While the increasing usage and new modification in next generation se­quencing, the third generation sequencing is coming out with new insight in the sequencing. Third-generation sequencing has two main character­istics. First, PCR is not needed before sequencing, which shortens DNA preparation time for sequencing. Second, the signal is captured in real time, which means that the signal, no matter whether it is fluorescent (Pacbio) or electric current (Nanopore), is monitored during the enzymatic reaction of adding nucleotide in the complementary strand.

Single-molecule real-time (SMRT) is the third-generation sequencing method developed by Pacific Bioscience (Menlo Park, CA, USA), which made use of modified enzyme and direct observation of the enzymatic reaction in real time. SMRT cell consists of millions of zero-mode wave­guides (ZMWs), embedded with only one set of enzymes and DNA tem­plate that can be detected during the whole process. During the reaction, the enzyme will incorporate the nucleotide into the complementary strand and cleave off the fluorescent dye previously linked with the nucleotide. Then the camera inside the machine will capture signal in a movie format in real-time observation [19]. This will give out not only the fluorescent signal but also the signal difference along time, which may be useful for the prediction of structural variance in the sequence, especially useful in epigenetic studies such as DNA methlyation [22].

Comparing to second generation, PacBio RS (the first sequencer launched by PacBio) has several advantages. First the sample preparation is very fast; it takes 4 to 6 hours instead of days. Also it does not need PCR step in the preparation step, which reduces bias and error caused by PCR. Second, the turnover rate is quite fast; runs are finished within a day. Third, the average read length is 1300 bp, which is longer than that of any second-generation sequencing technology. Although the throughput of the PacBioRS is lower than second-generation sequencer, this technology is quite useful for clinical laboratories, especially for microbiology research. A paper has been published using PacBio RS on the Haitian cholera out­break [19].

We have run a de novo assembly of DNA fosmid sample from Oyster with PacBio RS in standard sequencing mode (using LPR chemistry and SMRTcells instead of the new version FCR chemistry and SMRTcells). An SMRT belt template with mean insert size of 7500 kb is made and run in one SMRT cell and a 120-minute movie is taken. After Post-QC fil­ter, 22,373,400 bp reads in 6754 reads (average 2,566 bp) were sequenced with the average Read Score of 0.819. The Coverage is 324x with mean read score of 0.861 and high accuracy (~99.95). The result is exhibited in Figure 2.

Nanopore sequencing is another method of the third generation se­quencing. Nanopore is a tiny biopore with diameter in nanoscale [23], which can be found in protein channel embedded on lipid bilayer which facilitates ion exchange. Because of the biological role of nanopore, any particle movement can disrupt the voltage across the channel. The core concept of nanopore sequencing involves putting a thread of single-strand­ed DNA across a-haemolysin (aHL) pore. aHL, a 33 kD protein isolated from Staphylococcus aureus [20], undergoes self-assembly to form a hep — tameric transmembrane channel [23]. It can tolerate extraordinary voltage up to 100 mV with current 100 pA [20]. This unique property supports its role as building block of nanopore. In nanopore sequencing, an ionic flow is applied continuously. Current disruption is simply detected by standard electrophysiological technique. Readout is relied on the size difference between all deoxyribonucleoside monophosphate (dNMP). Thus, for giv­en dNMP, characteristic current modulation is shown for discrimination. Ionic current is resumed after trapped nucleotide entirely squeezing out.

Nanopore sequencing possesses a number of fruitful advantages over existing commercialized next-generation sequencing technologies. First­ly, it potentially reaches long read length >5 kbp with speed 1 bp/ns [19]. Moreover, detection of bases is fluorescent tag-free. Thirdly, except the use of exonuclease for holding up ssDNA and nucleotide cleavage [24], involvement of enzyme is remarkably obviated in nanopore sequencing [22]. This implies that nanopore sequencing is less sensitive to tempera­ture throughout the sequencing reaction and reliable outcome can be main­tained. Fourthly, instead of sequencing DNA during polymerization, single DNA strands are sequenced through nanopore by means of DNA strand depolymerization. Hence, hand-on time for sample preparation such as cloning and amplification steps can be shortened significantly.