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

By B. Flusberg, Dale R. Webster, Jessa Lee, K. Travers, Eric C. Olivares, Tyson A. Clark, J. Korlach, S. Turner. Nature Methods, 2010

1,389 citations recorded by the source corpus.

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.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/5eb309b5fbdc028abdde9dacd3b417d3e32b0b1a

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Citations in the corpus

• Long-read human genome sequencing and its applications (2020, neutral): “…for sequencing, the DNA and RNA molecules retain base modifications, allowing epigenomic changes to be detected through polymerase kinetics 96,125,126,137 or current changes, respectively 86,126,127 . the development of these technologies, the most common base modification that could be…”
• Computational solutions to large-scale data management and analysis (2010, background): “that will enable us to scan entire genomes, microbiomes and transcriptomes and to assess epigenetic changes directl”
• Piercing the dark matter: bioinformatics of long-range sequencing and mapping (2018, neutral): “Part A is adapted from ReF.(112), Macmillan”
• De novo Identification of DNA Modifications Enabled by Genome-Guided Nanopore Signal Processing (2017, background): “In particular, pioneering work 7, 8 demonstrated the capacity to identify DNA methylation marks via the comparison of native versus amplified DNA through supervised machine learning.”
• methylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles (2012, methodology): “Our data framework is also extensible to emerging methods in quantization of other base modifications, such as 5hmC [14], or sites discovered through single molecule sequencing [17,18].”
• Analysing and interpreting DNA methylation data (2012, background): “This limitation may eventually be overcome by single-molecule sequencing using nanopores or real-time monitoring of DNA polymerases 140,143 .”
• The Landscape of long non-coding RNA classification (2015, neutral): “Hopefully, existing [127] and emerging technologies [128] will enable progress in cataloguing RNA modifications.”
• Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine (2010, background): “A single-molecule, real-time sequencing technology has been applied to distinguish between cytosine, 5-mC and 5-hmC, but further improvements are necessary to affinity-enrich 5-hmC–containing DNA and to achieve base-resolution sequencin”
• The sequence of sequencers: The history of sequencing DNA (2016, methodology): “As sequencing occurs at the rate of the polymerase it produces kinetic data, allowing for detection of modi fi ed bases [84].”
• Next‐generation sequencing technologies for environmental DNA research (2012, background): “cence moiety with the nucleotide incorporation (Pushpendra 2008; Flusberg et al. 2010).”
• PacBio Sequencing and Its Applications (2015, methodology): “More importantly, it cannot discriminate between C, m(5)C, and 5hmC [67].”
• A window into third-generation sequencing. (2010, background): “Via the SMRT sequencing process, changes in the kinetics of incorporation associated with chemical modifications to bases, such as methylation, can be detected in the normal course of collecting sequence data (2).”
• The advantages of SMRT sequencing (2013, methodology): “Now a new technology, SMRT sequencing from Pacifi c Biosciences [1], has been developed that not only produces considerably longer and highly accurate DNA sequences from individual unamplifi ed molecules, but can also show where methylated bases occur [2] (and thereby provide functional information ”
• Mapping single molecule sequencing reads using basic local alignment with successive refinement (BLASR): application and theory (2012, background): “It has been shown that the additional information one may gain by observing the signal from single-molecule events in real time may indicate DNA modifications such as methylation [25,31].”