenetic band ideogram with centromeres shaded in red. Heading inwards, the next track displays the difference in methylation of colorectal cell lines relative to normal leukocytes in a 10 color purple-green scale, with deep purple bins the most hypermethylated in colorectal cell lines. The next four tracks denote, from outermost in, the mean normalized methylated CpG per “sequenceable” CpG across normal leukocytes, HCT-116, HT-29 and SW-480. Data are presented in a 10 color red-yellow-blue scale with dark red and dark blue denoting hyper- and hypomethylation, respectively. The innermost histogram yields the ‘sequenceable’ CpG sites per bin (green) and a line graph (black) of CpG sites significant in at least two pairwise comparisons with normal leukocytes.
Figure 4. Boxplot of normalized sum of methylation by cell line across the 500 bp proximal to the cTSS for published known methylated (M) and unmethylated (U) biomarkers.

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