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Reduced Representation Bisulfite Sequencing

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CD Genomics is offering reduced representation bisulfite sequencing (RRBS) service at single-nucleotide resolution by reducing the amount of sequencing required while capturing the majority of promoters and other relevant genomic regions. RRBS is favorable for biomarker discovery by the identification and analysis of differentially methylated regions (DMRs) between samples.

The Introduction of RRBS

DNA methylation of cytosine is an epigenetic modification involved in gene expression regulation. DNA methylation occurs predominantly in CpG contexts, and these CpG di-nucleotides are more abundant in selected regions of the genome. RRBS is a powerful approach to genome-wide DNA methylation analysis that combines restriction enzyme digestion with bisulfite sequencing to enrich a CpG-dense fraction of the genome. This combination makes RRBS improve the efficiency of the utilization of samples and the perfect platform for pilot studies and clinical applications.

RRBS only sequences the fragments pertaining to CpG-rich regions at a significantly reduced cost when compared with whole genome bisulfite sequencing (WGBS). RRBS is very cost-effective, given that about 1-5% of the genome is sequencing, covering about 12% of genome-wide CpG sites and about 84% of CpG islands in promoters. Plants exhibit a different CpG distribution: (i) lacking characteristic CpG islands in promoters; (ii) occuring at cytosine bases within all sequence contexts. Therefore, while enzyme MspI is commonly used in animals and human, enzyme SacI/MseI is often used in plants.

Advantages of RRBS

  • Low input DNA requirement
  • Methylation pattern of CpG rich region across the genome
  • Covering up to 5 million CpG sites in Human
  • Simultaneously detection of DNA methylation and SNPs.
  • Epigenetic biomarker discovery
  • Single-nucleotide resolution and cost-efficiency

RRBS Workflow

The general workflow for RRBS sequencing is outlined below. RRBS utilizes the MspI restriction enzyme to cut at CCGG sites across the genome, enriching CpG-dense regions. The regions containing CpGs are then recovered by gel purification. After methylated adaptor ligation, end repair and dA tailing, theses fragments are then converted by bisulfite, amplified, and sequenced. Our highly experienced expert team executes quality management, following every procedure to ensure confident and unbiased results.

Service Specifications

Sample requirements and preparation
  • Sample type: Total DNA without degradation or RNA contamination or severe degradation
  • Starting amount of total DNA ≥ 5 µg
  • Sample concentration ≥ 50 ng/µl; OD = 1.8~2.0
  • All DNA samples are validated for purity and quantity
Sequencing
  • RRBS library preparation
  • Illumina HiSeq X Ten, paired-end 150 bp
  • Sequencing depth > 50M clean reads
  • More than 80% of bases with a ≥Q30 quality score
Bioinformatics Analysis
We provide customized bioinformatics analysis including:
  • Alignment against reference genome
  • Promoter and CpG islands coverage analysis
  • Promoter and CpG island methylation level analysis
  • DMR (differentially methylated region) analysis
  • Functional annotation of differentially expressed genes
  • Relation of methylation level and functional elements’ length
  • CpG region distribution
  • Regional distribution of genes

By combining enzyme digestion, bisulfite conversion and NGS technology, CD Genomics is able to provide RRBS as an alternative way to generate and profile genome-wide methylomes at a reduced cost. If you have additional requirements or questions, please feel free to contact us.

References:
1. Guo H, Zhu P, Guo F, et al. Profiling DNA methylome landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing. Nature protocols, 2015, 10(5): 645.
2. Meissner A, Gnirke A, Bell G W, et al. Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic acids research, 2005, 33(18): 5868-5877.

1. What is the workflow for RRSB?

The workflow of RRBS is demonstrated in Figure 1 below. Briefly, the DNA is first digested, typically with MspI which is methylation insensitive and cuts at CCGG sites, enriching CpG rich regions of the genome. The fragment ends are then repaired and dA-tailed and adapters are ligated. Subsequently, the fragments are selected by size, converted by bisulfite, amplified and sequenced.

The schematic workflow for RRBS
Figure 1. The schematic workflow for RRBS.

2. What are the requirements for sequencing samples?

You can submit cells (at least 5*106), fresh tissues (at least 2~3 g), or DNA (at least 5 μg; concentration ≥ 100 ng/µl; OD = 1.8~2.0; without degradation or RNA contamination or severe degradation). Before shipping, the cells should be stored at -80°C, and the DNA should be dissolved in water and stored at -20°C. Please avoid repeated freeze-thaw cycles. When shipping, the samples should be sealed with parafilm and kept in a frozen state probably with ice packs.

3. What species are suitable for whole genome bisulfite sequencing?

The species subjected to RRBS should meet three requirements: (i) eukaryotes; (ii) its reference genome has been assembled to the scaffold level at least; (iii) relatively complete genome annotations.

Case Study DNA methylation changes induced by long and short photoperiods in Nasonia

Journal: Genome Research
Impact factor: 11.922
Published: December 15, 2015

Abstract

Wasp Nasonia vitripennis is an emerging model organism that exhibits a strong photoperiodic response, and can be used to study the molecular mechanisms underlying photoperiodic timing. The authors attempted to figure out how female Nasonia control the developmental trajectory of their offspring to survive the winter by taking advantage of RRBS method. It showed that knocking down DNA methyltransferase or blocking DNA methylation largely disrupts the photoperiodic diapause response of the wasps, which suggests the role of DNA methylation in insect photoperiodic timing.

Results

1. The Nasonia methylome

The authors profiled DNA methylation in genomic DNA extracted from female N. vitripennis kept in either long- or short-day conditions by utilizing RRBS. CpG methylation accounts for a large amount of overlap between the two samples in contrast to other sequence contexts (Figure 1B), which may suggest that non-CpG methylation largely represents experimental noise. CpGs seem to be either heavily or lightly methylated (Figure 1C). The majority of the methylated CpGs were located in exons, and the majority were located in introns, promoter regions and intergenic regions (Figure 1D, E).

The Nasonia methyloma
Figure 1. The Nasonia methyloma.

2. Identification of differentially methylated genes

The authors identified 51 differentially methylated CpG sites (DMCs) that were mapped to 37 genes. Approximately half of the DMCs (23/51) exhibited hypomethylation in long day compared with short day, whereas the others exhibited the opposite trend. qPCRs were conducted to validate the RRBS analysis.

Differential DNA methylation associated with photoperiod in <em>Nasonia</em>
Figure 2. Differential DNA methylation associated with photoperiod in Nasonia.

3. Functional assays demonstrating a causative role of DNA methylation

The authors knocked down Dnmt1a-c and Dnmt3 by injecting dsRNA into female pupae of the N. vitripennis strain AsymC. The knockdown was verified by qPCR. Knockdown of Dnmt1a led to females producing diapause offspring regardless of day length. Silencing Dnmt3 resulted a nonsignificant (but close to significance) difference in diapause response, suggesting that the effect of Dnmt3 is much weaker or harder to resolve.

They also tested the impact of DNA methylation pharmacologically by utilizing DNA methylation inhibitor 5-aza-2’-deoxy-cytidine (5-aza-dC), and observed a reduction of diapause in the progeny of wasps maintained in short day, as well as an increase in long-day progeny.

DNA methylation is required for photoperiodic-mediated diapause response Figure 3. DNA methylation is required for photoperiodic-mediated diapause response.

Pharmacological tests by using 5-aza-dC.
Figure 4. Pharmacological tests by using 5-aza-dC.

Reference Pegoraro M, Bafna A, Davies N J, et al. DNA methylation changes induced by long and short photoperiods in Nasonia. Genome research, 2016, 26(2): 203-210.

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