Utilization of MBD-seq to Elucidate Differentially Methylated Regions incfDNA Across Healthy and Cancer Patients
Camille Michelon1, Shuxiong Wang1, Vidyodhaya Sundaram2, Brian Egan1, Benjamin Delatte1
1
Active Motif, Inc., Carlsbad, CA 92008 2BioChain, Inc., Newark, CA 94560

Poster # 4415

Enabling Epigenetics Research

Introduction

QC on Spike-In DNA

Enrichment of Methylated DNA by cfMethylCollector

5-Methylcytosine (5-mC) is the most abundant epigenetic DNA modification in eukaryotes. The presenceof 5-mC alters gene expression and genome metabolism, and its deregulation is associated with many
human diseases. Cell-Free DNA (cfDNA) released from apoptotic cells has recently gained attention, asmutations or epimutations (e.g. 5-mC variations) detected in cfDNA were shown to have high diagnostic
potential to assess the presence, stage and outcome of several cancers. However, 5-mC detection incfDNA remains a challenge as input material is often very scarce. MBD-Seq is a method that leverages theability of Methyl-CpG-binding domain protein 2 (MBD2) to capture and detect highly methylated regions ofDNA genome-wide. We evaluated MBD-Seq for the detection of aberrant methylation patterns in cfDNAfrom healthy and diseased patients. We found MBD-seq on cfDNA to be specific (> 100-fold mC:C detectionratio) and sensitive (5 ng of input cfDNA). Applying MBD-seq to samples from healthy vs colorectal (CRC-IV)
and breast (B-IIIC, B-IV) cancer patients showed striking differences in methylation of key tumor-associatedgenes. Hence, MBD-Seq can elucidate aberrant methylation patterns from cfDNA, highlighting its potential
as a diagnostics tool for the detection of cancer.

AA“pET28a”

BCFigure 6 A. NBR2. IGV at canonical methylated locus, NBR2, arrow indicates CpG island. cfDNA, normal gDNA, andtumor gDNA across four cancer patients are displayed. Signal at methylated locus indicates specificity for enrichment ofmethylated DNA. Red (patient 1, CRC-IV), Green (patient 2, CRC-IV), Yellow (patient 3, B-IIIC), Purple (patient 4, B-IV).
Figure 6 B. GAPDH. Lack of signal at canonical unmethylated locus, GAPDH, indicates cfMethylCollector specificity forenrichment of methylated DNA.

Schematic Workflow of Active Motif’s cfMethylCollector Technology
Extract and purify
cfDNA from plasma.

Incubate sheared DNA withHis-tagged MBD2b protein.

Capture the methylated DNA/
MBD2b complexes withnickel-coated magnetic beads.

Wash to remove theunmethylated DNA fragments.

Elute the bound, methylated
DNA while, simultaneously
degrading the MBD2b protein.

The purified, methylated DNA
is ready for PCR analysis.

Figure 3 A. Spike-In DNA Production. Methylated spike-in DNA originates from v5 plasmid propagated in dcm+/
dam+ (DNA methyltransferases) E. coli, extracted, then sonicated using PIXUL™. M.SssI incubation was performed tomethylate all cytosine residues in CpG dinucleotide context. Unmethylated Spike-In DNA was produced from pET28
plasmid propagated in dcm-/dam- E. coli, extracted, sonicated, and purified using same parameters. After quantification,
methylated and unmethylated DNA were combined equimolarly and spiked at 1%.

Figure 1. cfDNA is extracted and purified from plasma using the Active Motif Cell-Free DNA (cfDNA) Purification Kit™,spiked with 1% (ng) spike-in DNA, then introduced into a binding reaction alongside His-MBD2b, nickel beads, and highsalt binding buffer for 1 hour. MBD2b binds to methylated spike-in DNA and methylated CpG islands (CGI) within cfDNAand forms DNA-Protein-Bead complexes. The complexes are pelleted on a magnet, unbound (unmethylated) material
is washed and discarded. The enriched pulldown is eluted in a Proteinase K (PK) digestion buffer for 30 minutes. Silicabeads are used to recover and purify DNA. The final pulldown undergoes qPCR QC on methylated and unmethylated
spike-in DNA targets. If % methylated input ÷ % unmethylated input ratio is ≥ 50x, there is a quality enrichment suitable toproceed with library prep and sequencing. Assay time before library prep is approximately 4 hours (including qPCR QC).

B

Differential Methylation Region (DMR) Analysis
AD

B-IV

Figure 3 B. Sample Overview. Cancer type and stage are specified across 4 patients. Red (patient 1, CRC-IV), Green(patient 2, CRC-IV), Yellow (patient 3, B-IIIC), Purple (patient 4, B-IV).
Figure 3 C. qPCR QC. Percent input and fold-enrichment for cfDNA, normal gDNA, and tumor gDNA are displayed
across 4 patients. Purple bars represent % input of methylated spike-in DNA, Black bars represent % input ofunmethylated spike-in DNA.

Unchanged
Increased Methylation
Decreased Methylation

Orange line represents fold-enrichment of spike-in DNA.
To meet QC criteria for a quality enrichment (depletion of unmethylated, enrichment of methylated spike-in DNA), foldenrichment must be ≥ 50x. All samples meet QC criteria.

BECRC-IV

Correlation with Published Low Input MBD-Seq DataCPearson correlation coefficient = 0.81

Sequencing Statistics

cfMethylCollector 5ng

A

Unchanged
Increased Methylation
Decreased Methylation

Figure 4. Sequencing Statistics. FRiP (Fraction of readsin peaks) and filtered peaks are plotted for each sample.
Red (patient 1, CRC-IV), Green (patient 2, CRC-IV), Yellow(patient 3, B-IIIC), Purple (patient 4, B-IV).

CHuang, et al.

B

Figure 7 A. DMR Analysis, B-IV. HRNR gene identified as significant (p-value = 1.91E-05, FDR = 0.01) DMR between
healthy cfDNA control and cfDNA from patient 4, B-IV (Purple).

CpG Island (CGI) Aggregation Plots

Figure 7 B. STRING Analysis. HRNR belongs to S100 gene family, its suppression is associated with breast cancer
progression in transformation from preinvasive to invasive carcinoma [2].
Figure 7 C. DMR Analysis, CRC-IV. GLIPR1L2 gene identified as significant (p-value = 2.17E-05, FDR = 0.01) DMR
between healthy cfDNA control and cfDNA from patient 1, CRC-IV (Red). GLIPR1L2 is a paralog of GLIPR1, a tumorsuppressor gene with apoptosis-inducing activities [3].
Figure 7 D, E. Volcano Plots. Distribution of significant (FDR) methylation fold-changes for D. B-IV (patient 4, Purple)
and E. CRC-IV (patient 1, Red) relative to healthy cfDNA control.

Figure 2 A. CGI Aggregation plot. Heatmap intensity indicates signal overlap between cfDNA sample and regions
within reference CpG islands (CGI) file. Each row (y-axis) is a CGI in the reference file spanned over +/- 2kb (x-axis).
Green is reference data [1], Purple is cfMethylCollector data.
Figure 2 B. IGV Tracks. Integrative genome viewer at canonical methylated locus, NBR2, arrow indicates CpG island.
Both samples have CpG-specific signal of similar amplitude.

SummaryFigure 2 C. Pearson Correlation. cfMethylCollector pulldown and reference cfDNA public data for low input MBD-seq
have a good correlation coefficient. Bin size is 5kb.

References1.

Huang J et al. 2021 Mar 16. Cell-free DNA methylome profiling by MBD-seq with ultra-low input. Epigenetics.:1–14. doi:https://doi.org/10.1080/15592294.2021.1896984.
SRR13040548

2.

Zhang S et al. 2017. Distinct prognostic values of S100 mRNA expression in breast cancer. Scientific Reports. 7(1). doi:https://doi.org/10.1038/srep39786.

3.

Torres-Martinez Z et al. 2021. Key genes and drug delivery systems to improve the efficiency of chemotherapy. Cancer drug resistance (Alhambra, Calif). 4:163–191. doi:https://doi.org/10.20517/cdr.2020.64.

activemotif.com

Figure 5. CpG Island (CGI) Aggregation Plots.

1. cfMethylCollector utilizes MBD2b to bind and pulldown methylated CpG-islands (CGI) and prepare highquality libraries with as little as 5ng cfDNA.

Top plot, Aggregate Signal. Mean signal overlap between sample and reference CGI file across the genome. Signal
intensity (y-axis) is centralized and spanned over +/- 2kb of CGI regions (x-axis).

2. qPCR on spike-in DNA targets enables post-enrichment QC to ensure quality enrichment before library
preparation and sequencing.

Bottom plot, Heatmap. Heatmap intensity indicates signal overlap between cfDNA sample and regions within reference
CpG islands (CGI) file. Each row (y-axis) is a CGI in the reference file spanned over +/- 2kb (x-axis).

3. cfMethylCollector elucidates differentially methylated regions (DMRs) in cfDNA. Analogous methylation
patterns and DMRs are found between cfDNA and tumor gDNA relative to normal gDNA and cfDNA controls.

Strong CGI-specific signal demonstrates assay specificity. Generally, tumor gDNA and cfDNA exhibits higher aggregate
signal than normal gDNA. Red (patient 1, CRC-IV), Green (patient 2, CRC-IV), Yellow (patient 3, B-IIIC), Purple (patient 4, B-IV).

4. cfDNA can be a surrogate molecule to assess aberrant methylation patterns in cancer patients without theneed for invasive tumor biopsy.

Patient genomic DNA was extracted using BioChain AnaPrep 12 Dx instrument. cfDNA was collected byconsenting donors courtesy of BioChain. Healthy cfDNA control obtained from BioChain Cat No. #Z2121001.

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Contact: bdelatte@activemotif.com