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TECH NOTE

Enabling Epigenetics Studies Using Methylated DNA-Seq

A single workflow for methylated-DNA enrichment and next-generation sequencing library preparation

Epigenetics is the study of heritable changes in gene expression that take place by chemically modifying the DNA without affecting the underlying sequence. One widely studied epigenetic modification is the methylation of DNA at cytosine residues, which usually occurs in dense clusters near transcriptional start sites and regulates gene expression. Using next-gen sequencing, the methylation profile can be captured from a genomic DNA sample of interest.

Overview of the EpiXplore Meth-Seq DNA Enrichment Kit workflow

The EpiXplore Meth-Seq DNA Enrichment Kit is a next-gen sequencing library preparation kit which separates the methylated and unmethylated genomic DNA fragments by utilizing the highly specific binding affinity of the MBD2 protein for methylated DNA. DNA enrichment is followed by library preparation using the DNA SMART method. This kit facilitates your methylated DNA-seq experiments with:

  • A simple protocol for enriching both methylated and unmethylated DNA from inputs of 25 ng–1 µg of fragmented genomic DNA
  • A streamlined workflow for generating Illumina-ready sequencing libraries from sheared genomic DNA inputs in just 6 hours
  • An efficient method for adding adapters without ligation or the associated cleanup, reducing the loss of limited input DNA
Generation of sequencing libraries from enriched methylated DNA

EpiXplore Meth-Seq DNA Enrichment technology workflow. His-tagged MBD2 protein (Methyl-CpG Binding Domain Protein 2) is used to capture the methylated DNA fraction from the sheared genomic DNA sample, enabling column separation of the methylated and unmethylated DNA fractions. The subsequent library preparation portion of the workflow allows users to generate Illumina-compatible libraries for meth-seq experiments.

Sensitivity and reproducibility of the enrichment protocol and downstream library preparation

The EpiXplore Meth-Seq DNA Enrichment Kit has the sensitivity to generate sequencing libraries from samples enriched for either methylated or unmethylated fractions of DNA, from inputs as low as 25 ng (see table below). Excellent and consistent mapping statistics are seen across all input levels. Library complexity was maintained in enriched samples when compared to unenriched samples: at all input levels >90% of the total reads mapped to the target genome, with >70% of the total reads mapping uniquely.

Sequencing Metrics Comparing Libraries from Methylated and Unmethylated Enrichment Fractions
Sheared genomic DNA input for enrichment (ng) 25 500 1,000
Enrichment fraction Unenriched Methylated Unmethylated Methylated Unmethylated Methylated Unmethylated
Post-enrichment DNA input for library prep (ng) 1 1 1 1
Total reads (in millions) 2.5 2.3 2.8 2.7 3.8 2.8 3.0
No. of reads on target (in millions) 2.3 2.2 2.6 2.6 3.5 2.7 2.8
% reads mapped 92 94 94 96 93 97 94
% useful reads mapped (without duplicates) 77 74 77 70 76 67 76
No. of peaks called 318 2,950 3,390 3,672 1,227 7,372 6,518

Sequencing metrics comparing libraries from methylated- or unmethylated-DNA fractions. Methylated and unmethylated fractions were enriched from fragmented Arabidopsis thaliana genomic DNA using his-tagged MBD2 protein. 1 ng of the enriched DNA fractions was used to generate DNA SMART libraries with 15 PCR cycles. 1 ng of unenriched DNA was used as a control. Reads were mapped to the Arabidopsis genome (TAIR14) using Bowtie2 (Langmead, et al., 2012). Peak calling was performed using MACS (Zhang, et al., 2008).

Distinct differences in methylated and unmethylated regions identified by next‑gen sequencing

The sequencing data obtained from methylated and unmethylated fractions with initial inputs of 25, 500, and 1,000 ng were compared to publically available bisulfite sequencing data. The peaks identified in the methylated and unmethylated libraries correspond to distinct regions of the genome and show good correlation with the methylation patterns identified in this bisulfite sequencing data.

Methylation patterns in aligned NGS libraries from enriched DNA

Distinct methylation patterns are observed in aligned NGS data of libraries generated from methylated and unmethylated DNA fractions. A representative region of Arabidopsis chromosome 3 (outlined in the green box) shows the peaks identified in the DNA enriched for methylated and unmethylated fractions. The methylated regions are outlined in red boxes and the unmethylated regions are outlined in blue boxes. The amount of initial input DNA is shown along the left-hand side of the alignments. The data show good concordance with the publicly available bisulfite sequencing data. Data were visualized using the Integrative Genomics Viewer (Broad Institute). Public data was obtained from GEO accession GSM980986; Stroud et al., 2013.

References:

  • Langmead, B., et al., (2012) Nat Methods. 9(4):357–359.
  • Stroud, H., et al., (2013) Cell 152(1–2):352–364.
  • Zhang, Y., et al., (2008) Genome Biol. 9(9)R137

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