Third-generation sequencing

All NGS platforms are hampered with two major limitations: (i) generation of short read length that requires bioinfonnatic tools for assembly (ii) requires PCR amplification of target gene that itself may introduce biases. In Third-Generation Sequencing (TGS), instead of sequencing of PCR amplified products, single molecule can be sequenced and therefore termed as Single Molecule Sequencing (SMS). SMS approach supersedes SBS and SBL by (i) observing single molecule of DNA polymerase to synthesize single molecule of DNA template that overcomes PCR biases, (ii) Nanopore sequencing technology facilitating the detection of individual bases when it passes through the nanopore that surmounts wash-and-scan system and finally (iii) sophisticated imaging system with advanced microscopy techniques that can directly see the integration of single nucleotide by solitary DNA polymerase defeating the need of fluorophore/terminators, scanning and imaging. TGS employs Single Molecule Real-Time (SMRT) approach to sequence DNA, which hires Zero- Mode Waveguide (ZMW) technology offering single base detection from a pile of bases carrying light signals. ZMW—a pore in 100 mu metal film, selectively detects the visible light of ~ 30 nm than 600 mn (actual wavelength of visible light) by simple diffusion of visible light through its pore that gets decayed to 30 nm. DNA polymerase attached to the metal film through biotin-streptavidin interaction aids the incorporation of correct nucleotide, thereby facilitating sequencing (Schadt et al. 2010). Beyond bacterial diversity identification, the raise of TGS gives an unprecedented picture of complex biological analysis with more accuracy and with great predictive power. Current technologies in TGS include few commercialized platforms such as SMAT by Pacific Biosciences and Oxford Nanopore’s MinlON.

Nanopore sequencing

Currently, Oxfords Nanopore’s MinlON anticipates the era of third generation sequencing technology. The core concept of sequencing through Nanopore’s MinlON is based on disruption in voltage of a tiny biopore (nano-scaled pore that facilitates ion exchange in biological membranes) dining particle movement. Single stranded DNA is electrophoretically passed through the nanopore of a-haemolysin (aHL) protein in a controlled maimer using motor protein. aHL (isolated from Staphylococcus aureus), a 33kD protein, self-assembles to form lieptameric transmembrane channel for a molecule to pass through. As the single stranded DNA is translocated across the membrane pore, changes in current (current shifts) are recorded in real-time and graphically represented as squiggle plot. Alternative to aHL, the MspA pore and the CsgG pore are currently used in Oxfords Nanopore technology. Sensing region of a pore determines the accuracy of base determination. The MspA pore and the CsgG pore have shorter sensing region that senses even smaller number of nucleotides, leading to more precise base detection. The advantage offered by Oxfords Nauopore technology is its USB size, palm portable DNA sequencer with higher read length and shorter run tune (> 5 kbp read length with speed of 1 bp/ns) to sequence. Moreover, the non-usage of PCR reduces the quantity of starting material and PCR- based biases. Oxfords Nanopore technology also sequences both strands of DNA that offers less error rate.

Concluding remarks and future prospective

Planet Earth harbors a large spectrum of microorganisms. For nearly 250 years, microbiologists have been straggling to catalog and understand then interaction as well as functionality in natural environment as the efforts were majorly hampered by technical development. Advances in DNA sequencing technologies such as high- throughput next-generation sequencing and progress in computational methods have made a tremendous upsurge in biological research.

As the advancements in sequencing platforms progress, the affordability and feasibility of examining total metagenome of any habitat are becoming reality. Diversity and functional analysis of habitat’s microbiome are attaining its peak of precision allowing best resolution of taxa. NGS has allowed to understand the intricate networks between the human society, environment and microbial communities. Today, we are in a position to define an environment from its microbial footprints. From the human body to the surrounding environment, the planet is filled with microbes and it is clear that personal microbiome sheds everywhere we move. Hence, one is curious to ask whether it is possible to design and/or rebuilt our own microbiome to keep ourselves healthy. Or can we make all the hunraus as gnotobiotic (germ- free)? Can we trace the lifetime of any individual through sequencing technologies? Answer to all these questions is possible through developing sequencing platforms. Tire upcoming sequencing generation developments will shed more light on the unculturable microorganisms and its biochemical advantages.

Despite the existing advancements in NGS technologies, the method progression and bioinfonnatic tools to analyze data are still in then infancy. Although the advanced new generation sequencing methods have stepped into the field of microbial diversity, Ilhtmina is highly preferred over other NGS platforms because of its high sequencing efficiency with least cost and well established data analysis pipelines. Moreover, it is certain that new sequencing technologies will evolve rapidly, resulting in powerful and cheaper platforms. It is in the hands of researchers to have informed choices on NGS platforms to fulfill their research objectives.

In this chapter, we discussed comprehensive overview of generation of sequencing technologies commenced by DNA sequencing history' followed by comparison of three sequencing generations, various platforms of NGS and application in microbial diversity analyses. Though there are associated challenges hr NGS such as intrinsic sequencing errors, PCR biases, counting the artifacts, difficulties in storing and analyzing the data generated, understanding and appreciation with exploitation of current methodologies will provide new insights to various fields of biology.

Acknowledgements

MR gratefully thank the financial support provided by RUSA Phase 2.0 [F.24- 51/2014-U, Policy (TN Multi-Gen), Dept, of Edn, Gol] in the form of Pli.D. Fellowship. Authors sincerely acknowledge the computational and bioinfomiatics facility provided by the Bioinfomiatics Infrastructure Facility (funded by DBT, GOI; File No. BT/BI/25/012/2012, BIF). The authors also thankfully acknowledge DST- FIST (Grant No. SR/FST/LSI-639/2015 (C)), UGC-SAP (Grant No. F.5-1/2018/ DRS-II (SAP-II)) and DST-PURSE (Grant No. SR/PURSE Phase 2/38 (G)) for providing instrumentation facilities.

 
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