New Generation Sequencing Technologies: Population Genetics

New Generation Sequencing Technologies: Population Genetics New generation sequencing technologies have the potential to rapidly accelerate population genetics research, allowing scientists to comprehensively understand complex evolutionary histories, as well as functional and ecological biodiversity (Shokralla, et al., 2012; Shendure Hanlee, 2008). Prior to 1977, sequence production involved the handling of toxic chemicals and radio-active isotopes, restricting sequencing to persons of high expertise and speciality (Hunkapiller, 1991; Swerdlow, et al., 1990; Sanger, et al., 1977). In 1977, Fred Sanger and Alan R. Coulson published two methodological papers describing a new form of DNA sequencing technology, which would lead to the method (capillary-based, semi-automated Sanger biochemistry) used almost exclusively in the field, for the next 30 years (Shendure Hanlee, 2008). Sanger sequencing transformed biology. It became a tool for deciphering complete genes and, later, entire genomes. Due to the unprecedented extent at which Sanger techn ology grew, factory-like enterprises, called sequencing centres, were established, housing hundreds of DNA sequencing instruments, operated by cohorts of personnel (Schuster, 2008; Hunkapilla, et al., 1991). Despite the dominance of Sanger sequencing in laboratories, for a number of decades, the technology had and continues to be hampered by inherent limitations in throughput, scalability, speed and resolution (Shendure Hanlee, 2008). To overcome these barriers, an entirely new technology was required, one that democratised the field, putting the technology of comprehensive genetic analysis into the hands of individual investigators, not only major genome research centres (Shendure Hanlee, 2008). The need for new technologies was pushed for by the facilitators of the Human Genome Project (HGP) (Ventor, et al., 2001). The excitement and successful completion of the HGP, by two competing research bodies, lead to collective hunger for more advanced, economical sequencing technologies. Next-generation sequencing (NGS), also known as massively parallel sequencing, was such a technology and has ignited a revolution in genomic science, similar to that seen when Sanger technology was presented in 1977, honing in on the era of ‘post-genomic’ research (Schuster, 2008). The revolutionary nature of NGS technologies first became apparent in 2005, in two separate publications, 454 Life Sciences (Marguiles, et al., 2005) and the Multiplex Polony Sequencing Protocol (Shendure, et al., 2005). The methodology of both research groups resulted in vast reductions in the necessary reaction volume, while dramatically extending the number of sequencing reactions (Schuster, 2008). Despite such advances, in sequencing technology, NGS had a slow uptake in the scientific community, with a number of scientists having reservations. According to Schuster (2008), scientists accustomed to Sanger sequencing, as well as the initial scepticism echoed by funding bodies, resulted in a fear that large financial investments into Sanger-sequencing technologies would not produce returns, due to the technologies becoming obsolete. Other concerns were also raised, regarding the sequencing fidelity, read length, infrastructure cost and the handling of the large data volumes produced by NGS (Zhang, et al., 2011). It was the process of combining ongoing Sanger sequencing projects with NGS technologies that promoted its acceptance, into the scientific community. Once the enormous potential of the technology had been realised, along with new and upcoming biology projects that required sequencing outside of what the current Sanger technology could feasibly produce, the concerns raised by NGS’s early sceptics started to be overlooked. A combination of both first and second generation technologies are now used in sequencing facilities and projects around the world, the implications of which, for the fields of evolutionary biology and population genetics is vast. Researchers now have the ability to observe small changes in ecological community structure that may occur following anthropogenic or natural environmental fluctuations (Hajibabaei, et al., 2011; Leininger, et al., 2006; Hunkapiller, et al., 1991). Such implications of NGS technologies has led to the generation of whole-genome sequence data, for thousands of individuals (Akey Shriver, 2011; Harismendy, et al., 2009). The availability of such data is leading to a better understanding of evolutionary processes, such as descriptions of sex-biased dispersal and mutation rate biases (e.g., Wilson Sayres, M. A., et al., 2011). Furthermore, the ability to sequence the genomes of species, that have been long extinct, is no longer nonsensical, provided the samples from which DNA is to be extracted is still viable (Green, et al., 2010; Reich, et al., 2010). The hope that such projects may help population geneticists better understand the process of extinction, whether anthropomorphically or n aturally induced, may help those endangered species whose current possibility of extinction, in the near future, is high (Akey Shriver, 2011; Miller, et al., 2011). However, despite such ambitious aspirations of population geneticists, one large area of research that remains surprisingly unanswered, within the literature, is the definition of a population or ‘the population concept’ (Waples Gaggiotti, 2006). Given the importance of such a concept, one might expect to find a commonly used definition, one that is applicable to wild species, to determine how many populations exist within a delineated geographic area and the relationships amongst them (Waples Gaggiotti, 2006). However, one does not exist, rather there is evidence that what makes a ‘population’ is based on the research question. NGS technologies are providing population geneticists with the opportunity to flesh out a detailed definition of a population, on the molecular level. For example, Waples Gaggiotti (2006) ask â€Å"How different must molecular units be before individuals can be considered a part of separate populations?† Different criteria can be established and assigned to individuals, in order to determine the answer. The interplay of different evolutionary forces (selection, migration, drift) will favour different species, with different forces being more obvious, at the molecular level, than others. The ability to pose a research question, pertaining to the individuals, within a particular habitat, is now possible due to the ability to sequence numerous samples with NGS technologies. The implications, in population genetics, for a new generation of sequencing technologies, are a greater focus on testing expectations. Such expectations, simultaneously, result in excitement and daunt to those undertaking evolutionary and population genetic research, at present. Excitement exists because fundamental questions, pertaining to the patterns of genetic variation, within and between species, can now be analysed, with new generation sequencing technologies, such as NGS. Although NGS technology may still be in its infancy, the powerful possibility of analysing massive data sets is within reach of the individual and large-scale sequencing facilities alike, at a highly reduced cost. However, the methodological tools and theoretical models needed to interpret such large data sets are equally daunting to both new, and experienced, evolutionary and population geneticists. Despite such present and future challenges, population genetics research is looking promising, thanks to adv ances in NGS adoption and computation. References Akey, J. M. Shriver, M. D. (2011). A grand challenge in evolutionary population genetics: new paradigms for exploring the past and charting the future in the post-genomic era. Frontiers in Genetics 2, 1-2. Green R. E., Krause J., Briggs A. W., Maricic T., Stenzel U., Kircher M., Patterson N., †¦ Pà ¤Ãƒ ¤bo S. (2010). A draft sequence of the Neanderthal genome. Science 328, 710–722. Hajibabaei, M., Shokralla, S., Zhou, X., Singer, G. A. C. Baird, D. J. (2011). Environmental barcoding: a next-generation sequencing approach for biomonitoring applications using river benthos. PLoS ONE 6, e17497. Harismendy, O., Ng, P. C., Strausberg, R. 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Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 1728-1732. Shokralla, S., Spall, J. L., Gibson, J. F. Hajibabaei, M. (2012). Next generation sequencing technologies for environmental DNA research. Molecular Ecology 21, 1794-1805. Swerdlow, H., Wu, S. L., Harke, H. Dovichi, N. J. (1990). Capillary gel electrophoresis for DNA sequencing. Laser-induced fluorescence detection with the sheath flow cuvette. Journal of Chromatography 516, 61-67. Ventor, J. C., Adams, M. D., Myers, E.W., Li, P. W., Mural, R. J., Sutton, G. G., Amanatides, P., †¦, Zhu, X. (2001). The sequence of the human genome. Science 291, 1304-1351. Waples, R. S. Gaggiotti, O. (2006). INVITED REVIEW: What is a population? An empirical evaluation of some genetic methods for identifying the number of gene pools and their degree of connectivity. Molecular Ecology 15 (6), 1419-1439. Wilson Sayres M. A., Venditti C., Pagel M., Makova K. D. (2011). Do variations in substitution rates and male mutation bias correlate with life history traits? A study of 32 mammalian genomes. Evolution 65 (10), 2800-2815. Zhang, J., Chiodini, R., Badr, A. Zhang, G. (2011). The impact of next-generation sequencing on genomics. Journal of Genetics and Genomics 38, 95-109. Influences of Greek Culture: History of the Spartans Influences of Greek Culture: History of the Spartans The Spartans In the summer of 480 B.C a battle took place that would forever change the history of the Greeks and that would eventually influence the way in which the Western world looked at war. The Spartans took their stand against the massive army of Persians in a three day battle which resulted in the Persians taking the win but may have very well led to the Greeks winning the Greco-Persian War. A culmination of strong tactical skill and bravery contributed to the Spartans making a stand much longer and stronger than anybody could have ever predicted. The Persian king Xerxes led his massive army through the narrow mountain pass known as Thermopylae expecting no considerable fight on the part of the Spartans. The Persians bid to conquer Greece was significantly halted because of the Spartan resistance, which was led by Leonidas,followed by a small army of Spartans, amounting to no more than 300. But no matter the size of Spartas fleet, Sparta if not the strongest, was one of the strongest mili tary powers in ancient Greece. And despite them being vastly outnumbered by the Persians at Thermopylae, they did indeed prove their military strength and sophistication which resulted in their near defeat of the Persian army. Greek culture was and still is up to today a heavy influence on the modern cultures of the Western world. It is because of this heavy influence of Greek culture in the western world, the knowledge of their history proves crucial to many aspects of our understanding of our own cultures. This heavy influence on the development of the western world could very easily be the reason that the Battle of Thermopylae and other battles surrounding it, have become of such importance and high level of study. The valiant stand of the Spartans at Thermopylae lead to the Greeks defeat of the Persians in the Greco-Persian war and enabled the further development of a culture from which the western world gains many of its principles and ideas. If the Spartans had not delayed the Persians at Thermopylae there may have been a very different ending to the Greco-Persian war. This being an important observation because the Greco-Persian war played such a crucial role in the history of Greece, a defeat could have resulted in a very different future for the western civilization. The culture of Greece was one that strived for perfection in every sense of the word, but there was a dark side to the culture that so many have grown to praise. This dark side can be seen in the Spartans treatment of the Helots, who were in essence a Greek culture in their own, the Messenia’s, but early on became enslaved by the power Spartans who were in desperate need to acquire more land to deal with the burden of overpopulation. The Spartans held true and easily demonstrated as what is seen as Greece’s inability to incorporate. The poor treatment of the Helots lead them to begin a 30 year revolt, in which the Spartans took twenty years to take control of the situation. Fear of more events like this, is what turned Sparta into the war state that it became. The attempt to suppress the Helots, by the Spartans, in many ways assisted the Spartans in their attempt to defeat the Persians. No longer willing to undergo a similar revolt, the Spartans devoted a considerable a mount of time and energy making certain to prevent all such events. It was because of these efforts that major components of Spartan culture, as we know it today, were all enforced. This can be easily seen in the devotion to physical perfection and warring techniques. And equally as important as their attempt to suppress the Helots was the contribution of the Helots in constructing their armor and warring tools. So despite the overwhelmingly poor treatment of the Helots, they played a crucial role in preparing the Spartans for the challenges to come and in the heat of battle. Even though they may have played an indirect role the affect that had on both the culture and the Battle of Thermopylae itself was indeed direct. The Greeks had a large influence on the development of western world in many respects. Whether it is an influence on science or art, to anyone who has studied Greece in the days of its glory the influences be easily pinpointed. These influences continue into the art of war. As previously stated, war was a constant in Greece so much so that it became imbedded in its very culture. Consequently the way in which war was conducted in ancient Greece has become a portrait of the way in which it should be conducted, and set a standard around the Western world for years to come. Despite war being one of the most immoral, barbaric, and most appalling of all human creations, the Greeks did the impossible, by successfully portraying war as something of beauty, patriotism, freedom and self-sacrifice. Therein lays a reason the significance of the Battle at Thermopylae. That one battle not only captured the spirit of the Greeks, more specifically the Spartans, in three days but became a turning poi nt of the art of war. But the Battle of Thermopylae more importantly defended the very future of the modern world. It was Spartan culture, which in many ways, influenced the Spartans ability to stand against the Persians as long as they did. To overlook the role of Spartan culture in relation to their stand at Thermopylae would be to overlook one of the most influential aspects of the battle. Spartan culture was one of great complexity having many intricate characteristics, which adapted to the situations that they held witness to. Spartans were people of extreme patriotic pride and military prowess, who sought perfection in every form. But equal to their patriotism was their oppressive tactics towards their captives. Spartans weren’t people who believed in the concept of freedom. The Spartans for several centuries, while in Laconia and Messenia, exercised a ruthless enslavement of other native Greeks, whose land they conquered. Sparta was a military aristocracy, who wasn’t a military state for the sake of being a military state. In many respects Sparta’s army, parallel to no t other, was created and maintained for the sole purpose of suppressing the Helots. In theory it was because of Sparta’s ‘inability to incorporate’ that lead to their standing army. Sparta’s military achievements are, no doubt, the most impressive of all their possible accomplishments. By the middle of the sixth century Sparta was already considered the strongest military force in Greece. Despite the brute strength the of the Spartan army, the Spartan were still worried that a revolt from their underclass (the Helots)would cripple their advancement as a society. â€Å"The Helot underclass were always threatening to rise up in significant numbers against their masters. So, at the beginning of each new civil year Sparta’s chief elected officials, the board of five ephors (overseers, supervisors), formally declared war on them. If any Helots did choose to rebel, they might then be killed with impunity†¦.† The awareness of a possible revolt kept the Spartans military forces extremely strong. This tension between the Spartans and Helots, strongly prepared the military forces for both the expected and unexpected, a beneficial trait which played to their advantage at the Battle of Thermopylae. Another trait that played to their advantage was the educational system of Sparta. The agoge was instated into Spartan culture to both develop the physical and mental maturity of all Spartan boys and was a requirement for all Spartan males.