In this program, we will enable direct DNA methylation profiling during real-time DNA sequencing, without bisulfite conversion. We will accomplish this by tailoring Pacific Biosciences'high throughput Single-Molecule Real-Time (SMRT) DNA sequencing technology for the detection of altered DNA polymerase kinetics due to the presence of 5-methylcytosine (5mC) in the DNA template. In addition, we will develop five-base SMRT sequencing, in which the 5th base indicates the presence of 5mC in the original sample. Our proposed technologies offer several advantages over current methylation profiling techniques. For example, direct methylation sequencing without bisulfite conversion and DNA amplification will reduce the time and effort required for sample preparation. In addition, long reads provided by SMRT sequencing (several kilobases) will enable methylation analysis in highly-repetitive genomic regions. Pacific Biosciences is uniquely positioned to develop this technology because of our existing infrastructure of SMRT sequencing instrumentation and polymerase engineering resources. This program will create several new jobs within the next fiscal quarter, and, if successful, has the potential to create hundreds of new jobs as Pacific Biosciences delivers the technology to the broader research community.

Public Health Relevance

Epigenomics is emerging as an ever more important aspect in the biology of development and disease processes such as cancer. We are proposing to develop a technique for direct, single base-pair resolution methylation profiling during real-time DNA sequencing, without bisulfite conversion. The ability to directly determine genomic DNA methylation patterns will dramatically speed up and reduce the cost of projects aiming to illuminate its role in human health.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
High Impact Research and Research Infrastructure Programs (RC2)
Project #
5RC2HG005618-02
Application #
7944006
Study Section
Special Emphasis Panel (ZHG1-HGR-N (O2))
Program Officer
Schloss, Jeffery
Project Start
2009-09-30
Project End
2011-07-31
Budget Start
2010-08-01
Budget End
2011-07-31
Support Year
2
Fiscal Year
2010
Total Cost
$595,011
Indirect Cost
Name
Pacific Biosciences of California, Inc.
Department
Type
DUNS #
149130325
City
Menlo Park
State
CA
Country
United States
Zip Code
94025
Schadt, Eric E; Banerjee, Onureena; Fang, Gang et al. (2013) Modeling kinetic rate variation in third generation DNA sequencing data to detect putative modifications to DNA bases. Genome Res 23:129-41
Lluch-Senar, Maria; Luong, Khai; LlorĂ©ns-Rico, VerĂ³nica et al. (2013) Comprehensive methylome characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at single-base resolution. PLoS Genet 9:e1003191
Kozdon, Jennifer B; Melfi, Michael D; Luong, Khai et al. (2013) Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle. Proc Natl Acad Sci U S A 110:E4658-67
Clark, Tyson A; Murray, Iain A; Morgan, Richard D et al. (2012) Characterization of DNA methyltransferase specificities using single-molecule, real-time DNA sequencing. Nucleic Acids Res 40:e29
Korlach, Jonas; Turner, Stephen W (2012) Going beyond five bases in DNA sequencing. Curr Opin Struct Biol 22:251-61
Song, Chun-Xiao; Clark, Tyson A; Lu, Xing-Yu et al. (2011) Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine. Nat Methods 9:75-7
Flusberg, Benjamin A; Webster, Dale R; Lee, Jessica H et al. (2010) Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods 7:461-5