Journal: Cell Reports Methods
Article Title: Genome-wide profiling of unmodified DNA using methyltransferase-directed tagging and enrichment
doi: 10.1016/j.crmeth.2025.101187
Figure Lengend Snippet: Schematic of the Active-Seq workflow and initial method validation using synthetic oligonucleotides (A) Schematic showing the single-tube, Active-Seq workflow. Purified DNA (cfDNA or fragmented gDNA) is tagged using a CpG-targeting methyltransferase (M.MpeI) and a synthetic cofactor analog. The enzyme catalyzes DNA alkylation with azide-terminated tags exclusively at unmodified CpG sites. Tagged DNA molecules are subsequently subject to standard library end repair and adapter ligation, followed by affinity tagging and isolation using streptavidin-coated magnetic beads. Tagged (unmodified CpG sites, Active-Seq) and untagged (5mCpG and other modified CpG sites and CpG-free fragments, unbound fraction) DNA can be separately amplified and sequenced. (B) Plot showing the efficiency for DNA binding and release (recovery) of tagged and affinity-labeled DNA, using 10 ng target DNA input, as a function of target DNA CpG site density. Binding efficiency is calculated based on the assumption that any reduction in DNA concentration after DNA incubation with beads is as a result of DNA binding to the beads. (C) Plot showing the efficiency for binding and release (recovery) of tagged and affinity-labeled DNA for a decreasing amount of input target DNA against a background of 24 ng of non-target DNA (containing no CpG sites). Note that the recovered DNA concentration was determined by qPCR, and reported recoveries are measured against DNA we expect to be bound to the beads (input DNA quantified via Qubit). (D) Plot showing number of reads of spiked-in DNA oligos (5, 25, and 50 pg in total) in the enriched (unmethylated) fraction with four unmethylated CpG sites (4 uMe CpG), four methylated CpG sites (4 Me CpG), and no target CpG sites (0 CpG) from a typical Active-Seq experiment. (E) Plot showing number of reads of spiked-in DNA oligos (5, 25, and 50 pg in total) in the unbound fraction with four unmethylated CpG sites (4 uMe CpG), four methylated CpG sites (4 Me CpG), and no target CpG sites (0 CpG) from a typical Active-Seq experiment. Data in bar-plots are presented as mean ± SD of three independent replicates. Two-way ANOVA test: ∗ p < 0.05, ∗∗ p < 0.01 , ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001.
Article Snippet: Human gDNA: HT29 , ATCC , cat#: HTB-38D.
Techniques: Biomarker Discovery, Purification, Adapter Ligation, Isolation, Magnetic Beads, Modification, Amplification, Binding Assay, Labeling, Concentration Assay, Incubation, Methylation
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