The Impact Of Next Generation Sequencer Biology Essay

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Recently, genome sequence has been revolutionalized the field of virology, bacteriology and most importantly infectious diseases outbreaks investigation. The invention of high through output sequencing, computational assembly of sequences and functional inferences give access for gain large amount of information along with genome analysis, which provide an irreplaceable research tools for the different prospective of clinical microbiology. The first genome sequenced was done in 1995, of a free living organism name Homophiles influenza. Now, there are number of bacterial and eukaryotes genomes have been sequenced. Moreover, genome of different bacterial strains from each of 55 species also has been sequenced for the investigation of diseases outbreaks (Pierre-Edouard Fournie et al, 2007). The sequenced genome includes all important human bacterial pathogen, which cover all phylogenetic domains of bacteria. Now, bacterial genome can be easily sequenced in a week or a day instead of month even in a cheapest cost due the microbial genomic advancement(Mark J Pallen 2010). The research for the bacterial genomic sequence has been led to unique advancement in pathogen diagnosis, genotyping virulence detection and antibiotic resistance, which providing significant role to prevent and control the burdon of infectious diseases outbreaks.

Genome sequencing

The amount of novel microbial genomic information that is being generated on a daily basis is so vast that multidisciplinary approaches that integrate bioinformatics, statistics and mathematical methods are required to assess it effectively. This information is inspiring a new understanding of microorganisms that appreciates the scale of microbial diversity and acknowledges that the microbial gene pool is considerably larger than expected(Duccio Medini 2008). Whole genome sequencing (WGS) promises to be transformative for the practice of clinical microbiology, and the rapidly falling cost and turnaround time mean that this will become a viable technology in diagnostic and reference laboratories in the near future. WGS in routine diagnostic microbiology requires consideration of the processes used in current laboratory practice. In very broad terms this is made up of four main stages, starting with detection (or not) of a pathogen in a sample. If a clinically relevant pathogen is detected, then this may be further tested for identification, drug susceptibility, and epidemiological typing. This simplified description best fits bacteria and fungi and is less accurate for viruses. Detecting the presence of a virus and species identification are often performed by the initial test (for example, a species-specific PCR), and susceptibility testing and typing are not performed for many of the viruses detected in the routine laboratory (Claudio U. Koser et al 2012).

History of genome sequencing

Knowledge of the sequence of deoxyribonucleic acid (DNA), the molecule that stores the genetic information of almost all known living organisms, has revolutionised biology and driven a massive acceleration in research and development. The most commonly used approach for DNA sequence determination has, until recently, been the chain termination methodology. This was published by Fredrick Sanger (University of Cambridge) and colleagues in 1977 and led to Sanger's second Nobel Prize for Chemistry in 1980. The Sanger sequencing methodology has remained conceptually unchanged for over 30 years, but in the decades following publication improvements were made in reaction expediency and speed (e.g. dye termination methodologies, increased read lengths), process parallelisation (e.g. capillary electrophoresis) and automation. Automated sequencing machines first became commercially available in 1987 (US company, Applied Biosystems) and the current mature technology (e.g. ABI3730xl) is considered to offer high quality, high-throughput DNA sequence generation.

Next generation sequencing

Next-generation sequencing (NGS) technologies have ushered in a new era of biodiversity surveillance, enabling high-throughput analysis of complex microbial communities via short amplicons, typically hypervariable domains of prokaryotic 16S rDNA. Given the scale of sequencing reactions possible in a single run of most NGS platforms, hundreds to thousands of samples may be multiplexed using short DNA sequence "barcodes", providing adequate sequencing depth in each sample to characterize the top 99.99% of the microbiota. This has facilitated comparative ecological analysis on a large scale and-with sensitivity well beyond that of first-generation profiling technologies-provides relatively quantitative comparisons of microbial communities across ecosystems at depths previously unattainable.

In the past few years, unprecedented efforts have therefore been made to develop and deploy new sequencing strategies (Hall, N. 2007; Schuster and S. C. 2008). Three new methods are currently being commercialized that are based on amplification strategies as alternatives to the standard cloning system and use different methods of sequence detection: high-throughput pyrosequencing on beads30, sequencing by ligation, also on beads31 and sequencing by synthesis on DNA that is amplified directly on a glass substrate (Bennett, S. Solexa 2004; Bennett et al 2005) . Most of these new methods use PCR to amplify individual DNA molecules that are immobilized on solid surfaces, either beads or a glass surface, such that all the identical molecules present can be sequenced in parallel using various sequencing. Pyrosequencing uses the light that is emitted by the release of pyrophosphates that are attached to the incorporated bases; the sequencing-bysynthesis method uses fluorescent reversible dye terminators (which is conceptually similar to Sanger sequencing, but with a single base extension); and the sequencingby-ligation method uses the ligation of a pool of partially random oligonucleotides that are labelled according to the discriminating base or bases. Although extremely fast, these methods still only give short sequence reads, making subsequent sequence assembly problematic approaches(Duccio Medini et al 2008).

Impact of sequencing on infectious diseases

Pathogenicity, like other microbial traits, is a reflection of a particular specialization, which could be as simple as living in the ocean or in soil. There are similar genetic processes at play and common themes of survival. The application of genetic and molecular methods to the study of microorganisms that cause infection and disease has been propelled by the fact that we now possess at least one complete genomic sequence of virtually all bacterial species that cause human infectious disease. Yet, one cannot simply examine the complete genome of a bacterium on a computer screen and deduce from its sequence whether or not it is a pathogen. There is no 'core' set of genes that defines pathogenicity for a particular bacterial species, and it is still necessary to do experiments to understand bacterial pathogenesis, even in the post-genomic era. Moreover, the distinction between what is a pathogen, as compared with a commensal species, is blurred. Many of the recognized pathogens, such as the pneumococcus and the meningococcus, are far more likely to be carried asymptomatically than to

cause clinical disease. What we have considered to be virulence factors are, in reality, colonization factors in these bacteria.Bacterial pathogenesis is not necessarily reflected in death and disease (except, perhaps, when writing a grant application). To understand what constitutes a pathogen and pathogenicity, one must take into account that the evolution of pathogenicity paralleled the evolution of the host. The initial adaptation of bacteria to humans took place at a time when human life expectancy was much shorter than it is today. The extension of human life, paradoxically through the control of microorganisms and infectious diseases, now places an extraordinary selective pressure on microorganisms to evolve their specialization to survive in humans. This entails more than antibiotic resistance. We have observed, for example, the evolution of staphylococcal pathogenicity and transmissibility in the past few decades in response to the selective pressure of antibiotics, changes in human demographics and the appearance of new microbial opportunities as a result of nongenetic

host changes. The host-pathogen dynamic in humans is still a 'work in progress', as reflected by the deluge of emerging infectious diseases(Duccio Medini et al 2008).There are number of genome sequencing have been done for many infectious disease outbreaks including Mycobacterium tuberculosis outbreaks, Escherichia coli outbreak, meticillin-resistant Staphylococcus aureus .

Conclusion and future research

Whole-genome sequencing could make an important contribution to infection-control investigation and practice. Implementation of routine whole-genome sequencing for different infectious diseases outbreaks would need the development of a central database for comparison of sequence data with previous local, national, and global isolates, and development of a system for automated interpretation and linking of genome sequence data. Nevertheless, such an approach holds great promise for rapid, accurate, and comprehensive identification of bacterial transmission pathways in hospital and community settings, with concomitant reductions in infections, morbidity and with the costs of DNA sequencing rapidly falling and its use becoming more and more widespread, genomics will revolutionize our understanding of the transmission of bacterial pathogens.

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