sequences at once. high-throughput sequencing is intended to lower the cost of dna sequencing beyond what is possible with standard dye-terminator methods. in ultra-high-throughput sequencing, as many as 500,000 sequencing-by-synthesis operations may be run in parallel. illumina genome analyzer ii system. illumina technologies have set the standard for high-throughput massively parallel sequencing. the illumina dye sequencing method is based on reversible dye-terminators and was developed in 1996 at the geneva biomedical research institute, by pascal mayer and laurent farinelli. in this method, dna molecules and primers are first attached on a slide and amplified with polymerase so that local clonal colonies, initially coined "dna colonies", are formed. to determine the sequence, four types of reversible terminator bases (rt-bases) are added and non-incorporated nucleotides are washed away. unlike pyrosequencing, the dna chains are extended one nucleotide at a time and image acquisition can be performed at a delayed moment, allowing for very large arrays of dna colonies to be captured by sequential images taken from a single camera. decoupling the enzymatic reaction and the image capture allows for optimal throughput and theoretically unlimited sequencing capacity; with an optimal configuration, the ultimate throughput of the instrument depends only on the a/d conversion rate of the camera. the camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the dna, allowing the next cycle. an alternative approach, ion semiconductor sequencing, is based on standard dna replication chemistry. this technology measures the release of a hydrogen ion each time a base is incorporated. a microwell containing template dna is flooded with a single nucleotide, if the nucleotide is complementary to the template strand it will be incorporated and a hydrogen ion will be released. this release triggers an isfet ion sensor. if a homopolymer is present in the template sequence multiple nucleotides will be incorporated in a single flood cycle, and the detected electrical signal will be proportionally higher. assembly overlapping reads form contigs; contigs and gaps of known length form scaffolds.paired end reads of next generation sequencing data mapped to a reference genome.multiple, fragmented sequence reads must be assembled together on the basis of their overlapping areas. sequence assembly refers to aligning and merging fragments of a much longer dna sequence in order to reconstruct the original sequence. this is needed as current dna sequencing technology cannot read whole genomes as a continuous sequence, but rather reads small pieces of between 20 and 1000 bases, depending on the technology used. third generation sequencing technologies such as pacbio or oxford nanopore routinely generate sequencing reads >10 kb in length; however, they have a high error rate at approximately 15 percent. typically the short fragments, called reads, result from shotgun sequencing genomic dna, or gene transcripts (ests). assembly approaches assembly can be broadly categorized into two approaches: de novo assembly, for genomes which are not similar to any sequenced in the past, and comparative assembly, which uses the existing sequence of a closely related organism as a reference during assembly. relative to comparative assembly, de novo assembly is computationally difficult (np-hard), making it less favourable for short-read ngs technologies. within the de novo assembly paradigm there are two primary strategies for assembly, eulerian path strategies, and overlap-layout-consensus (olc) strategies. olc strategies ultimately try to create a hamiltonian path through an overlap graph which is an np-hard problem. eulerian path strategies are computationally more tractable because they try to find a eulerian path through a debruijn graph. finishing finished genomes are defined as having a single contiguous sequence with no ambiguities representing each replicon. annotation the dna sequence assembly alone is of little value without additional analysis. genome annotation is the process of attaching biological information to sequences, and consists of three main steps: identifying portions of the genome that do not code for proteins identifying elements on the genome, a process called gene prediction, and attaching biological information to these elements. automatic annotation tools try to perform these steps in silico, as opposed to manual annotation (a.k.a. curation) which involves human expertise and potential experimental verification. ideally, these approaches co-exist and complement each other in the same annotation pipeline (also see below). traditionally, the basic level of annotation is using blast for finding similarities, and then annotating genomes based on homologues. more recently, additional information is added to the annotation platform. the additional information allows manual annotators to deconvolute discrepancies between genes that are given the same annotation. some databases use genome context information, similarity scores, experimental data, and integrations of other resources to provide genome annotations through their subsystems approach. other databases (e.g. ensembl) rely on both curated data sources as well as a range of software tools in their automated genome annotation pipeline. structural annotation consists of the identification of genomic elements, primarily orfs and their localisation, or gene structure. functional annotation consists of attaching biological information to genomic elements. sequencing pipelines and databases the need for reproducibility and efficient management of the large amount of data associated with genome projects mean that computational pipelines have important applications in genomics. research areas functional genomics functional genomics is a field of molecular biology that attempts to make use of the vast wealth of data produced by genomic projects (such as genome sequencing projects) to describe gene (and protein) functions and interactions. functional genomics focuses on the dynamic aspects such as gene transcription, translation, and protein–protein interactions, as opposed to the static aspects of the genomic information such as dna sequence or structures. functional genomics attempts to answer questions about the function of dna at the levels of genes, rna transcripts, and protein products. a key characteristic of functional genomics studies is their genome-wide approach to these questions, generally involving high-throughput methods rather than a more traditional “gene-by-gene” approach. a major branch of genomics is still concerned with sequencing the genomes of various organisms, but the knowledge of full genomes has created the possibility for the field of functional genomics, mainly concerned with patterns of gene expression during various conditions. the most important tools here are microarrays and bioinformatics. structural genomics main article: structural genomics an example of a protein structure determined by the midwest center for structural genomics. structural genomics seeks to describe the 3-dimensional structure of every protein encoded by a given genome. this genome-based approach allows for a high-throughput method of structure determination by a combination of experimental and modeling approaches. the principal difference between structural genomics and traditional structural prediction is that structural genomics attempts to determine the structure of every protein encoded by the genome, rather than focusing on one particular protein. with full-genome sequences available, structure prediction can be done more quickly through a combination of experimental and modeling approaches, especially because the availability of large numbers of sequenced genomes and previously solved protein structures allow scientists to model protein structure on the structures of previously solved homologs. structural genomics involves taking a large number of approaches to structure determination, including experimental methods using genomic sequences or modeling-based approaches based on sequence or structural homology to a protein of known structure or based on chemical and physical principles for a protein with no homology to any known structure. as opposed to traditional structural biology, the determination of a protein structure through a structural genomics effort often (but not always) comes before anything is known regarding the protein function. this raises new challenges in structural bioinformatics, i.e. determining protein function from its 3d structure. epigenomics main article: epigenomics epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. epigenetic modifications are reversible modifications on a cell's dna or histones that affect gene expression without altering the dna sequence (russell 2010 p. 475). two of the most characterized epigenetic modifications are dna methylation and histone modification. epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis. the study of epigenetics on a global level has been made possible only recently through the adaptation of genomic high-throughput assays. metagenomics environmental shotgun sequencing (ess) is a key technique in metagenomics. (a) sampling from habitat; (b) filtering particles, typically by size; (c) lysis and dna extraction; (d) cloning and library construction; (e) sequencing the clones; (f) sequence assembly into contigs and scaffolds. main article: metagenomics metagenomics is the study of metagenomes, genetic material recovered directly from environmental samples. the broad field may also be referred to as environmental genomics, ecogenomics or community genomics. while traditional microbiology and microbial genome sequencing rely upon cultivated clonal cultures, early environmental gene sequencing cloned specific genes (often the 16s rrna gene) to produce a profile of diversity in a natural sample. such work revealed that the vast majority of microbial biodiversity had been missed by cultivation-based methods. recent studies use "shotgun" sanger sequencing or massively parallel pyrosequencing to get largely unbiased samples of all genes from all the members of the sampled communities. because of its power to reveal the previously hidden diversity of microscopic life, metagenomics offers a powerful lens for viewing the microbial world that has the potential to revolutionize understanding of the entire living world. viruses and bacteriophages bacteriophages have played and continue to play a key role in bacterial genetics and molecular biology. historically, they were used to define gene structure and gene regulation. also the first genome to be sequenced was a bacteriophage. however, bacteriophage research did not lead the genomics revolution, which is clearly dominated by bacterial genomics. only very recently has the study of bacteriophage genomes become prominent, thereby enabling researchers to understand the mechanisms underlying phage evolution. bacteriophage genome sequences can be obtained through direct sequencing of isolated bacteriophages, but can also be derived as part of microbial genomes. analysis of bacterial genomes has shown that a substantial amount of microbial dna consists of prophage sequences and prophage-like elements. a detailed database mining of these sequences offers insights into the role of prophages in shaping the bacterial genome: overall, this method verified many known bacteriophage groups, making this a useful tool for predicting the relationships of prophages from bacterial genomes. cyanobacteria at present there are 24 cyanobacteria for which a total genome sequence is available. 15 of these cyanobacteria come from the marine environment. these are six prochlorococcus strains, seven marine synechococcus strains, trichodesmium erythraeum ims101 and crocosphaera watsonii wh8501. several studies have demonstrated how these sequences could be used very successfully to infer important ecological and physiological characteristics of marine cyanobacteria. however, there are many more genome projects currently in progress, amongst those there are further prochlorococcus and marine synechococcus isolates, acaryochloris and prochloron, the n2-fixing filamentous cyanobacteria nodularia spumigena, lyngbya aestuarii and lyngbya majuscula, as well as bacteriophages infecting marine cyanobaceria. thus, the growing body of genome information can also be tapped in a more general way to address global problems by applying a comparative approach. some new and exciting examples of progress in this field are the identification of genes for regulatory rnas, insights into the evolutionary origin of photosynthesis, or estimation of the contribution of horizontal gene transfer to the genomes that have been analyzed. applications schematic karyogram of a human, providing a simplified overview of the human genome. it is a graphical representation of the idealized human diploid karyotype, with annotated bands and sub-bands. it shows dark and white regions on g banding. each row is vertically aligned at centromere level. it shows 22 homologous autosomal chromosome pairs, both the female (xx) and male (xy) versions of the two sex chromosomes, as well as the mitochondrial genome (at bottom left). further information: karyotype genomics has provided applications in many fields, including medicine, biotechnology, anthropology and other social sciences. genomic medicine next-generation genomic technologies allow clinicians and biomedical researchers to drastically increase the amount of genomic data collected on large study populations. when combined with new informatics approaches that integrate many kinds of data with genomic data in disease research, this allows researchers to better understand the genetic bases of drug response and disease. early efforts to apply the genome to medicine included those by a stanford team led by euan ashley who developed the first tools for the medical interpretation of a human genome. the genomes2people