The SM(A)RT Sequencing: Leading the way for latest sequencing innovation.

Decoding DNA constituted within the human genome has been widely anticipated for the contribution it will make toward understanding human evolution, the causation of disease, and the interplay between the environment and heredity in defining the human condition. Fast progress in DNA sequencing technology has made a substantial leap towards understanding human conditions at genomic frontier with the reduction in costs and a substantial increase in throughput and accuracy. Along with humans, greater number of organisms being sequenced, a flood of genetic data is inundating the world every day primarily due to a revolution in sequencing technology. Evolution in this technology is dynamic and leaps towards diverse technological aspects. Multiple platforms for massive parallel sequencing indicates the need of massive analysis with greater analysis and accuracy with immediate and applicability.
The third generation sequencing is a step closer to have faster, cheaper genome sequencing. Single-molecule real-time (SMRT) sequencing, developed by Pacific BioSciences (PacBio), offers an alternative approach to overcome many of the limitations posed by second generation methods. PacBiosequencing captures sequence information during the replication process of the target DNA molecule. The template, called a SMRTbell, is a closed, single-stranded circular DNA that is created by ligating hairpin adaptors to both ends of a target double-stranded DNA (dsDNA) molecule.20, A chip called SMRT chip is loaded with a sample with SMRTbell, it diffuses into a sequencing unit called Zero-mode Waveguide (ZMW) providing smallest available volume for light detection. Every ZMW consists of a single polymerase immobilized at the bottom binding to either of hairpin adaptors of SMRTbell and commences the replication. The SMRT cell is added with four fluorescent labelled nucleotides each generating distinct emission spectrum. Every time the nucleotide base interacts with the polymerase a light pulse is produced identifying the base. The replication processes in all ZMWs of aSMRT cell are recorded by a ‘‘movie” of light pulses, andthe pulses corresponding to each ZMW can be interpreted to be a sequence of bases (called a continuous long read, CLR).20 As the SMRTbell forms closed circle after the polymerase replicates one strand of the target dsDNA, it can continue incorporating bases of the adapter and then the other strand. If the lifecycle of the polymerase is long enough, both strands will have a chance to be sequenced multiple times (called ‘‘passes”) in a single CLR. In this scenario, the CLR can be split to multiple reads (called subreads) by recognizing and cutting out the adaptor sequences. The consensus sequence of multiple subreads in a single ZMW yields a circularconsensus sequence (CCS) read with higher accuracy. If atarget DNA is too long to be sequenced multiple times in aCLR, a CCS read cannot be generated, and only a single subreadis output instead.20 Because PacBio sequencing takes placein real time, kinetic variation interpreted from the light-pulsemovie can be analysed to detect base modifications, such as methylation.
The long read length advantage of Pac Biosequencing also make the technology adept at identification and quantification of isoforms, including novel isoforms, particularly when used in conjunction with SGS. The latest platform, PacBio RS II, typically produces sequencing movies 0.5–4 h in length, While the original PacBio RS system with the first generation of chemistry (C1 chemistry) generated mean read lengths around 1500 bp, the PacBio RS II system with the current C4 chemistry boasts average read lengths over 10 kb, with an N50 of more than 20 kb (that is, over half of alldata are in reads longer than 20 kb) and maximum read lengths over 60 kb

References

-Travers K, Chin CS, Rank D, Eid J, Turner S. A flexible andefficient template format for circular consensus sequencing andSNP detection. Nucleic Acids Res 2010;38:e159.
-Pacific Biosciences. Media Kit, <http://www.pacb.com/company/newsevents/media-resources/page/3/> (May 19, 2015, date last accessed).