SMRT Sequencing for Epigenomics

In bacterial genomes, N6-methyladenine (m6A), N4-methylcytosine (m4C) and 5-methylcytosine (m5C) act as components of restriction-modification (RM) systems. Along with m6A and m5C, modified bases such as 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) are also present in the eukaryotic genome. However, next-generation sequencing (NGS) technology lacks simple methods to determine the location of most DNA modifications, so many DNA modifications are generally ignored. The recently developed single molecule real time (SMRT) DNA sequencing technology directly detects natural epigenetic modifications through measuring kinetic changes during base incorporation. By capturing these modifications simultaneously with the sequenced data, the method eliminates the need for special sample preparation and additional sequencing.

Creative BioMart is providing SMRT sequencing service for epigenetic research to complement our NGS technology-based research methods.

What Is SMRT Sequencing?

SMRT sequencing technology applies the idea of sequencing while synthesizing. This technology utilizes four-color fluorescence labeled dNTPs and zero-mode waveguides (ZMWs) to sequence single DNA molecules. When the DNA template is captured by DNA polymerase, dNTPs with four different fluorescence enter the detection area randomly through Brownian motion and bind to the polymerase. The bonding time of the base matched with the template is much longer than that of other bases. Therefore, the matching base and free base can be distinguished by counting the duration of the fluorescence signal.

Four kinds of bases (dNTP) are labeled with four-color fluorescence respectively. In the base pairing phase, different bases will emit different fluorescent light when added, and the type of base entering can be determined according to the wavelength and peak value of fluorescent light. In addition, since the fluorescence signal is attached to the phosphate group of dNTP, when the previous dNTP is synthesized, the phosphate group is automatically detached, which ensures the continuity of detection, improves the detection speed and cooperates with the high-resolution optical detection system to realize real-time detection. Moreover, the difference in interpulse duration (IPD) between normal and modified bases can be used as a signal to detect base modification.

Schematic illustration of the principle behind SMRT sequencing.

Figure 1. Schematic illustration of the principle behind SMRT sequencing. (Rhoads A.; Au K F. 2015). A. A SMRTbell (gray) diffuses into a ZMW, and the adaptor binds to a polymerase immobilized at the bottom. B. Each of the four nucleotides is labeled with a different fluorescent dye (indicated in red, yellow, green, and blue, respectively for G, C, T, and A) so that they have distinct emission spectrums. C. SMRT sequencing can detect modified bases (e.g. m6A).

Features of SMRT Sequencing

Features Description
Single molecule
  1. Single-molecule resolution
  2. No need for PCR amplification during sequencing
  3. Can easily cover high GC and high repeat regions
  4. Quantify low abundance/low-frequency mutations more accurately
Extra-long read length
  1. Extremely long read length (> 20 kb)
  2. Accurate localization, improved assembly efficiency, and complete genome assembly with low coverage
Direct recognition of base modification
  1. Genomic and epigenetic data can be analyzed simultaneously by using the dynamic changes of polymerase reaction during sequencing, directly detecting base modifications
  2. Suitable for the detection of DNA or RNA modifications and overcomes many limitations of bisulfite sequencing, enabling simultaneous detection of multiple types of epigenetic modifications
  3. The location of adenine and cytosine methylation is found in a DNA strand-specific and non-hypothetical manner and the methyltransferase recognition motif can be identified

Our Advantages

  1. One-stop service: customers only need to provide samples, and we will complete full SMRT sequencing from sample and library preparation, sequencing to data analysis. The complete report is provided to you with both raw and analyzed data, graphs, detailed protocols, and a summary of findings.
  2. Highly customized service: since both SMRT sequencing and NGS have their own pros and cons, combined sequencing has become a more popular method to take full advantage of the advantages of the two platforms. We further explore the workflow and tools tailored to your research focus.
  3. Reliable data quality: we have accumulated rich project experience, with mature library construction and data analysis pipeline. Our experienced team of experts performs quality control after each procedure to ensure confident and fair results.
  4. Professional data analysis: Creative BioMart utilizes specialized software to characterize epigenetic markers. We provide you with both standard data analyses and customized bioinformatics analyses.

Workflow of SMRT Sequencing at Creative BioMart

Workflow of SMRT Sequencing at Creative BioMart

Figure 2. Workflow of SMRT Sequencing at Creative BioMart

Creative BioMart pursues a strong commitment to service and unparalleled data quality to help our customers achieve research goals in the rapidly evolving world of epigenetics. contact us for more information about our SMRT sequencing service.

1. Davis B M.; et al. Entering the era of bacterial epigenomics with single molecule real time DNA sequencing. Current Opinion in Microbiology. 2013, 16(2): 192-198.
2. Rhoads A.; Au K F. PacBio sequencing and its applications. Genomics, Proteomics & Bioinformatics. 2015, 13(5): 278-289.

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