RNA-Seq called whole transcriptome shotgun sequencing, uses next-generation sequencing to reveal the presence and quantity of RNA in a biological sample at a given moment. RNA-Seq is used to analyze the continuously changing cellular transcriptome. RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression over time, or differences in gene expression in different groups or treatments. In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as miRNA, tRNA, ribosomal profiling. RNA-Seq can be used to determine exon/intron boundaries and verify or amend annotated 5' and 3' gene boundaries. Recent advances in RNA-seq include in situ sequencing of fixed tissue. Prior to RNA-Seq, gene expression studies were done with hybridization-based microarrays. Issues with microarrays include cross-hybridization artifacts, poor quantification of lowly and expressed genes, needing to know the sequence a priori.
Because of these technical issues, transcriptomics transitioned to sequencing-based methods. These progressed from Sanger sequencing of Expressed Sequence Tag libraries, to chemical tag-based methods, to the current technology, next-gen sequencing of cDNA; the general steps to prepare a complementary DNA library for sequencing are described below, but vary between platforms. RNA Isolation: RNA is isolated from tissue and mixed with deoxyribonuclease. DNase reduces the amount of genomic DNA; the amount of RNA degradation is checked with gel and capillary electrophoresis and is used to assign an RNA integrity number to the sample. This RNA quality and the total amount of starting RNA are taken into consideration during the subsequent library preparation and analysis steps. RNA selection/depletion: To analyze signals of interest, the isolated RNA can either be kept as is, filtered for RNA with 3' polyadenylated tails to include only mRNA, depleted of ribosomal RNA, and/or filtered for RNA that binds specific sequences.
The RNA with 3' poly tails are mature, coding sequences. Poly selection is performed by mixing RNA with poly oligomers covalently attached to a substrate magnetic beads. Poly selection ignores noncoding RNA and introduces 3' bias, avoided with the ribosomal depletion strategy; the rRNA is removed because it represents over 90% of the RNA in a cell, which if kept would drown out other data in the transcriptome. CDNA synthesis: DNA sequencing technology is more mature, so the RNA is reverse transcribed to cDNA. Reverse transcription results in loss of strandedness, which can be avoided with chemical labelling. Fragmentation and size selection are performed to purify sequences that are the appropriate length for the sequencing machine; the RNA, cDNA, or both are fragmented with sonication, or nebulizers. Fragmentation of the RNA reduces 5' bias of randomly primed-reverse transcription and the influence of primer binding sites, with the downside that the 5' and 3' ends are converted to DNA less efficiently.
Fragmentation is followed by size selection, where either small sequences are removed or a tight range of sequence lengths are selected. Because small RNAs like miRNAs are lost, these are analyzed independently; the cDNA for each experiment can be indexed with a hexamer or octamer barcode, so that these experiments can be pooled into a single lane for multiplexed sequencing. When sequencing RNA other than mRNA, the library preparation is modified; the cellular RNA is selected based on the desired size range. For small RNA targets, such as miRNA, the RNA is isolated through size selection; this can be performed with a size exclusion gel, through size selection magnetic beads, or with a commercially developed kit. Once isolated, linkers are added to the 3' and 5' end purified; the final step is cDNA generation through reverse transcription. As converting RNA into cDNA using reverse transcriptase has been shown to introduce biases and artifacts that may interfere with both the proper characterization and quantification of transcripts, single molecule Direct RNA Sequencing technology was under development by Helicos.
DRSTM sequences RNA molecules directly in a massively-parallel manner without RNA conversion to cDNA or other biasing sample manipulations such as ligation and amplification. A variety of parameters are considered when designing and conducting RNA-Seq experiments: Tissue specificity: Gene expression varies within and between tissues, RNA-Seq measures this mix of cell types; this may make it difficult to isolate the biological mechanism of interest. Single cell sequencing can be used mitigating this issue. Time dependence: Gene expression changes over time, RNA-Seq only takes a snapshot. Time course experiments can be performed to observe changes in the transcriptome. Coverage: RNA harbors the same mutations observed in DNA, detection requires deeper coverage. With high enough coverage, RNA-Seq can be used to estimate the expression of each allele; this may provide insight into phenomena such as cis-regulatory effects. The depth of sequencing required for specific applications can be extrapolated from a pilot experiment.
Data generation artifacts: The reagents, personnel involved, type of sequencer can result in technical artifacts that might be mis-interpreted as meaningful results. As with any
In the fields of molecular biology and genetics, a genome is the genetic material of an organism. It consists of DNA; the genome includes both the genes and the noncoding DNA, as well as mitochondrial DNA and chloroplast DNA. The study of the genome is called genomics; the term genome was created in 1920 by Hans Winkler, professor of botany at the University of Hamburg, Germany. The Oxford Dictionary suggests the name is a blend of the words chromosome. However, see omics for a more thorough discussion. A few related -ome words existed, such as biome and rhizome, forming a vocabulary into which genome fits systematically. A genome sequence is the complete list of the nucleotides that make up all the chromosomes of an individual or a species. Within a species, the vast majority of nucleotides are identical between individuals, but sequencing multiple individuals is necessary to understand the genetic diversity. In 1976, Walter Fiers at the University of Ghent was the first to establish the complete nucleotide sequence of a viral RNA-genome.
The next year, Fred Sanger completed the first DNA-genome sequence: Phage Φ-X174, of 5386 base pairs. The first complete genome sequences among all three domains of life were released within a short period during the mid-1990s: The first bacterial genome to be sequenced was that of Haemophilus influenzae, completed by a team at The Institute for Genomic Research in 1995. A few months the first eukaryotic genome was completed, with sequences of the 16 chromosomes of budding yeast Saccharomyces cerevisiae published as the result of a European-led effort begun in the mid-1980s; the first genome sequence for an archaeon, Methanococcus jannaschii, was completed in 1996, again by The Institute for Genomic Research. The development of new technologies has made genome sequencing cheaper and easier, the number of complete genome sequences is growing rapidly; the US National Institutes of Health maintains one of several comprehensive databases of genomic information. Among the thousands of completed genome sequencing projects include those for rice, a mouse, the plant Arabidopsis thaliana, the puffer fish, the bacteria E. coli.
In December 2013, scientists first sequenced the entire genome of a Neanderthal, an extinct species of humans. The genome was extracted from the toe bone of a 130,000-year-old Neanderthal found in a Siberian cave. New sequencing technologies, such as massive parallel sequencing have opened up the prospect of personal genome sequencing as a diagnostic tool, as pioneered by Manteia Predictive Medicine. A major step toward that goal was the completion in 2007 of the full genome of James D. Watson, one of the co-discoverers of the structure of DNA. Whereas a genome sequence lists the order of every DNA base in a genome, a genome map identifies the landmarks. A genome map is less detailed than aids in navigating around the genome; the Human Genome Project was organized to sequence the human genome. A fundamental step in the project was the release of a detailed genomic map by Jean Weissenbach and his team at the Genoscope in Paris. Reference genome sequences and maps continue to be updated, removing errors and clarifying regions of high allelic complexity.
The decreasing cost of genomic mapping has permitted genealogical sites to offer it as a service, to the extent that one may submit one's genome to crowdsourced scientific endeavours such as DNA. LAND at the New York Genome Center, an example both of the economies of scale and of citizen science. Viral genomes can be composed of either RNA or DNA; the genomes of RNA viruses can be either single-stranded or double-stranded RNA, may contain one or more separate RNA molecules. DNA viruses can have either double-stranded genomes. Most DNA virus genomes are composed of a single, linear molecule of DNA, but some are made up of a circular DNA molecule. Prokaryotes and eukaryotes have DNA genomes. Archaea have a single circular chromosome. Most bacteria have a single circular chromosome. If the DNA is replicated faster than the bacterial cells divide, multiple copies of the chromosome can be present in a single cell, if the cells divide faster than the DNA can be replicated, multiple replication of the chromosome is initiated before the division occurs, allowing daughter cells to inherit complete genomes and partially replicated chromosomes.
Most prokaryotes have little repetitive DNA in their genomes. However, some symbiotic bacteria have reduced genomes and a high fraction of pseudogenes: only ~40% of their DNA encodes proteins; some bacteria have auxiliary genetic material part of their genome, carried in plasmids. For this, the word genome should not be used as a synonym of chromosome. Eukaryotic genomes are composed of one or more linear DNA chromosomes; the number of chromosomes varies from Jack jumper ants and an asexual nemotode, which each have only one pair, to a fern species that has 720 pairs. A typical human cell has two copies of each of 22 autosomes, one inherited from each parent, plus two sex chromosomes, making it diploid. Gametes, such as ova, sperm and pollen, are haploid, meaning they carry only one copy of each chromosome. In addition to the chromosomes in the nucleus, organelles such as the chloroplasts and mitochondria have their own DNA. Mitochondria are sometimes said to have their own genome referred to as the "mitochondrial genome".
The DNA found within the chloroplast may be referred to as the "plastome". Like the bacteria they originated from and chloroplasts have a circular chromosome
Nucleotides are organic molecules that serve as the monomer units for forming the nucleic acid polymers deoxyribonucleic acid and ribonucleic acid, both of which are essential biomolecules within all life-forms on Earth. Nucleotides are the building blocks of nucleic acids. A nucleoside is a 5-carbon sugar, thus a nucleoside plus a phosphate group yields a nucleotide. Nucleotides play a central role in metabolism at a fundamental, cellular level, they carry packets of chemical energy—in the form of the nucleoside triphosphates Adenosine triphosphate, Guanosine triphosphate, Cytidine triphosphate and Uridine triphosphate —throughout the cell to the many cellular functions that demand energy, which include: synthesizing amino acids and cell membranes and parts, moving the cell and moving cell parts, dividing the cell, etc. In addition, nucleotides participate in cell signaling, are incorporated into important cofactors of enzymatic reactions. In experimental biochemistry, nucleotides can be radiolabeled with radionuclides to yield radionucleotides.
A nucleotide is composed of three distinctive chemical sub-units: a five-carbon sugar molecule, a nitrogenous base—which two together are called a nucleoside—and one phosphate group. With all three joined, a nucleotide is termed a "nucleoside monophosphate"; the chemistry sources ACS Style Guide and IUPAC Gold Book prescribe that a nucleotide should contain only one phosphate group, but common usage in molecular biology textbooks extends the definition to include molecules with two, or with three, phosphates. Thus, the terms "nucleoside diphosphate" or "nucleoside triphosphate" may indicate nucleotides. Nucleotides contain either a purine or a pyrimidine base—i.e. The nitrogenous base molecule known as a nucleobase—and are termed ribonucleotides if the sugar is ribose, or deoxyribonucleotides if the sugar is deoxyribose. Individual phosphate molecules repetitively connect the sugar-ring molecules in two adjacent nucleotide monomers, thereby connecting the nucleotide monomers of a nucleic acid end-to-end into a long chain.
These chain-joins of sugar and phosphate molecules create a'backbone' strand for a single- or double helix. In any one strand, the chemical orientation of the chain-joins runs from the 5'-end to the 3'-end —referring to the five carbon sites on sugar molecules in adjacent nucleotides. In a double helix, the two strands are oriented in opposite directions, which permits base pairing and complementarity between the base-pairs, all, essential for replicating or transcribing the encoded information found in DNA. Unlike in nucleic acid nucleotides, singular cyclic nucleotides are formed when the phosphate group is bound twice to the same sugar molecule, i.e. at the corners of the sugar hydroxyl groups. These individual nucleotides function in cell metabolism rather than the nucleic acid structures of long-chain molecules. Nucleic acids are polymeric macromolecules assembled from nucleotides, the monomer-units of nucleic acids; the purine bases adenine and guanine and pyrimidine base cytosine occur in both DNA and RNA, while the pyrimidine bases thymine and uracil in just one.
Adenine forms a base pair with thymine with two hydrogen bonds, while guanine pairs with cytosine with three hydrogen bonds. Nucleotides can be synthesized by a variety of means both in vitro and in vivo. In vitro, protecting groups may be used during laboratory production of nucleotides. A purified nucleoside is protected to create a phosphoramidite, which can be used to obtain analogues not found in nature and/or to synthesize an oligonucleotide. In vivo, nucleotides can be recycled through salvage pathways; the components used in de novo nucleotide synthesis are derived from biosynthetic precursors of carbohydrate and amino acid metabolism, from ammonia and carbon dioxide. The liver is the major organ of de novo synthesis of all four nucleotides. De novo synthesis of pyrimidines and purines follows two different pathways. Pyrimidines are synthesized first from aspartate and carbamoyl-phosphate in the cytoplasm to the common precursor ring structure orotic acid, onto which a phosphorylated ribosyl unit is covalently linked.
Purines, are first synthesized from the sugar template onto which the ring synthesis occurs. For reference, the syntheses of the purine and pyrimidine nucleotides are carried out by several enzymes in the cytoplasm of the cell, not within a specific organelle. Nucleotides undergo breakdown such that useful parts can be reused in synthesis reactions to create new nucleotides; the synthesis of the pyrimidines CTP and UTP occurs in the cytoplasm and starts with the formation of carbamoyl phosphate from glutamine and CO2. Next, aspartate carbamoyltransferase catalyzes a condensation reaction between aspartate and carbamoyl phosphate to form carbamoyl aspartic acid, cyclized into 4,5-dihydroorotic acid by dihydroorotase; the latter is converted to orotate by dihydroorotate oxidase. The net reaction is: -Dihydroorotate + O2 → Orotate + H2O2Orotate is covalently linked with a phosphorylated ribosyl unit; the covalent linkage between the ribose and pyrimidine occurs at position C1 of the ribose unit, which contains a pyrophosphate, N1 of the pyrimidine ring.
Orotate phosphoribosyltransferase catalyzes the net reaction yielding orotidine monophosphate: Or
The phenotype of an organism is the composite of the organism's observable characteristics or traits, including its morphology or physical form and structure. An organism's phenotype results from two basic factors: the expression of an organism's genetic code, or its genotype, the influence of environmental factors, which may interact, further affecting phenotype; when two or more different phenotypes exist in the same population of a species, the species is called polymorphic. A well-documented polymorphism is Labrador Retriever coloring. Richard Dawkins in 1978 and again in his 1982 book The Extended Phenotype suggested that bird nests and other built structures such as caddis fly larvae cases and beaver dams can be considered as "extended phenotypes"; the genotype-phenotype distinction was proposed by Wilhelm Johannsen in 1911 to make clear the difference between an organism's heredity and what that heredity produces. The distinction is similar to that proposed by August Weismann, who distinguished between germ plasm and somatic cells.
The genotype-phenotype distinction should not be confused with Francis Crick's central dogma of molecular biology, a statement about the directionality of molecular sequential information flowing from DNA to protein, not the reverse. The term "phenotype" has sometimes been incorrectly used as a shorthand for phenotypic difference from wild type, bringing the absurd statement that a mutation has no phenotype. Despite its straightforward definition, the concept of the phenotype has hidden subtleties, it may seem that anything dependent on the genotype is a phenotype, including molecules such as RNA and proteins. Most molecules and structures coded by the genetic material are not visible in the appearance of an organism, yet they are observable and are thus part of the phenotype, it may seem that this goes beyond the original intentions of the concept with its focus on the organism in itself. Either way, the term phenotype includes inherent traits or characteristics that are observable or traits that can be made visible by some technical procedure.
A notable extension to this idea is the presence of "organic molecules" or metabolites that are generated by organisms from chemical reactions of enzymes. Another extension adds behavior to the phenotype. Behavioral phenotypes include cognitive and behavioral patterns; some behavioral phenotypes may characterize psychiatric syndromes. Phenotypic variation is a fundamental prerequisite for evolution by natural selection, it is the living organism as a whole that contributes to the next generation, so natural selection affects the genetic structure of a population indirectly via the contribution of phenotypes. Without phenotypic variation, there would be no evolution by natural selection; the interaction between genotype and phenotype has been conceptualized by the following relationship: genotype + environment → phenotype A more nuanced version of the relationship is: genotype + environment + genotype & environment interactions → phenotype Genotypes have much flexibility in the modification and expression of phenotypes.
The plant Hieracium umbellatum is found growing in two different habitats in Sweden. One habitat is rocky, sea-side cliffs, where the plants are bushy with broad leaves and expanded inflorescences; these habitats alternate along the coast of Sweden and the habitat that the seeds of Hieracium umbellatum land in, determine the phenotype that grows. An example of random variation in Drosophila flies is the number of ommatidia, which may vary between left and right eyes in a single individual as much as they do between different genotypes overall, or between clones raised in different environments; the concept of phenotype can be extended to variations below the level of the gene that affect an organism's fitness. For example, silent mutations that do not change the corresponding amino acid sequence of a gene may change the frequency of guanine-cytosine base pairs; these base pairs have a higher thermal stability than adenine-thymine, a property that might convey, among organisms living in high-temperature environments, a selective advantage on variants enriched in GC content.
Richard Dawkins described a phenotype that included all effects that a gene has on its surroundings, including other organisms, as an extended phenotype, arguing that "An animal's behavior tends to maximize the survival of the genes'for' that behavior, whether or not those genes happen to be in the body of the particular animal performing it." For instance, an organism such as a beaver modifies its environment by building a beaver dam. When a bird feeds a brood parasite such as a cuckoo, it is unwittingly extending its phenotype.