We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph-based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.

We report here on the genome sequence of Pasteurella multocida Razi 0002 of avian origin, isolated in Iran. The genome has a size of 2,289,036 bp, a GC content of 40.3%, and is predicted to contain 2,079 coding sequences.

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-methyladenosine, 5-methylcytosine, and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of the primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We utilize these kinetic signatures to identify adenosine methylation in genomic samples and show 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 within even highly repetitive genomic regions.

Optical approaches for observing the dynamics of single molecules have required pico- to nanomolar concentrations of fluorophore in order to isolate individual molecules. However, many biologically relevant processes occur at micromolar ligand concentrations, necessitating a reduction in the conventional observation volume by three orders of magnitude. We show that arrays of zero-mode waveguides consisting of subwavelength holes in a metal film provide a simple and highly parallel means for studying single-molecule dynamics at micromolar concentrations with microsecond temporal resolution. We present observations of DNA polymerase activity as an example of the effectiveness of zero-mode waveguides for performing single-molecule experiments at high concentrations.

We studied the passage of DNA molecules, driven by an electric field, through a microfabricated channel with 90 nm size constrictions. DNA molecules were entropically trapped at the constriction and escaped with a characteristic lifetime. Counterintuitively, longer DNA were found to escape entropic traps faster than shorter ones. DNA molecules overcome the entropic barrier by stretching their monomers into the constriction, which results in the fact that the energy barrier for DNA escape is independent of the chain length.PACS numbers: 87.14. Gg, 83.10.Nn Recently, the concept of entropic trapping was introduced as a new dynamic regime in gel electrophoresis [1]. A long polymer molecule can be trapped entropically in a random restrictive environment such as a gel, and this effect becomes important when the dimension of pores in a retarding matrix is comparable to the radius of gyration (R 0 ) of the polymer. So far, entropic trapping has been demonstrated by computer simulation [2] and experiment in gel [3]. However, the lack of information on the structure of gel has hindered researchers from obtaining detailed microscopic understanding of the effect. Volkmuth and Austin [4] suggested the use of an artificial structure as a substitute for gel in electrophoresis. Aside from the application to polymer separation [5,6], microfabricated structures provide an ideal environment for studying these polymer dynamics problems, because one can easily control the dimension of obstacles or retarding matrix.The object of this Letter is to characterize the motion of long DNA polymer in an artificial channel with entropic traps. As a model pore-constriction system, we designed a channel with regions of two different depths. Figure 1(a) shows a schematic diagram of the device. The thick regions of the channel are as deep as 1 mm, comparable to the R 0 of the double stranded DNA molecules we used in this experiment. However the depth of the thin region is 90 nm, which is much smaller than R 0 . These thin and thick regions alternate along the channel, and DNA molecules in thick regions are entropically hindered from entering thin regions. Therefore at low electric fields DNA molecules are trapped at the entrance of the thin region and are unable to overcome the trapping barrier. We designed four different channels with different spatial periods of structure (4, 10, 20, and 40 mm). One period is divided by equal lengths of a thick and a thin region, which ensures that the fluid (electrical) resistance of the channel, as well as E s and E l (the electric field in the thin and thick region, respectively), are the same in all four channels. DNA molecules are free to relax while they are traveling in thick regions. By changing the period we can vary the time for DNA to relax before it meets another constriction.The channel was fabricated by standard photolithography techniques on Si substrate, and the front surface was anodically bonded to a Pyrex coverslip. The bonded channels were filled with a buffer solution containing DNA mole...