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If you glance around, you’ll see different species of animals that have different shapes and structures. With more than one million species of animal have been identified to date the need to classify them is all the more crucial. The classification aids in assigning a logical position to new species. 4.1 The premise of CLASSIFICATION Although there may be the differences in structure and shape of species, there are some basic characteristics shared by all species in terms of cell arrangement as well as body symmetry, the character of coelom, and patterns of digestive, circulatory , or reproductive systems. These characteristics are the basis of classification of animals and a few of them are described here. 4.1.1 The levels of organisation Although the majority of animals in Animalia can be multicellular each are not able to display the same patterns of organization of cells.
For instance in sponges, the cells are laid out in loose aggregates of cells, i.e. they show the cellular level of organization. There is a certain division of work (activities) are observed among cells. In coelenterates arrangements of the cells are more intricate. In coelenterates, cells performing similar functions are organized into tissues, which it is referred to as tissue level of organization. Another level of organisation i.e. the organ level, is demonstrated by the members of Platyhelminthes and the other higher phylas where tissues are joined to form organs with a specific function. In mammals like Annelids Arthropods, Molluscs, to a central cavity, called a spongocoel and then out through the orifice. Best NEET Coaching in Silchar
This water-transport pathway assists in food collection as well as respiratory exchange and elimination of waste. Collar cells or Choanocytes are found in the spongocoel as well as the canals. Digestion happens intracellularly. Your body’s skeleton is that is made from spongin or spicules fibres. Sexual sexes aren’t distinct (hermaphrodite), i.e. eggs and sperms can be produced by the same. Sponges reproduce sexually through fragmentation, and sexually through the creation of gametes. The process of reproduction is internal. Development is indirect. It has the larval stage that differs morphologically in morphology from adult. The Echinodermata has bilateral or radial co-symmetry, dependent on the stage. The figure 4.4 A broad classification for Kingdom Animalia with common basic characteristics.
Some examples: Sycon (Scypha), Spongilla (Fresh water sponge) and Euspongia (Bath sponge). 4.2.2 Phylum 4.2.2 Phylum Coelenterata (Cnidaria) The species is aquatic, usually marine as well as free-swimming or sessile, animals that are radially symmetrical (Figure 4.6). The name “cnidaria” comes of the 2022-23 ANIMALANIMAL KINGDOM KINGDOM 51 51 4.2.3 Ctenophora Ctenophores. Generally referred to as sea walnuts, or comb jellies, are marine-specific diloblastic, radially symmetrical organisms that have a tissue-level of organization. The body has eight rows of ciliated external plates for combs that aid in the process of locomotion (Figure 4.8). Digestion is both intracellular and intracellular. Bioluminescence (the ability of a living creature to release light) is clearly identified in Ctenophores. The sexes do not have separate identities. Reproduction occurs only through sexual methods. Best NEET Coaching in Silchar
Fertilisation occurs externally and is accompanied by an indirect process of development. Example: Pleurobrachia and Ctenoplana. 4.2.4 The phylum of Platyhelminthes have a dorso-ventrally flattened body and thus they are known as flatworms (Figure 4.9). They are mostly endoparasites that are found in mammals, including humans. Flatworms are bilaterallysymmetrical, triploblastic, and acoelomate species with organ-level organisation. Hooks and suckers can be found in the parasitic form. They may take in nutrients from the host directly by absorbing them through their body’s surface. Flame cells, which are special cells, aid in osmoregulation as well as excretion. The sexes do not have distinct. Fertilisation is internal while the development of the larvae goes through many stages. Certain species like Planaria have a high capacity for regeneration.
Examples: Taenia (Tapeworm), Fasciola (Liver fluke). Figure 4.8 Illustration of Ctenophora (Pleurobrachia) (a) (b) Figure 4.9 Examples of Platyhelminthes : (a) Tape worm (b) Liver fluke 2022-23 Biology 4.2.5 Aschelminthes, a phylum Aschelminthes’ body is circular in cross-sections, which is why they are called circular worms (Figure 4.10). They can be free-living or terrestrial, as well as aquatic. They can also be parasitic to animals and plants. Roundworms are organ-system-based in their body structure. These are bilaterally symmetrical triploblastic and pseudocoelomate creatures. The alimentary canal is fully developed with a muscular pharynx that is well-developed. The excretory tube is used to remove bodily waste from the cavity via the excretory pore. Both genders are distinct (dioecious), i.e. males and females have distinct. The length of females is usually greater than males.
Fertilisation happens internally and growth can be direct (the youngsters resemble adults) as well as indirect. Examples: Ascaris (Roundworm), Wuchereria (Filaria worm), Ancylostoma (Hookworm). 4.2.6 Phylum The phylum is Annelida They can have water-based (marine or fresh water) or terrestrial, they are free-living and often parasitic. They show organ-system levels of body structure and bilateral symmetry. They are metamerically segmented and coelomate creatures. Their body surfaces are clearly divided into metameres or segments and that is why they are referred to as a classification of phylum Annelida (Latin Annulus small rings) (Figure 4.11). They have both circular and longitudinal muscles that assist in the process of locomotion. Aquatic annelids, like Nereis have appendages that are lateral parapodia, that assist when swimming. An open circulatory system can be also present.
Nephridia (sing. nephridium) assist in osmoregulation and excretion. Neural system comprises paired the ganglia (sing. Ganglion) connected via two lateral nerves that connect to a double ventral cord of nerve. Nereis, a form that is aquatic is dioecious. leeches and earthworms are monoecious. Sexual reproduction is sexual. Examples: Nereis and Pheretima (Earthworm) along with Hirudinaria (Blood sucker). Figure 4.11 Examples of Annelida (a) Nereis (b) Hirudinaria Male Female Figure 4.10 Illustration of Aschelminthes: Roundworm 2022-23 ANIMALANIMAL KINGDOM KINGDOM 53 53 4.2.7 The phylum is Arthropoda This is the biggest phylum in Animalia that includes insects. Two-thirds of all the named species in the world comprise arthropods (Figure 4.12). They have an organ system levels of organization. The species is bilaterally homogeneous, triloblastic segmented, and coelomate creatures.
The arthropod’s body is covered by a chitinous exoskeleton. The body is comprised of thorax, head and abdomen. They are joined by appendages (arthros-joint and poda-appendages). The respiratory organs are gills book gills, book lung or tracheal systems. The circulatory system is an open kind. Sensory organs such as eyes, antennae (compound and basic) and statocysts as well as the organs that balance are in place. The excretion process is carried out via malpighian tubules. They are usually dioecious. Fertilisation tends to be internal. They are usually fertile. It is possible to develop in the form of indirect or direct. Examples: Insects that are economically important such as Apis (Honey Bee), Bombyx (Silkworm), Laccifer (Lac insect) Vectors Anopheles, Culex and Aedes (Mosquitoes) Gregarious pest Locusta (Locust) living fossil Limulus (King crab). Best NEET Coaching in Silchar
4.2.8 Mollusca phylum is the second most important animals family (Figure 4.13). Molluscs are aquatic or terrestrial (marine as well as fresh water) that have an organ system level of organization. They are bilaterally homologous, triploblastic, and coelomate creatures. The body is covered by the calcareous shell. The body is unsegmented , with distinct head, muscle foot and visceral-hump. A smooth and spongy layer skin is formed as a mantle on top of that visceral area. In the space that is between the hump and mantle is known as the mantle’s cavity. It is where feather-like gills exist. They are responsible for the ability to breathe and also excretory function. The head’s anterior region is home to sensory tentacles. The mouth has the file-like organ of feeding called the radula. The figure 4.12 Illustrations of Arthropoda : (a) Locust (b). Best NEET Coaching in Silchar.
Butterfly (c) Scorpion (d) Prawn (a) (c) (b) (d) Figure 4.13 Examples of Mollusca : (a) Pila (b) Octopus (b) (a) 2022-23, 54 BIOLOGY They are typically dioecious and oviparous , with indirect development. Some examples: Pila (Apple snail), Pinctada (Pearl oyster), Sepia (Cuttlefish), Loligo (Squid), Octopus (Devil fish) Aplysia (Seahare), Dentalium (Tusk shell) and Chaetopleura (Chiton). 4.2.9 Phylum 4.2.9 Phylum Echinodermata These animals possess an endoskeleton made of calcareous ossicles which is why they have they are known as Echinodermata (Spiny body in Figure 4.14). All are marine and have an organ system degree of organization. Echinoderms that are adults are radially symmetrical, whereas the their larvae have bilaterally symmetrical.
They are triploblastic, coelomate species. The digestive system is comprised of an upper and upper (ventral) face and anus to the higher (dorsal) aspect. The most distinct characteristic of echinoderms is their presence of the water vessels that aid in the movement, capture and the transport of food as well as respiration. A system for excretory elimination is absent. Sexuality is a separate issue. The process of reproduction can be described as sexual. Fertilisation usually happens externally. The development process is indirect, through free-swimming, solitary larvae. For example: Asterias (Star fish), Echinus (Sea urchin), Antedon (Sea lily), Cucumaria (Sea cucumber) and Ophiura (Brittle star). 4.2.10 Physical Phylum: Hemichordata Hemichordata was initially thought of as a sub-phylum within the Chordata phylum. However, it’s now classified as a distinct phylum within non-chordata.
Hemichordates possess a basic structure within the collar region known as the stomochord. It is a similar structure to that of the notochord. This phylum is comprised of tiny creatures that resemble worms, with organ system degree of organization. These are bilaterally symmetrical triploblastic and coelomate creatures. The body is cylindrical , and comprises an anterior proboscis collar, and a long trunk (Figure 4.15). The circulatory system is open kind. It is a way to breathe through the Gills. The organ that excretes is the proboscis gland. Both genders are separate. Fertilisation is an external process. The development process is indirect. For example: Balanoglossus and Saccoglossus. 4.2.11 The phylum Chordata Animals that belong to the phylum Chordata are distinguished through the existence of a nochord, an oblique figure 4.14 examples of Echinodermata : (a) Asterias (b) Ophiura (a) (b) Figure 4.15 Balanoglossus Proboscis Collar Trunk
ANIMALANIMAL KINGDOM KINGDOM 55 55 hollow nerve cord, and the paired pharyngeal gillslits (Figure 4.16). They are bilaterally symmetrical, triploblastic and coelomate to organ-system degree of organisation. They have a post anal tail as well as an open circulatory system. Table 4.1 provides a comparative analysis of important characteristics of non-chordates and chordates. The phylum Chordata is subdivided into three subphylas: Urochordata or Tunicata, Cephalochordata, and Vertebrata. Subphyla Urochordata, and Cephalochordata are frequently known as protochordates (Figure 4.17) and are only marine. In Urochordata the notochord is found only in the larval tail and in Cephalochordata the tidal zone extends from the head to the the tail area and persists throughout their entire lives.Best NEET Coaching in Silchar.
Example: Urochordata – Ascidia, Salpa, Doliolum; Cephalochordata – Branchiostoma (Amphioxus or Lancelet). Vertebrata is a subphylum that have a notochord in the embryonic phase. The notochord gets replaced by an osteoporotic or bony vertebral column when an adult is born. Therefore, all vertebrates are considered to be chordates, however all chordates aren’t vertebrates. In addition to the chordate basic characters vertebrates also possess a ventral muscle heart, which has two, three or more chambers. There are also kidneys to aid in Osmoregulation and excretion, and paired appendages, which could be limbs or fins. Nerve cord Notochord Postanal portion Gill slits 4.16. Chordata characteristics Figure 4.16 Chordata features Figure 4.17 Ascidia Table 4.1 The comparison of non-chordates with Chordates S.No. No. 1 Chordates. Notochord present.
Notochord absent. 2. The Central Nervous System is dorsal. the central nervous system ventral hollow and solid. and it is double. 3. Pharynx is perforated by Gill openings. Gill slits are not present. 4. The heart is ventral. The heart is dorsal (if there is one). 5. A post-anal portion (tail) can be found. Post-anal tails are absent. 2022-23 Biology Figure 4.18 Jawless vertebrate Petromyzon Figure 4.19 A typical example of Cartilaginous fishes: (a) Scoliodon (b) Pristis (a) (b) 184.108.40.206 Class 220.127.116.11 Class Cyclostomata The living species of Cyclostomata are ectoparasites found on a few fishes. They possess an elongated body with 6-15 pairs of respiration slits on their gills. Cyclostomes have sucking mouth and a circular one without jaws (Fig. 4.18). Best NEET Coaching in Silchar.
The body of the fish is free of scales and fins paired together. The vertebral column and the cranium are cartilaginous. Circulation is closed. Cyclostomes are marine , but they move to spawn in fresh water. After spawning, after just a few days, they are dead. Their larvae, upon metamorphosis, are returned back to the sea. For example: Petromyzon (Lamprey) and Myxine (Hagfish). 18.104.22.168 Classes – Chondrichthyes These are sea mammals with a slimmer body and the cartilaginous part of their endoskeleton (Figure 4.19). The mouth is located ventrally. Notochord persists throughout the life. Gill slits are distinct and are not covered by the operculum (gill covering). The skin is hard with tiny scales called placoids. Dental teeth have modified placoid scales that are oriented backwards. The jaws of these animals are extremely robust. They are predaceous. Due to the lack of an air bladders, they need to swim continuously to stay afloat.
22.214.171.124 Class 126.96.36.199 Class Cyclostomata Every living member of class Cyclostomata are ectoparasites that live on certain fishes. They possess an elongated body that has 6-15 pairs respiratory slits in the gills. Cyclostomes possess circular mouth with a sucking feature and no jaws (Fig. 4.18). Their body is devoid scales and fins paired together. The vertebral column and its cranial column are cartilaginous. Circulation is closed kind. Cyclostomes live in marine waters but move to spawn in fresh water. After spawning, after several days, they are dead. The larvae of their larvae, upon metamorphosis return back to the sea. For example: Petromyzon (Lamprey) and Myxine (Hagfish). 188.8.131.52 The class consists of Chondrichthyes The Chondrichthyes are marine mammals with a slim body and a an endoskeleton with cartilaginous joints (Figure 4.19).
Mouth is located in the ventral region. Notochord persists throughout the life. Gill slits are distinct and do not have the operculum (gill covering). The skin is strong with tiny scales called placoids. Dental teeth have modified placoid scales that are directed backwards. Their jaws are robust. They are predaceous. Because they lack an air bladders, they are required to swim frequently to keep from sinking. Vertebrata Division Agnatha (lacks jaw) Class 1. Cyclostomata Gnathostomata (bears jaw) Super Class Pisces (bear fins) Tetrapoda (bear limbs) Class 1. Amphibia 2. Reptilia 3. Aves 4. Mammals Class 1. Chondrichthyes 2. Osteichthyes Subphylum Vertebrata further divided in the following manner 2022-23 ANIMALANIMAL KINGDOM KINGDOM 57 57 The heart is two-chambered (one ventricle and one auricle). Some have electronic organs (e.g. Torpedo) and some have a poison stings (e.g., Trygon).
These are cold blooded (poikilothermous) species, i.e., they are unable to regulate body temperature. Both genders are distinct. In males pelvic fins bear claspers. They contain internal fertilisation, and a large proportion of them are viparous. For example: Scoliodon (Dog fish), Pristis (Saw fish), Carcharodon (Great white shark), Trygon (Sting ray). 184.108.40.206 Classes – Osteichthyes It comprises fresh and marine fishes with bony endoskeletons. Their bodies are smooth. The mouth is typically the terminal (Figure 4.20). They have four pairs Gills, which are covered with an operculum that runs along each side. The skin is covered by Ctenoid and cycloid scales. There is an air bladder that regulates buoyancy. The heart is twochambered (one ventricle and an auricle). They are cold-blooded creatures. Both genders are distinct. Fertilisation usually happens externally. Best NEET Coaching in Silchar.
They are mostly oviparous , and the development process is direct. Examples: Marine – Exocoetus (Flying fish), Hippocampus (Sea horse); Freshwater – Labeo (Rohu), Catla (Katla), Clarias (Magur); Aquarium – Betta (Fighting fish), Pterophyllum (Angel fish). 220.127.116.11 Class The class is Amphibia Like the name implies (Gr., Amphi : dual bios, bios Life) amphibians are able to reside in terrestrial and aquatic ecosystems (Figure 4.21). The majority of amphibians have two sets of legs. Body is split into trunk and head. Tails may be present in certain species. The skin of amphibians is damp (without the scales). Eyelids cover the eyes. The tympanum symbolizes the ear. The canal for the alimentary, urinary, and reproductive tracts all open up to a common chamber known as Cloaca, which is opened to the outside. Respiration occurs through gills lung and skin. The heart has three chambers (two ventricles and two auricles).
These are cold-blooded species. Both genders are distinct. Fertilisation happens externally. They are oviparous. Development is indirect. For example: Bufo (Toad), Rana (Frog), Hyla (Tree Frog), Salamandra (Salamander), Ichthyophis (Limbless amphibia). 4.21 Examples of Amphibia: 4.21 Illustrations of Amphibia : (a) Salamandra (b) Rana (a) (b) Figure 4.20 Examples of Bony fishes: (a) Hippocampus (b) Catla (a) (b) 2022-23 58 BIOLOGY 18.104.22.168 Class – Reptilia class name refers to their crawling or crawling method of movement (Latin repere, reptum or repere that means to crawl or crawl). They are primarily terrestrial animals and are surrounded by dry and dehydrated skin, epidermal scales and scuts (Fig. 4.22). They don’t have external ears. The ear is represented by the Tympanum. Limbs, in the event that they are present, have two pairs. The heart is typically three-chambered however, crocodiles have four chambers.
Reptiles are called poikilotherms. Lizards and snakes shed scales by casting skin. Both genders are distinct. The process of fertilization is internally. They are oviparous, and their development is direct. Examples: Chelone (Turtle), Testudo (Tortoise), Chameleon (Tree lizard), Calotes (Garden lizard), Crocodilus (Crocodile), Alligator (Alligator). Hemidactylus (Wall lizard), Poisonous snakes – Naja (Cobra), Bangarus (Krait), Vipera (Viper). 22.214.171.124 Class Aves The most distinctive features for Aves (birds) are feathers, and the majority of them fly, with the exception of the birds that are not flightless (e.g., Ostrich). They also have beaks (Figure 4.23). The forelimbs can be transformed to wings. The hindlimbs usually include scales and are designed to walk, swim or for securing the tree branches. The skin is dry, without glands, except for the oil gland located at the top of the tail. Best NEET Coaching in Silchar.
The endoskeleton is completely Ossified (bony) while the longer bones have hollow, with air-filled cavities (pneumatic). Birds’ digestive tract contains additional chambers: the crop and the gizzard. Heart is entirely fourchambered. The reptiles have warm-blooded (homoiothermous) species, i.e., they are able to keep an unchanging body temperature. The respiratory system is represented by figure 4.22 Reptiles (a) Chameleon (b) Crocodilus (c) Chelone (d) Naja (a) (b) (c) (d) 2022-23 ANIMALANIMAL KINGDOM KINGDOM 59 59 lung. Air sacs that connect to the lung lungs enhance breathing. Both genders have separate sexes. The process of fertilization is internally. They are oviparous, and their development is direct. Examples: Corvus (Crow), Columba (Pigeon), Psittacula (Parrot) Struthio (Ostrich), Pavo (Peacock), Aptenodytes (Penguin) Neophron (Vulture).
Class – Mammalia can be found in many habitats, including deserts, polar ice caps forests, mountains, grasslands and caves in darkness. Certain species have evolved to fly, or even live in the water. One of the most distinctive characteristics of mammals is the presence of milk-producing glands (mammary glands) through which young animals are fed. They possess two pairs of limbsthat are adapted to running, walking or climbing, burrowing flying or swimming (Figure 4.24). Its skin (a) figure 4.23 Certain birds : (a) Neophron (b) Struthio (c) Psittacula (d) Pavo (b) (c) (d) Figure 4.24 A few mammals : (a) Ornithorhynchus (b) Macropus (c) Pteropus (d) Balaenoptera (a) (b) (c) (d) 2022-23 60 BIOLOGY mammals is the only species in that they have hair. External ears, or pinnae, are present. Different kinds of teeth are found in the jaw. Heart is a fourchambered. It is homoiothermic. Best NEET Coaching in Silchar.
The lungs are the source of respiration. The sexes are distinct and fertilization is internal. They are viviparous, with a few exceptions. Development is direct. Examples: Oviparous-Ornithorhynchus (Platypus); Viviparous – Macropus (Kangaroo), Pteropus (Flying fox), Camelus (Camel), Macaca (Monkey), Rattus (Rat), Canis (Dog), Felis (Cat), Elephas (Elephant), Equus (Horse), Delphinus (Common dolphin), Balaenoptera (Blue whale), Panthera tigris (Tiger), Panthera leo (Lion). The main distinguishing traits of all phyla in the animal kingdom are outlined in Table 4.2.The fundamental characteristics like level of organisation and symmetry, cell organization and coelom as well as segmentation, notochord and more. These have allowed us to categorize animals into a variety of kingdoms. In addition to the basic characteristics that are mentioned above, there are other distinctive characteristics that are specific for every phyla or class.
Porifera comprises multicellular animals that display a cellular level of organization and have distinctive flagellated Choanocytes. The coelenterates possess tentacles and bear Cnidoblasts. They are predominantly aquatic, sessile, or floating free-floating. Ctenophores are marine animals that have comb plates. The platyhelminths sport a flat bodies and show bilateral the symmetry. The parasitic species have distinct hooks and suckers. Aschelminthes are pseudocoelomates that include non-parasitic and parasitic roundworms. Annelids are metamerically separated animals that are true coeloms. Arthropods are by far the largest group of animals characterized by the presence of joined appendages. Molluscs have an elongated body that is surrounded by a calcareous shell that is external to the. They are covered by an external skeletons composed from chitin.
Echinoderms have a spherical skin. Their most distinct feature is their water blood vessels. The hemichordates comprise a small species of marine animals that resemble worms. They possess a cylindrical body with a collar, proboscis and trunk. The phylum Chordata comprises animals that have an ochochord, either during or even in the first stages of embryonic growth. Other typical features found in the chordates include the dorsal hollow nerve cord, the paired pharyngeal-gill slits. Certain vertebrates lack jaws (Agnatha) while the majority have jaws (Gnathostomata). Agnatha is identified by the classification, Cyclostomata. They are among the earliest chordates, and they are ectoparasites of fishes. Gnathostomata is one of two superclasses, Pisces as well as Tetrapoda.
Classifications Chondrichthyes and Osteichthyes are characterized by fins that allow moving and are placed under Pisces. Chondrichthyes are Chondrichthyes are fishes with a cartilaginous endoskeletons, that are also marine. Classifications, Amphibia, Reptilia, Aves and Mammalia have two pairs limbs , and therefore are placed under Tetrapoda. Amphibians have evolved to live on both land and in water. Reptiles are distinguished by the dry, cornified and drying skin. Limbs are not present in snakes. Reptiles, amphibians, and fishes are all poikilothermous (coldblooded). Aves are warm-blooded creatures that have feathers covering their body, and front limbs that are transformed into wings for flying. Hind limbs are designed to walking, swimming and closing. The most distinctive features of mammals is the mammary glands, hairs and glands in the face. They typically exhibit viviparity.
Abstract High-throughput-next Generation Sequencing (HT-NGS) technology is currently the most talked about area of research in animal and human genomics research that can generate more than 100 times more information when compared to the highest-quality capillary sequencers that are based upon the Sanger method. With the continuous development of high-throughput sequencing machines and the development of advanced bioinformatics tools at a rapid pace the ultimate goal of sequencing each genome of living organisms for a price of just $1,000 each appears to be achievable within the next few years. In the brief timeframe since 2005, HT-NGS technologies have revolutionized research of the human genome and that of animals.
Studies through the analysis of an immunoprecipitation of chromatin that is linked with the DNA microarray (ChIPchip) (also known as the sequencing (ChIP-seq), RNA sequencing (RNAseq) which is a entire genome sequencing, genome-wide structural variation, de novo assembly and re-assembling genomes mutation detection, carrier screening, identification of genetic disorders and more complex human diseases DNA library preparation as well as paired ends and genome captures, as well as the sequencing of mitochondrial genomes as well as personal genomics. In this overview we will discuss the most important aspects of HTNGS such as, the first generation of DNA sequencers, the development of HTNGS, second generation HT-NGS platforms third generation HT-NGS platforms comprising single molecule Heliscope(tm), SMRT(tm) and RNAP sequencers, Nanopore, Archon Genomics X PRIZE foundation as well as a comparison between the third and second HT-NGS platforms, applications, advancements and the future of sequencing technologies in the human and animal genome research. Best NEET Coaching in Silchar.
Chip-seq . De novo assembling . High-throughput next-generation sequencing . Personal genomics. Re-sequencing . RNA-seq Introduction. The publication of the first drafts of the human genome (Yamey 2000) was just the beginning of the modern era of DNA sequencing that led to further innovation advancements and development of advanced methods of high-throughput sequencing, also known as “high-throughput next-generation sequencing” (HT-NGS). The strategies that were developed for HT-NGS focused on our future requirements of high-throughput sequencing and cost in a manner that enabled the potential for a multitude of applications both in the present and the future to mammalian genomics research. Furthermore, in these sophisticated methods of laboratory, a range of the latest generation of bioinformatics instruments has also emerged as a vital requirement to allow for future strategic advancements and improvements to output results. The HT-NGS is among the major challenges facing current genomic research.
In the near future we will require in-depth analysis of genome sequences and genome sequences for all mammals, including humans, to better understand the genetic variations in economic characteristics, genetic susceptibility to diseases, and pharmacogenomics as a the drug response. The most important genomics C. S. Pareek (*) : R. Smoczynski Laboratory of Functional Genomics, Institute of General and Molecular Biology, Faculty of Biology and Earth Science, Nicolaus Copernicus University at the address is. Gagarina 11, 11 87 100 Torun, Poland e-mail: firstname.lastname@example.org A. Tretyn Department of Plant Physiology and Biotechnology, Faculty of Biology and Earth Science, Nicolaus Copernicus University in Gagarina 11. Gagarina 11, 87 100 Torun, Poland J Appl Genetics (2011) 52:413-435 DOI 10.1007/s13353-011-0057-x research centers and scientists have publicly recognized that these are the core enabling goals for the next decade genomics research. In the National Human Genome Research Institute (NHGRI) has also echoed this requirement through its mission for research in genomics (Collins and colleagues. 2003).
The NHGRI has classified the latest sequencing methods in terms of offering short-term and long-term benefits that include an increase of 100 times the cost by base pairs (bp) in the five years to come. To extend the short-term, i.e., of in the next 5-10 years, the breakthrough advantages should help improve the field by an increase of 10,000 times the cost per base pair. This will can lead to the “US$1000 genome”. Year 2011 is celebrated as the 10th anniversary since the human genome was first sequenced (www.nature.com/ natureconferences/hg10years/index.html). In this time, a remarkable achievements have been achieved in the areas of decoding human genome, advancement in technology of a new era in the human genome, towards personalized genomes and the discovery in rare mutations and leveraging the power of genome sequencing to improve mammalian evolutionary research and cancer and the structure of populations. The last decade has seen an era of change within the realm of human genome research.
In the present, a more international approach to research is taken that has not just given the rise to the science of systems biology but also has impacted all fields of medical and biological research, making them more integrated and blurring the boundaries that used to define them as distinct research disciplines. The perspectives and possibilities are expanding due to technological advancements that have taken place in the field of genomics, particularly the HT-NGS and its broad array of applications like the chromatin immunoprecipitation process coupled with the DNA microarray (ChIPchip) or sequencing (ChIP-seq), RNA sequencing (RNAseq) and entire genome genome analysis, de novo assembly and re-assembling genomes genomic structural variation as well as mutation detection and screening for carriers, detection of inherited diseases and complicated human illnesses DNA library preparation genome captures, paired ends and sequencing of the mitochondrial genome and personal genome (for the full description read Table 2.).
Alongside the advances in sequencing techniques, the previous decade will be known for the decade that was genome research. Since the release of the first genomes that were composite of human (Lander and colleagues. 2001; Venter et al. 2001) – many draft genomes from other organisms have been published (www.ensembl.org/info/about/species.html). The speed at that new genomes are now able to be sequenced has been made possible through the development of technology and assembly methods for HT-NGS. The ability to build in a single step a massive genome. A good example is the genome assembly process for the huge panda (Li et and al. 2010b) that relied on the only short reads offered through next-generation DNA sequencing. The first generation of DNA sequencers in 1975, Sanger presented the concept of DNA sequencing in his pioneering Croonian presentation (Sanger 1975) and, later published a method of determining the sequences of DNA by primed synthesis DNA polymerase (Sanger and Coulson 1975). Best NEET Coaching in Silchar.
In 1977, two key research papers on DNA sequencing appeared, i.e., the Frederick Sanger’s enzyme-based dideoxy DNA sequencing method based on chain-terminating dideoxynucleotide analogs (Sanger and Coulson. 1977) and Allan Maxam and Walter Gilbert’s chemical degradation DNA sequencing method that used terminally labeled DNA pieces were then chemically cut at specific bases, and then separated using the gel electrophoresis (Maxam and Gilbert 1977). These two renowned labs were the ones responsible for the development of the first automated DNA sequencers , led by Caltech (Smith and co. 1986) and later developed via Applied Biosystems (ABI), the European Molecular Biology Laboratory (EMBL) (Ansorge and colleagues. 1986 1987) and PharmaciaAmersham which was then General Electric (GE) healthcare. The improvement and the commercialization technique led to its wide spread across the entire research community.
In the first automated fluorescent DNA sequencing equipment, a complete gene locus for the hypoxanthineguanine phosphoribosyltransferase (HPRT) gene was sequenced, using for the first time the paired-end sequencing approach (Edwards et al. 1990). The year 1996 was the time ABI released the very first DNA sequencer commercially which used electrophoresis on slab gels by ABI Prism 310. ABI Prism 310. A few years after, the tedious work involved in placing slab gels into the capillaries was eliminated by automated reloading of capillaries using polymer matrix. This was done by the ABI Prism 3700 that has more than 96 capillaries. The automated DNA sequencer was used successfully for the sequencing of the human genome for the first time in 2003, taking into account 13years of effort from the human genome consortium and at the estimated price in the range of $2.7 billion. Best NEET Coaching in Silchar.
In the years following another milestone was reached through the DNA sequence of the earliest bacterium genome (5386 bases) and the sequencing for the entire human genome, which is 3 billion bases or more (Lander and others. 2001; Venter et al. 2001). It’s amazing that such advancements have been made with methods that have been refined from the original ‘dideoxy’ approach developed by Sanger in 1977. The birth of HT-NGS in 2000, Jonathan Rothberg founded 454 Life Sciences, which further developed the first commercially-available NGS platform, called the GS 20. Its GS tool was the four14 J Appl Genetics (2011) 52:413-435 was introduced in 2005 through 454 Life Sciences (www.454.com), as the first NGS platform available on the market. The technique was tested by combining single-molecule DNA PCR and Pyrosequencing (shotgun sequencing) of the complete 580 069 base pairs of Mycoplasma genitalia’s genome with 96 percent coverage and 99.96 percent accuracy within a single GS 20 run (Margulies et and al. 2005).
In the years following, Roche applied science acquired 454 Life sciences , and developed a new model of 454’s instrument i.e. the GS FLX titanium. It shares the same principle of technology for each of the GS 20 and GS FLX titanium the flow cell is known in the form of “picotiter as well” plate that is constructed from the fiber-optic bundle that is fused. In a different direction the single-molecule PCR technique in microcompartments made up of oil-water emulsions was also created by the Roche the HT-NGS technology (Tawfik as well as Griffiths 1998). In general, the premise of pyrosequencing techniques is based on “sequencing through synthesizing”. It is different from Sanger sequencing in that it is based on the determination of pyrophosphate release following nucleotide incorporation rather than chain termination using dideoxynucleotides. This technique was developed through collaboration between a Swedish team (the teams comprised of M. Ronaghi, M. Uhlen, and P. Nyren) in Stockholm (Ronaghi and Nyren. 1996). Best NEET Coaching in Silchar.
They first reported the sequencing method that relies on chemiluminescent detection the release of pyrophosphate during polymerase-mediated desoxynucle triphosphate (dNTP) incorporation (Nyren and co. 1993; Nyren 2007) and the real-time DNA sequencing using the release of pyrophosphate detection (Ronaghi and colleagues. 1998). When pyrosequencing DNA, the synthesizing process is carried out by a complex process that involves ATP sulfurylase as well as luciferase enzymes as well as the adenosine 5′-phosphosulfate and Luciferin substrates, in such an approach that, the pyrophosphate groups release the nucleotide upon addition which results in the generation of visible light. The HT-NGS strategies provide new possibilities and have a significant impact on research in mammalian genomics were chosen as the most effective methods of 2007 (Schuster and colleagues. 2008). But, the path to the acceptance of these innovative techniques was not an simple one.
The initial phase of the HTNGS technique was to detect the next fluorescently labeled DNA base (reversible terminator) within the expanding DNA chain with CCD cameras that are sensitive. This was done on a number of DNA samples simultaneously connected to either an elongated support or beads on DNA chips, which reduces the amount of reaction in a tiny microsystem. In the next step , the terminator was transformed into an ordinary nucleotide, and the dye removed. This process were repeated in order to identify the next base of the sequence. The concept of this application is almost identical to the method employed in today’s next-generation devices developed through Roche, Illumina-Solexa, ABI, Helicos and other companies. The principle of HT-NGS is DNA molecules that are sequenced in an enormously parallel manner in the flowing cells (Mardis 2008a, and Metzker 2010.). The sequence is performed using either a stepwise procedure or in an ongoing, real-time way.
Because of this multi-dimensional process, each individual molecule, or clonal template will be “individually” recorded and may be included in the total sequences created. The high-throughput blend of quantitative and qualitative sequence information has enabled advanced genome analysis that was previouslynot feasible or expensive. Second generation HTNGS platforms second generation HTNGS platforms can generate around 5 billion bases sequence raw (Roche)
and billions of bases one cycle (Illumina, SOLiD). These innovative methods are based on cyclic, parallel interrogation of sequences using the spatially separated amplicons of clonal amplicons (26 mm oil-aqueous-emulsion beads [Roche: pyrosequencing Chemistry 1 mm clonal beads [SOLiD sequencing using sequential binding of oligonucleotide probes]], clonal bridge [Illumina: sequencing using dye terminators that are reversible). Presently, the (above discussed) three most popular second generation technology platforms for HT-NGS (Fig. 1) are available commercially and the race to develop other platforms is always in the near future (for extensive reviews of all lab procedures, technical agreements of sample preparation and sequencing data analysis for Roche, Illumina, SOLiD platforms, refer to: Mardis 2008a, b, 2009, 2010; Metzker 2010).
In 2008, the US National Human Genome Research Institute (NHGRI) has initiated funding for a series of projects as part of its revolutionary genome sequencing technologies program and aimed toward its target goal of sequencing a human genome for $1000 or less (http://www.genome.gov/27527585). In December the NHGRI consortium released the largest study of genetic variations in humans made using next-generation DNA sequencing techniques to identify the genetic differences between the 179 individuals of four populations, and 697 people from seven groups in three preliminary studies (Durbin and co. 2010). These pilot studies of the “1000 genomes project” laid a critical foundation for studying human genetic variation, and aimed to create a comprehensive, publicly available map of genetic variation, that will ultimately collect sequence from 2,500 people from multiple populations worldwide and underpin future genetics research (http://www.genome.gov/27541917).
J Appl Genetics (2011) 52:413-435 415 Third Generation HT-NGS platforms. In the previous discussion of the second generation of HTNGS platforms the concept was based on the emulsion-based PCR amplification of DNA fragments to ensure that the light signal is strong enough to ensure reliable detection of bases using CCD cameras. Although PCR amplifying technique is revolutionizing DNA analyses however, in certain instances, it can create errors in the base sequence or favor of particular sequences over other which can alter the amount and frequency of different DNA fragments prior to the amplification. To combat this issue, the ultimate reduction to the nanoscale, and the use of biochemicals in a minimal manner could be achieved in the event that the sequence could be identified from one DNA molecule without the requirement for PCR amplifying and the possibility of causing distortions in the abundance levels. Best NEET Coaching in Silchar.
The sequencing of one DNA molecule is now being referred to”HT-NGS3″ or the “third technological generation for HTNGS” (Schadt and co. 2010). The concept of sequencing-bysynthesis without a prior amplification step, i.e., singlemolecule sequencing is currently pursued by a number of companies and described below in Sects. 5.1 up to 5.7. Heliscope(tm) single-molecule sequencer One of the earliest methods for sequencing one DNA molecule was developed by Braslavsky and colleagues. 2003, and granted a license by Helicos biosciences as the first commercial single-molecule DNA sequencing technology in 2007. The basic principle behind Heliscope sequencer is based on “true single-molecule sequence” (tSMS) technique. The tSMS method begins with the preparation of a DNA library by DNA shearing and the introduction of poli-(A) tails to created DNA fragments (Ozsolak and colleagues.
2010) and then the hybridization of DNA fragments with the poli-(T) Oligonucleotides that are fixed with the flow cells and sequentially sequenced during parallel reaction. The sequence cycle is comprised of DNA extension by using one of the four nucleotides fluorescently labeled followed by detection of nucleotides using the Heliscope sequencer. The following chemical cleavage of fluorophores allows the following cycle of DNA elongation with a second fluorescently labeled nucleotide which allows for the determination of DNA’s sequence (Harris and colleagues. 2008). The Heliscope sequencer can handle processing as much as 28 Gb in one sequence and can take around 8 days. It can produce shorter reads, with a maximum duration of up to 55 bases. A recent announcement, Helicos announced that it has created a modern version of “one-base-at-a-time” nucleotides that allow greater accuracy in homopolymerization and direct RNA sequence (Ozsolak and Milos 2011a, b).
Single molecule real-time (SMRT(tm)) sequencer underlying principle behind SMRT sequencer is based on single-molecule real-time sequencing using synthesizing method that is available on the chip for sequencing that houses hundreds of Zero-Mode Waveguides (ZMWs). The sequence of DNA fragments is executed by the DNA polymerase molecule. It is connected at the base of every ZMW to ensure that every DNA polymerase is in the zone of detection ZMW (Fig. 2). During the sequence reaction it is extended through DNA polymerase, using DNTP’s as shown in. 1. The advanced technological capabilities of three leading second-generation HT-NGS platforms. Appl Genetics (2011) 52:413-435 fluorescently labeled (each nucleotide is marked with the fluorophore of a different colors) at the moiety that is the terminal of phosphate. This DNA sequence can be identified by using CCD arrays using the detection of nucleotides by fluorescence, which is carried out prior to nucleotide incorporation in the same way that the dNTP labeled creates a cognate connection in the DNA template.
The flash of light is stopped following the formation of phosphodiester bonds. This results in an emission of fluorescent which diffuses out of ZMW. Following that, the labeled nucleotide incorporation and detection allows us to identify what sequence of DNA is present (Levene and colleagues. 2003; Eid et al. 2009). It is believed that the SMRT sequencer was created and is currently being developed by Pacific Biosciences (www.pacific biosciences.com). While the SMRT instrument is only recently put on sale and is claimed by the company that the SMRT analyzer has the capability of producing 100 Gb per hour, with readings that are more than 1000 in one run. Single molecule real-time (RNAP) sequencer A distinct single-molecule DNA sequencing technique, i.e., RNA polymerase (RNAP) was developed in the work of (Greenleaf Block 2006) and Greenleaf Block 2006) where it is believed that the RNAP can be attached to a polystyrene beadand the distal end of the DNA fragment can be attached to a different bead.Best NEET coaching in Silchar.
Each bead is put in an optical trap, and the traps are able to lift the beads. They are able to move as RNAP reacts to the DNA fragment, and the transcriptional movement of RNAP across the template alters the length of the DNA between both beads. This causes movement of two beads which can be detected with precision within the Angstrom range, which results in one-base resolution of the DNA molecule. By aligning four displacement recordings each one with smaller concentrations in one nucleotide as a way to mimic the primers utilized in Sanger sequencing, and also for calibration using sequences that are known and that flank the unidentified sequenced fragment it is possible to discern what sequence data is contained. The method demonstrates the nucleic acid enzyme, and the highly precise optical trap method can allow the extracting the sequence data directly from the DNA molecule itself.
Nanopore DNA sequencer Contrary to the other DNA sequencers described previously, the process of the process of sequencing DNA molecules using this sequencer is devoid of nucleotide labeling and detecting. This method was developed through research on the DNA translocation through various artificial nanopores. The DNA sequencing using Nanopore instrument is based on conversion of the electrical signal of nucleotides via Nanopores, which are A-hemolysin-like pore, which is covalently connected to the cyclodextrin-containing molecule that is the nucleotide binding site. The principle behind this technique is founded on the modulation the ionic current that flows through the pore when the DNA molecule passes through it, revealing the characteristics and parameters (diameter length, size, and shape) of the DNA molecule (Fig. 2). During the sequence process, the ionic charge that flows across the nanopore gets blocked nucleotide i.e. it is the nucleotide that was previously cut by exonuclease. DNA strands that interact with the cyclodextrin.
The duration of the block of current is unique for every base in Fig. 2. Advanced technological features of the top four 3rd generation platforms for HT-NGS J Appl Genetics (2011) 52:413-435 417. This allows that the sequence of DNA to be identified (Astier and co. 2006. Rusk 2009). However, further enhancements and adjustments to the method such as expanding the amount of variables that are measured when translocating DNA, enabling single-base resolution could result in a speedy nanopore-based DNA sequencing method. Real-time single-molecule DNA sequencer platforms created by VisiGen Biotechnologies VisiGen Biotechnologies (www.visigenbio.com) has introduced a specially engineered DNA polymerase functions as a “real-time detector for nucleotides that have been modified using an donor fluorescent dye located near the active site in the selection of nucleotides in synthesis (Fig. 2). Best NEET coaching in Silchar.
The four nucleotides that were to be integrated were altered each using a different acceptor dye. When the correct nucleotide was identified was selected, it was inserted into the active area of the enzyme. During this process, the dye used by the donor within the polymerase came into close proximity to the dye used to accept the nucleotides. The energy was transferred from donor dye to acceptor dye resulting in the fluorescent resonant energy transfer (FRET) luminescence signal (Selvin 2000). The frequency of the signal was dependent on the dye used in the nucleotides. Thus, by recording the frequency of FRET signals emitted could be used to identify bases, in the rate at which the polymerase is able to incorporate the nucleotides in the process of synthesis (usually around a few hundred times each second). The fluorophore that is the acceptor is eliminated when nucleotide integration is completed, which makes sure that there aren’t DNA changes that could hinder the polymerase’s the synthesis process.
It is working to develop the first version of the tool that could generate up to four Gb of data daily. The single-molecule method does not require for cloning or amplification which means that it will eliminate a substantial portion of the expense, compared to the current technology. Furthermore, the read lengths on the device are anticipated to be approximately 1 kb. This is more than the current platforms. Multiplex polony technology Run by the privately-funded personal genome project (PGP) and lead by Prof. G Church’s research group (www.personalgenomes.org), has developed and introduced the multiplex polony technology (Mitra et al. 2003; Shendure et al. 2005). This technique involves a number of hundreds of sequencing templates are put onto thin layers of agarose and sequences are then determined in parallel.
This method allows for an the possibility of increasing several orders of magnitude in the quantity of samples that can be analysed simultaneously. It offers the benefit due to the significant reduction of the volume of reactions and requiring less chemicals and resulting in an affordable cost. The instrument designed, i.e., Danaher Motion Polonator model G.007 is capable of between 10 and 35 Gbp for each module for a 2.5 days run. The instrument can be paired with 200 modules to obtain 100 diploid genomes with 30X coverage in five days and the remaining five days used to repeat any weak runs in order to guarantee 98% coverage , with 1E-5 precision. Due to the significantly reduced amount of reagents used, the price per unit is reduced than 10x and the company plans to achieve its goal of $1000 for each genome within the next few months. Ion Torrent sequencing technology Ion Torrent sequencing technology In the last few years the initial PostLightTM sequencer (Ion Torrent) was introduced (http://www. iontorrent.com/). Best NEET coaching in Silchar.
This technology establishes an immediate connection between the chemical element and digital data, which allows for fast simple, scalable, and easily sequencing. It uses the basic nucleic acid Watson’s chemistry incredible power, exclusive of semiconductor technology – The Moore’s law (Moore 1965). The premise of Ion Torrent semiconductor technology is founded on a well-studied biochemical process that occurs when nucleotides are incorporated into DNA strands through a polymerase. The result is in the release of hydrogen Ion as a byproduct (Fig. 2). The technology uses the highest density of micro-machined, wells to carry out this biochemical procedure in a massively parallel manner, with each well having a distinct DNA template. Under the wells is an ion-sensitive layer, and below that is an exclusive Ion sensor. The massive parallel sequencing in the Ion Personal Genome Machine (PGM(tm)) sequencer operates by using the “base” concept.
For example, if a nucleotide A is added into a template DNA, it is integrated into a DNA strand and the hydrogen ion is released, then it is released. The charge that is released by the Ion will alter in the pH level of the liquid. It is detected by an ion sensor, without scanning, cameras or light. In this manner it is possible to use it is possible to use the PGM(tm) sequencer is able to flood each chip’s nucleotide and then another. The specially designed PGM(tm) system is able to carry out a wide array of applications for sequencing, including multixing amplicons, transcriptome, small RNA and ChIP-Seq. paired-end sequencing and the methylation. Concerning concerns regarding quality of genomics data as well as analysis, a massive $10 million in funding has been provided via The Archon Genomics X PRIZE (AGXP) to produce fast, precise and complete human DNA sequences for the worldwide research communities (editorial discussion: A step towards an medical-grade Human genome sequence.
Nat Genet. 2011 Mar, 43 : 173). Since a lot of genome researchers are in the game, AGXP offers to help through a process of consultation in order to develop equitable and effective methods to verify contestant genomes with the highest numbers (418 J Appl Genetics (2011) 52:413-435 degree of completeness and accuracy (Kedes and co. 2011). Since the introduction AGXP in 2006, there have been significant advances. AGXP in 2006 significant advancements in the validation protocols of DNA sequencing techniques both in terms speed and cost reduction (Sutton and colleagues. 2011). But, there is no evidence that a humans genome sequencing is precise, complete or guaranteed to contain any rearrangements or other information about the chromosome and phasing (haplotype). Highly repetitive regions and other genome-wide areas are still difficult to sequence, however they are likely to be crucial in determining heritable traits. Thus, the ideals associated with the X Prize remain as critical to our future in genetic medicine and human genes just as they were in the past.
A comparison of the second and third HT-NGS platforms. The third HT-NGS technology, which relies on PCR for the growth of clusters of DNA template and attach DNA templates’ clusters to a surface that is then imaged after these clusters get sequenced through synthesizing in a phased manner Third HT-NGS techniques analyze DNA molecules in a single step in such a way that there is no synchronization (a issue with the second HTNGS) is needed (Whiteford and colleagues. 2009) which means that they can overcome issues with the biases created through PCR dephasing and amplification. Additionally, third HT-NGS technology can exploit more fully the high catalytic efficiency and the high speed of processing of DNA polymerase or even omitting chemical or biology completely to dramatically increase the length of reads (from 10 bases to 10s of thousands of bases in a single read) and time to achieve (from days to hours or minutes).
Additionally, the third generation HT-NGS technology could offer the advantages over second HTNGS technologies which include: first,) greater throughput, ii) quicker turnaround times (e.g. sequencing metazoan genomes in high fold in just minutes) and iii) longer lengths of reads to increase the de novo assembly process and allow the direct detection of haplotypes, or even whole chromosomes phase-shifting and fourth) greater accuracy in consensus to allow rare variant detection and the last) tiny amounts of beginning material (theoretically only one molecules could be needed to sequence) and finally,) affordable costs, and the sequencing of human genomes at high fold coverage at less than $1000 seems to be an achievable objective for the community. Over the last six years, a flood of both original and extensive review papers on the second and third generation HT-NGS platforms has been published. Best NEET coaching in Silchar.
This is why the differences between the second HT-NGS platforms (Roche/454, SOLiD, and Illumina) and third HTNGS platforms (Helicos and Pacific Biosciences etc.) are summarized in Table 1 which highlights how similarities and distinctions exist between these platforms, based on different indicators. In terms of technical features they both work through synthesis. The differences the second HTNGS platform focuses on the washing and scanning of many copies of DNA molecules as opposed to a direct physical inspection of the DNA molecular as well as its resolution in real-time (i.e. there are no lengthy cycles of hybridization, or repeated enzyme processes) on third HT-NGS platforms. Other dissimilarities include RNA sequencing, however, the Second HT-NGS platform can only perform the cDNA sequencing, whereas there is direct RNA sequencing is possible on the third-party HTNGS platform. Best NEET coaching in Silchar.
Concerning the analysis of data both platforms are complex because of the huge volume of data. For the second HT-NGS platform, the biggest challenges are short reads that can be difficult to assembly genomes and alignment algorithms. On the other hand, new issues with signal processing are still prevalent in third HTNGS platforms. With the rapid development of HT-NGS technology, DNA sequencing costs have decreased dramatically (Table 1.). It is now possible to sequence hundreds or thousands of genes in a single person with a suspicion of genetic illness or a complicated predisposition to disease. In addition to the advantages offered by these new technologies however, there are a variety of issues that need to be overcome before large-scale sequencing is adopted into the practice of genome research. Best NEET coaching in Silchar.
Diagnosticians in the field of molecular medicine must get familiar with, and confident using these new technologies, that are built on completely different technologies to the conventional DNA sequencers used employed in routine diagnostics. Since 2001 when the technology was able to sequence the human genome was developed on the base of capillary electrophoresis was developed, which is an the individual, fluorescently labeled Sanger sequencing methods The advent technology for next generation sequencing has drastically increased the speed with that DNA sequences are obtained, while also reducing cost by several orders of magnitude in comparison to their predecessors (Fig. 3). This is because the fundamental mechanisms of data generation have changed dramatically and resulted in a greater number of sequencing reads for each instrument and at a much lower cost.
Figure 3 shows how the information resulting from HT-NGS has made us more knowledgeable and has increased the potential impact of the genome in research in biomedicine (Mardis 2011,). These platforms are able to produce shorter reads, but with less quality in comparison to Sanger platform. The decrease in length and quality required the creation of bioinformatics software to aid in the mapping of the short reads with reference sequences, or assembly de novo. The creation of these innovative methods is aimed at meeting the requirement for sequence information in a variety of areas of study including research into the study of genetics and evolution as well as forensics, epidemiology, therapeutics applied and diagnostics. Applications and advancements of sequencing technology for human genome research important milestone of the sequencing of the human genome was carried out by two organizations, i.e., the publically supported Human Genome Group (HGP) and Celera Groups. Best NEET coaching in Silchar.
Both of them employed different methods. The HGP group created an unfinished draft version of our human genome using the map-based approach, while Celera was able to analyze the human genome, using The wholegenome shotgun (WGS) method (Fig. 4). The accessibility of sequence information from various methods helped all scientists to understand the information. The method of HGP was initially developed by the publically funded project was based upon the localizing of bacterial artificial genomes (BACs) with massive fragments of human DNA in the context of a physical map based on landmarks. The ideal situation would have been that sequencing would have taken place using a clone by clone approach using clones that were selected from the least of BACs that had a tiling path. The main element of the HGP’s method was the mapping process in which the BACs were positioned in the genome’s chromosomes searching for distinct marker sequences, also known as sequence tags sites (STSs) which whose locations had been identified.
By doing this the BACs offered a high-resolution image of the whole genome (Fig. 4). The draft, while with some ambiguities and gaps according to order, is highly helpful in the identification of genes associated with disease. The ideal method that was employed by Celera was to steer clear of the mapping process in the beginning by subcloning random pieces of humans’ genomes directly. Sequencing both ends of fragments of libraries with different dimensions made it easier to order. While reducing time and effort in initial, Celera’s strategy led to the assembly process becoming dependent on algorithms as well as the time of computers. In the pursuit of their objectives, their best-designed strategies were transformed into hybrids where the HGP chose more clones randomly and Celera employed BAC sequences and maps generated from HGP HGP (Fig. 4). Best NEET coaching in Silchar.
Since the launch of the HTNGS platform back in 2005 the generation of large quantities of reads at a low cost made NGS platforms useful for a variety of applications related to human genome research, including genome sequencing done de novo and whole-genome resequencing, or specific sequencing, which cataloguing the transcriptomes of tissues and cells in organisms (RNA-seq) and genomic mutation detection and variation as well as genome-wide profiling of epigenetic markers and chromatin structure by using DNase-seq, methyl-seq as well as ChIP-seq (chromatin immunoprecipitation coupled with the DNA microarray) and personal genomics (Table 2). De Novo, resequencing and targeted sequencing In general the HT-NGS platforms make the assembly de novo of many species, including humans which was a costly and lengthy process. Best NEET coaching in Silchar.
For humans, this project had already begun by the release of several full genomes, including using Roche 454 technology Roche 454 technology, which provides 7.5x humans’ genomes (Wheeler and colleagues. 2008) Human genome sequences from Chinese (Wang and co. 2008) and an African (Pushkarev and co. 2009) and two Korean individuals (Ahn and co. 2009; Kim et al. 2009; Kim et al.) All were performed with The Illumina Genome Analyzer. They were able to sequence approximately 20x haploid genome coverage except for Kim et al. 2009). African male’s genome, which was also resequenced using the ABI SOLiD (McKernan and Kim. 2009). Then James Lupski’s DNA was sequenced to a 30x coverage of bases by the SOLiD System (Lupski et al. 2010). Human genome sequencing is not restricted to second-generation platforms. Best NEET coaching in Silchar.
Steven Quake’s genome for instance has been sequenced with 90% coverage of the genome using Helicos Single-molecule Sequencing Platform (Pushkarev and colleagues. 2009). Whole genome-wide genotyping method that is based on HTNGS allows for unlimited multiplexing and free one nucleotide variant (SNP) selection such as typing HLA genotypes in humans (Lind and colleagues. 2010) and genome-wide fetal genotyping with non-invasive HTNGS from maternal blood (Burgess 2011). It is also being used in studying small RNAs. For instance, a thorough investigation of miRNAs in acute myeloidleukaemia carried out using HT-NGS revealed differentially expressed miRNA binding sites in acute myeloid leukaemia (Ramsingh and co. 2010).
Recent studies have revealed that various efficient methods are being developed to conduct RNA-Seq with Illumina sequencing (Buermans et al. Illumina Sequencing Platform (Buermans and co. 2010; Nagalakshmi et al. 2010; Nagalakshmi and colleagues.) including the technical aspects (Marguerat 2010; Nagalakshmi et al. Bahler 2010) Building a an extensive miRNA repertoire databases (Lee and al. 2010) and preparation of small RNA libraries, and analysis of the sequence data to measure the abundance of microRNAs (Morin and co. 2010) in addition to analysis and discovery of small RNAs using transcriptomic data (Yang and co. 2011). RNA seq that utilizes Illumina and 454 technologies has been shown to be an effective instrument for detecting new genes fusions within cancer cells as well as tissues (Maher and colleagues. 2009). The understanding of the transcriptome is vital to understand the functional components of the genome as well as uncovering the molecular components of tissues and cells as well as for understanding the diseases and development. Best NEET coaching in Silchar.
The primary goals of transcriptomics are (1) to catalogue the transcripts of all of different cell types within an organism, which includes non-coding RNAs, mRNAs, and small RNAs (2) to identify the gene’s transcriptional structure according to their starting points 5′-, 3′-ends as well as splicing patterns and other post-transcriptional changes and (3) to measure the expression levels of every transcript during development , or under various physiologic and pathological conditions. With the advent of more efficient and more affordable HT-NGS systems, more studies on transcriptomics are being conducted with a recently developed deep-sequencing approach (Wang et al. 2009). The short reads generated from HTNGS technologies, specifically Illumina and SOLiD could be useful for profiling gene expression. The RNA-Seq technique has been utilized to monitor the precise expression of particular genes in order to assess the differences in splicingof allele-specific transcripts , and other biological problems that arise in RNA-Seq studies (Costa and colleagues. 2010b).
Epigenetics The HTNGS technologies have the possibility of significantly speeding up epigenomic studies (the research into heritable genes and their regulation which is not based on the DNA sequence however its higher order structures and modifications) that includes posttranslational changes of histones and the interaction with transcription factor and immediate target, nucleosome position on large scales across the genome and the study of DNA pattern of methylation (Bormann and colleagues. 2010; Fouse et al. 2010; Bhaijee et al. 2011). Histone modification and DNA methylation are two key epigenetic mechanisms that control the transcriptional state of genes. Utilizing ChIP-Seq (chromatin immunoprecipitation as well as direct sequencing) technique, the posttranslational changes of histones as well as the locations for transcription factors may be examined on a global scale (Neff and Armstrong 2009) while the methylated DNA immunoprecipitation (meDIP) as well as bisulphite protocol can be utilized to investigate the methylation process of DNA itself (Popp and others. 2010).
In particular, using ChIP-seq using the HT-NGS platform the binding sites of the transcription factor (TF) and for the GABP alpha of the human growth associated protein (GABP beta) have been directly sequenced, instead of being hybridized using a chip-array in order to unravel the many and complex gene pathways controlled by the PPARG gene (Costa et al. 2010.a) which predicted de novo discovery of the motif (Jiao et al. 2010). The ChIP-Seq technology on the HT-NGS platform allows researchers to increase the quality and quantity of the data they generate. In addition to other high-throughput methods that are widely used that have studied protein-DNA interactions, these have been investigated using the use of chromatin immunoprecipitation (ChIP) and DNA microarrays (ChIP-chip). However, ChIP-seq has two advantages over the HT-NGs platforms. Best NEET coaching in Silchar.
First it is not restricted by the microarray’s content, and, secondly it is not dependent on the performance of the probe hybridization. The ChIP-seq method was recently employed to find binding sites for two transcription factors: STAT1 as well as NRSF on human cells (Robertson and colleagues. 2007; Euskirchen et al. 2007). Both studies compared their results with those produced by ChIP-chips, showing that ChIP-seq was more precise and required fewer replications. Table 2. (continued) Abstract References to Sequencing of the mitochondrial genome annotation of the mitochondrial genome HTNGS: study suggested a high-throughput sequencing and bioinformatics pipeline to mt genomics. It has implications for analysis and annotation of other organelles (e.g. the apicoplast genome or plastid genomes) and viruses’ genomes and also long, contiguous areas in nuclear genomes.
HT NGS in the mitochondrial genome study developed and suggested an approach to sequencing as well as de novo assembly many mitochondrial genomes without the expense of indexing. McComish et al. 2010 Sequencing of the complete four mitochondrial genomes of the F-type (15 761 Bp) of the European freshwater bivalve Unio Pictorum (Unionidae) Comparing mitochondrial genomes showed very little nucleotide variation within the species that could be important for the environmental management of policies. Soroka Burzynski and Burzynski The 2010 personal genomics study explores the human genome using a the total integrated archive of short-read as well as array (TIARA) Establishment of a new database that is more precise in identification of individual genomic variations, including SNPs, short-indels and structural variations (SVs). Hong et al. 2011. Best NEET coaching in Silchar.
Genetics (2011) 52:413-435 Genomic variations and identification of genetic mutations NGS promises to make it easier for genome-wide structural variation of the human population research (Xi et al. 2010; Henn et al. 2010; Henn et al.) in revealing all the genetic variation among the human population (Bowne and colleagues. 2011). In fact”10000 Genomes “1000 Genomes Project” has made tremendous advancements towards this target (Durbin and co. 2010). With a detailed genome map that covers all the human variations produced by NGS Researchers will now be able conduct more in-depth studies to identify genetic variations that underlie the responses to drugs. The HT-NGS forms have also found use in high-throughput analysis of mutations as well as carrier screen using the method of Functional genetic fingerprinting (FGF). Best NEET coaching in Silchar.
The technique involves a targeted enhancement of specific genomic areas (the promoterome, the exome, or exon enhancers of splice) approach to respond to the identification of the causal mutations responsible for the development of disease or drug responses (Senapathy and co. 2010). The target enrichment method based on microarrays also enabled simultaneous, large-scale analysis of entire genomic regions for multiple genes in the disease process and for multiple samples at once which makes it an effective instrument for the comprehensive testing of genetic mutations (Amstutz and co. 2011). The HT-NGS carrier screening is also applicable for the general population suffering from severe recessive disorders of childhood (Bell and co. 2011) and in the detection of mutations related to autosomal-recessive cerebellar aphasia through the combination of SNP Linkage analyses based on arrays with targeted sequencing of relevant sequences within links in the interval (Vermeer and colleagues. 2010).
Biomarkers for cancer research and research- NGS technological advances are leading to the development of new ways to diagnose and treat combating cancer (Meyerson and co. 2010) As researchers reverse-sequence cancer and normal genomes, and compare them to certain kinds of tumors (Pfeifer and Hainaut, 2011). The cancer genome allows rapid identification of specific patient changes in the genome of solid tumors (Ding and co. 2010, McBride et al. 2010). Another intriguing application of this technology is the creation of ‘personalized biomarkers that have been created recently to determine the presence of specific genomic rearrangements within plasma samples taken from patients in order to create a tumor-specific biomarker. Rearrangements in the genome were found to be specific to the cancer that were not found in the normal tissue. Digital PCR tests were created across the breakpoints of rearrangement to create a specific biomarker specific to the tumour that was utilized to track the residual disease after treatment (Leary and co. 2010).
Additionally, HTNGS technology helps to determine the cause of mutations that cause metastasis or cancer initiation. It also has significant hopes for better outcomes in oncology (Katsios et al. 2010) and the identification for 4883 SOX2 binding regions within the Glioblastoma (GBM) cancer genome (Fang et al. 2011). Small RNAs could be an additional application for NGS in the discovery of biomarkers (Lee and colleagues. 2010). “Mirror RNAs” (miRNAs) are involved in the regulation of translational processes and are found within blood plasma. NGS can be used to analyze blood plasma and tissues to produce wholegenome miRNA profile that can then be analyzed for biomarker signatures. Applications and developments of sequencing technology on animal genome research Discussion related to the economic importance of farm animals and their impact on the development of HT-NGS technology also is the subject of this paper.
The updated assembled animal genome server is available for the following species: cat (Felis catus), chicken (Gallus gallus), cow (Bos taurus), dog (Canis lupus familiaris), horse (Equus caballus) and pig (Sus scrofa) (http://www.ensembl.org/info/about/ species.html). In addition the preview of genome assembly is available for sheep (Ovis aries) (http://pre. ensembl.org/Ovis_aries/Info/Index) and turkey (Meleagris gallopavo) genome (http://pre.ensembl.org/Meleagris_ gallopavo/Info/Index). The key characteristics of these assembled genomes are listed in Table 3. The initial effort towards sequences of the animal genome began right before the sequencing of the human genome was conducted in 2001 (Lander and colleagues. 2001; Venter et al. 2001; Venter et al.) to promote public funding for research on animal genomics from various universities, private industry producers and Animal Genome Research (AGR) scientific group.
In the beginning, AGR under the National Academy of Sciences (NAS) hosted a public conference about “exploring possibilities for genetics of animals in the domestic environment” (Pool and Waddell 2002). The goal of the workshop was to determine research goals, both public and private funds to create draft genome sequences with high coverage of the main domestic animals, i.e., cattle and pigs, horses and sheep, as well as chicken as well as cat and dog. In the first phase there were 2 “white paper” were published in support of sequencing and assembly the genome of cattle (Gibbs and co. 2002) and pig genome (Rohrer et al. 2002). Since then, significant advancements have been made towards the entire DNA sequence for domestic animals. Best NEET coaching in Silchar.