Direct detection of DNA methylation during single-molecule, real-time sequencing
Benjamin Flusberg,Dale R. Webster,Jessica Lee,Kevin Travers,Eric Olivares,Tyson A. Clark,Jonas Korlach,Stephen Turner +7 more
TLDR
The direct detection of DNA methylation, without bisulfite conversion, through single-molecule, real-time (SMRT) sequencing is described and is amenable to long read lengths and will likely enable mapping of methylation patterns in even highly repetitive genomic regions.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.read more
Citations
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STAR: ultrafast universal RNA-seq aligner
Alexander Dobin,Carrie A. Davis,Felix Schlesinger,Jorg Drenkow,Chris Zaleski,Sonali Jha,Philippe Batut,Mark Chaisson,Thomas R. Gingeras +8 more
TL;DR: The Spliced Transcripts Alignment to a Reference (STAR) software based on a previously undescribed RNA-seq alignment algorithm that uses sequential maximum mappable seed search in uncompressed suffix arrays followed by seed clustering and stitching procedure outperforms other aligners by a factor of >50 in mapping speed.
Journal ArticleDOI
Coming of age: ten years of next-generation sequencing technologies
TL;DR: These and other strategies are providing researchers and clinicians a variety of tools to probe genomes in greater depth, leading to an enhanced understanding of how genome sequence variants underlie phenotype and disease.
Journal ArticleDOI
A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers.
Michael A. Quail,Miriam Smith,Paul Coupland,Thomas D. Otto,Simon R. Harris,Thomas R. Connor,Anna Bertoni,Harold Swerdlow,Yong Gu +8 more
TL;DR: All three fast turnaround sequencers evaluated here were able to generate usable sequence, however there are key differences between the quality of that data and the applications it will support.
Journal ArticleDOI
PacBio Sequencing and Its Applications.
Anthony Rhoads,Kin Fai Au +1 more
TL;DR: Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research.
Journal ArticleDOI
Comparison of Next-Generation Sequencing Systems
TL;DR: Technologies of next-generation sequencing systems are reviewed, and first-hand data from extensive experience is summarized and analyzed to discuss the advantages and specifics associated with each sequencing system.
References
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Principal Component Analysis
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Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals
Rudolf Jaenisch,Adrian Bird +1 more
TL;DR: Advances in the understanding of the mechanism and role of DNA methylation in biological processes are reviewed, showing that epigenetic mechanisms seem to allow an organism to respond to the environment through changes in gene expression.
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Peter A. Jones,Stephen B. Baylin +1 more
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Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1
Mamta Tahiliani,Kian Peng Koh,Yinghua Shen,William A. Pastor,Hozefa S. Bandukwala,Yevgeny Brudno,Suneet Agarwal,Lakshminarayan M. Iyer,David R. Liu,L. Aravind,Anjana Rao +10 more
TL;DR: It is shown here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro.
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