May 11, 2010

Direct detection of DNA methylation during single-molecule, real-time sequencing

Nature Methods
Benjamin A FlusbergStephen Turner

Abstract

We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule, real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenine, 5-methylcytosine and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We used these kinetic signatures to identify adenine methylation in genomic samples and found that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns in even highly repetitive genomic regions.

  • References32
  • Citations396

Citations

Mentioned in this Paper

Hydrogen sulfite
DNA Methylation [PE]
Polymerase
Physiologic Pulse
NCOR2 wt Allele
Alkalescens-Dispar Group
Protein Methylation
Genome
DNA Methylation
Study of Epigenetics

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