BASE MODIFICATION ANALYSIS

BASE MODIFICATION ANALYSIS

Overview - What are base modifications and why are they important?

Epigenetic modifications include DNA methylation, histone modification and non-coding RNA- (ncRNA)-associated gene silencing. Methylated DNA carries a methyl group (-CH3) at a DNA base. Well-known modifications are 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and N6-methyladenosine (m6A). Due to technical limitations, only the 5mC epigenetic mark has been thoroughly investigated. 

DNA methylation plays a role in essential cellular processes like regulation of gene expression and metabolic pathways, DNA repair mechanisms, microbial virulence and maintenance of chromosome stability. Due to their involvement in vital cellular pathways, abnormal epigenetic modifications are often implicated in genetic diseases, cancer and the development of drug resistance.  

Applications - What are the advantages of direct detection of epigenetic modifications?

Base modification analysis with the PacBio technology platform is a one-of-a-kind method for exploring the methylome, as it provides: 

  • Profiling of base modifications in unamplified source material with single-base resolution
  • Strand-specific detection of 5-methylcytosine, 4-methylcytosine (4-mC), 6-methyladenine (6-mA) and glucosylated 5-hydroxymethylcytosine
  • One library for sequencing and for detection of modified DNA
  • Simultaneous analysis of SNPs, InDels and multiple types of base modifications
  • Discovery of novel or unexpected modifications
  • More complete understanding of regulation of biological processes and phenotypic variability

Workflow - Methods & technologies for detecting DNA modifications

Analysis of common epigenetic methylation is based on indirect detection of DNA methylation through bisulfite sequencing. Some of the limitations inherent to bisulfite-based techniques include poor data quality from incomplete bisulfite conversion or from DNA degradation during bisulfite treatment, as well as the inability to discriminate between 5mC and 5hmC. 

To address these questions, GATC Biotech uses PacBio technology. With SMRT technology, it is possible to go one step beyond the DNA sequence and gain in-depth epigenetic information from a single sequencing run. Several modifications can be directly profiled with single-base resolution from unamplified, untreated source material.

Molecular structures of selected base modifications

Base modification detection with PacBio’s SMRT technology involves analysis of polymerase kinetics during sequencing. When a polymerase encounters a modification, the enzyme is slower to incorporate the nucleotide on the opposite strand. This slow-down is detected computationally as a longer interpulse duration (IPD). The resulting IPDs are compared to a control, and an “IPD ratio” is calculated to pinpoint the modified nucleotides as well as the modifications in the sequenced genome.

Scientific expertise: modification analysis

GATC Biotech is a pioneer in implementing new technologies for the improvement of epigenetic profiling. As a certified PacBio service provider, the company is fully dedicated to the exploration of the modified nucleotides beyond 5mC in an unbiased manner. By providing a direct method of DNA-modification profiling of 5mC, 5hmC and m6A nucleotides, GATC Biotech provides an exciting new option for exploring more modifications across the entire genome. 

Publications

Find here, a list of selected research articles supported by GATC Biotech’s sequencing products.

Projects

The KLEBSICURE Consortium

INVIEW MODIFICATION ANALYSIS is applied for the KLEBSICURE Consortium, a cooperation project with GATC Biotech AG, the Max Planck Institute for Infectious Biology (Berlin, Germany) and the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy (Wroclaw, Poland) as consortium members and Arsanis Biosciences GmbH (Vienna, Austria) as consortium leader. The main objective of the project is the identification and characterisation of the pathogen Klebsiella pneumoniae, which causes severe infections. The study uses de novo sequencing in combination with direct detection of base modifications to study the virulence of the pathogen to and subsequently guide the generation of monoclonal antibody therapeutics and the establishment of a test method for clinical diagnostics.

Related products to modification analysis

Did you know that analysis of base modifications can be achieved at single-base resolution without chemical conversion or DNA amplification steps? To discover more modifications with reduced bias, take advantage of our complete service package including DNA isolation, expert library preparation, sequencing on the leading PacBio RS II platform, professional BioIT analysis and a final comprehensive GATC Data Analysis report. See how our INVIEW MODIFICATION ANALYSIS product can help advance your epigenetics project.

Are you interested in analysing the 5-methylcytosines across the genome of nearly any organism? Then whole genome bisulfite sequencing (BS-Seq) with INVIEW EPIGENETICS BS-SEQ might be the right product for you. 

Are you studying the DNA methylome of a mammalian model organism? Then our INVIEW EPIGENETICS RRBS-SEQ product can help advance your research. 

If you are currently exploring DNA-protein interactions, then discover how you can benefit from our established NGSELECT

Further reading on base modification analysis

Flusberg, B.A. et al. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods. 7(6), 461 – 5 (2010).

Zillner, K., Németh, A. Single-molecule, genome-scale analyses of DNA modifications: exposing the epigenome with next-generation technologies. Epigenomics 4(4), 403 – 14 (2012).

Clark, T.A. et al. Characterization of DNA methyltransferase specificities using single-molecule, real-time DNA sequencing. Nucleic Acids Res. 40(4):e29 (2012).