non methylated control dna  (Zymo Research)

Journal: bioRxiv

Article Title: Genome-wide extraction of differentially methylated DNA regions using adapter-anchored proximity primers

doi: 10.1101/2025.06.29.660377

Figure Lengend Snippet: Overview of Methylation-Specific Amplification using Proximity Primers (MAPP). a) Schematic representation of the proximity primer-PCR concept. Proximity primers consist of two components: “Backbone” and “Probe”. The backbone functions as a universal primer complementary to the adapter sequence, while the probe (6-12 bp) is variable and targets a nearby sequence. The DNA strand can fold, allowing the probe to bind to its fully matched target, resulting to a deletion in the folded region. b) Demonstration of Proximity Primer-PCR using a proximity primer with a 9-bp probe (CGACCCGCG) to amplify different fragments from a single target, representing cfDNA or randomly fragmented gDNA. Converted methylated ultramers are depicted with solid lines and converted unmethylated ultramers with dashed lines. The probe targets are located at varying distances from the adapter. c) Plot illustrating the efficiency of Proximity Primer-PCR as a function of the distance between the probe target and the adapter. d) Application of Proximity Primer-PCR for methylation-specific amplification, showcasing a novel approach for extracting and enriching methylated, or un-methylated, CpG-containing genomic regions. The probe can bind to both the top and bottom strands, depending on target availability.

Article Snippet: Human fully-methylated and fully-nonmethylated control DNA was obtained from ZYMO Research (Catalog No. D5014).

Techniques: Methylation, Amplification, Sequencing

Journal: bioRxiv

Article Title: Genome-wide extraction of differentially methylated DNA regions using adapter-anchored proximity primers

doi: 10.1101/2025.06.29.660377

Figure Lengend Snippet: aMAPP efficiently captures both methylated and unmethylated CpG Islands (CGIs) using ultra-low depth sequencing. Methylation enrichment via aMAPP was performed on EM-seq-converted DNA, while un-methylation enrichment was conducted on TAPS-converted DNA. a) CpG methylation percentage and distribution in aMAPP libraries compared to standard libraries using sheared NA12878 gDNA. aMAPP selectively enriches methylated and unmethylated CpGs depending on the conversion method applied. b) Number of distinct CGIs covered using ultra-low depth sequencing in aMAPP libraries compared to standard control libraries of methylated and unmethylated control DNA. c) Ratio of CGI-associated reads to total reads in aMAPP libraries versus standard control libraries of methylated and unmethylated control DNA.

Article Snippet: Human fully-methylated and fully-nonmethylated control DNA was obtained from ZYMO Research (Catalog No. D5014).

Techniques: Methylation, Sequencing, CpG Methylation Assay, Control

Journal: bioRxiv

Article Title: Genome-wide extraction of differentially methylated DNA regions using adapter-anchored proximity primers

doi: 10.1101/2025.06.29.660377

Figure Lengend Snippet: Detection of differentially methylated regions (DMRs) using aMAPP in a colorectal tumor sample through ultra-low depth sequencing, ∼300×10 3 reads. a) Heatmap illustrating the methylation levels of detected DMRs, which annotated close a gene, in tumor tissue versus adjacent normal tissue. b) Total number of DMRs identified using aMAPP compared to standard library methods under ultra-low depth sequencing conditions. aMAPP applied to EM-seq-converted DNA detected 293 hypermethylated DMRs, and aMAPP applied to TAPS-converted DNA identified 55 hypomethylated DMRs. While standard EM-seq and standard TAPS libraries detected 16 and 3 DMRs, respectively. c) Proportion of DMRs associated with CpG Islands (CGIs), detected using aMAPP on EM-seq-converted DNA (hypermethylated DMRs) and aMAPP on TAPS-converted DNA (hypomethylated DMRs).

Article Snippet: Human fully-methylated and fully-nonmethylated control DNA was obtained from ZYMO Research (Catalog No. D5014).

Techniques: Methylation, Sequencing

Journal: bioRxiv

Article Title: Genome-wide extraction of differentially methylated DNA regions using adapter-anchored proximity primers

doi: 10.1101/2025.06.29.660377

Figure Lengend Snippet: aMAPP detects DMRs at a 0.01% frequency in both solid tumor (a) and cfDNA (b) samples using ultra-low-depth sequencing, 3×10 3 reads. Integrated Genome Viewer, IGV visualization of a highly methylated CGI (chr10:132783854-132789145; CpG: 642) identified in DMR analyses of colon tumor and adjacent normal tissue (referenced in ). This CGI is located near the NKX6-2 gene. a) In a 0.01% spike-in tumor DNA sample within adjacent normal tissue, aMAPP successfully detected methylated tumor DNA. No amplification was observed in normal tissue, confirming the unmethylated state of this CGI in normal samples. b) In a 0.01% spike-in tumor DNA sample within normal cfDNA, aMAPP effectively captured methylated tumor DNA in 2 out of 4 sequence reads. The same region in normal cfDNA showed completely unmethylated CpGs.

Article Snippet: Human fully-methylated and fully-nonmethylated control DNA was obtained from ZYMO Research (Catalog No. D5014).

Techniques: Sequencing, Methylation, Amplification