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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 methylations play important roles in essential cellular processes such as 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. The methylation modifications of the genome is called methylome.


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. 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.

In order to address these questions, Eurofins Genomics utilises the PacBio technology that is based on single-molecule real-time sequencing (SMRT) technology. This allows scientists to go 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 and untreated source material.

Base modification detection with PacBio’s SMRT technology involves analysis of polymerase kinetics during sequencing. When a polymerase encounters a base modification, the enzyme is slower to incorporate the nucleotide on the complimentary strand. This slowdown 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.

Did you know that we at Eurofins Genomics offer broad range of next generation sequencing products, such as the INVIEW Human Exome?

 

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