Определение последовательности ДНК: методы и применение

Process of determining the nucleic acid sequence

DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery. Knowledge of DNA sequences has become indispensable for basic biological research, DNA Genographic Projects and in numerous applied fields such as medical diagnosis, biotechnology, forensic biology, virology and biological systematics. Comparing healthy and mutated DNA sequences can diagnose different diseases including various cancers, characterize antibody repertoire, Having a quick way to sequence DNA allows for faster and more individualized medical care to be administered, and for more organisms to be identified and cataloged. DNA sequencing has become easier and orders of magnitude faster.

The field of metagenomics involves identification of organisms present in a body of water, sewage, dirt, debris filtered from the air, or swab samples from organisms. Knowing which organisms are present in a particular environment is critical to research in ecology, epidemiology, microbiology, and other fields. Sequencing enables researchers to determine which types of microbes may be present in a microbiome, for example.

As most viruses are too small to be seen by a light microscope, sequencing is one of the main tools in virology to identify and study the virus. During the 1990 avian influenza outbreak, viral sequencing determined that the influenza sub type originated through reassortment between quail and poultry. This led to legislation in Hong Kong that prohibited selling live quail and poultry together at market. Viral sequencing can also be used to estimate when a viral outbreak began by using a molecular clock technique.

Medical technicians may sequence genes (or, theoretically, full genomes) from patients to determine if there is risk of genetic diseases. This is a form of genetic testing, though some genetic tests may not involve DNA sequencing. As of 2013 DNA sequencing was increasingly used to diagnose and treat rare diseases. As more and more genes are identified that cause rare genetic diseases, molecular diagnoses for patients become more mainstream. DNA sequencing allows clinicians to identify genetic diseases, improve disease management, provide reproductive counseling, and more effective therapies. Gene sequencing panels are used to identify multiple potential genetic causes of a suspected disorder. Also, DNA sequencing may be useful for determining a specific bacteria, to allow for more precise antibiotics treatments, hereby reducing the risk of creating antimicrobial resistance in bacteria populations.

DNA sequencing may be used along with DNA profiling methods for forensic identification and paternity testing. DNA testing has evolved tremendously in the last few decades to ultimately link a DNA print to what is under investigation. The DNA patterns in fingerprint, saliva, hair follicles, etc. uniquely separate each living organism from another. Testing DNA is a technique which can detect specific genomes in a DNA strand to produce a unique and individualized pattern.

The canonical structure of DNA has four bases: thymine (T), adenine (A), cytosine (C), and guanine (G). DNA sequencing is the determination of the physical order of these bases in a molecule of DNA. However, there are many other bases that may be present in a molecule. In some viruses (specifically, bacteriophage), cytosine may be replaced by hydroxy methyl or hydroxy methyl glucose cytosine. In mammalian DNA, variant bases with methyl groups or phosphosulfate may be found. Depending on the sequencing technique, a particular modification, e. g., the 5mC (5 methyl cytosine) common in humans, may or may not be detected. In almost all organisms, DNA is synthesized in vivo using only the 4 canonical bases; modification that occurs post replication creates other bases like 5 methyl C. However, some bacteriophage can incorporate a non standard base directly. In addittion to modifications, DNA is under constant assault by environmental agents such as UV and Oxygen radicals. At the present time, the presence of such damaged bases is not detected by most DNA sequencing methods, although PacBio has published on this https://www. pacb. com/publications/direct detection and sequencing of damaged dna bases/

Deoxyribonucleic acid (DNA) was first discovered and isolated by Friedrich Miescher in 1869, but it remained under studied for many decades because proteins, rather than DNA, were thought to hold the genetic blueprint to life. This situation changed after 1944 as a result of some experiments by Oswald Avery, Colin MacLeod, and Maclyn McCarty demonstrating that purified DNA could change one strain of bacteria into another. This was the first time that DNA was shown capable of transforming the properties of cells. In 1953, James Watson and Francis Crick put forward their double helix model of DNA, based on crystallized X ray structures being studied by Rosalind Franklin. According to the model, DNA is composed of two strands of nucleotides coiled around each other, linked together by hydrogen bonds and running in opposite directions. Each strand is composed of four complementary nucleotides – adenine (A), cytosine (C), guanine (G) and thymine (T) – with an A on one strand always paired with T on the other, and C always paired with G. They proposed that such a structure allowed each strand to be used to reconstruct the other, an idea central to the passing on of hereditary information between generations. The foundation for sequencing proteins was first laid by the work of Frederick Sanger who by 1955 had completed the sequence of all the amino acids in insulin, a small protein secreted by the pancreas. This provided the first conclusive evidence that proteins were chemical entities with a specific molecular pattern rather than a random mixture of material suspended in fluid. Sanger's success in sequencing insulin spurred on x ray crystallographers, including Watson and Crick, who by now were trying to understand how DNA directed the formation of proteins within a cell. Soon after attending a series of lectures given by Frederick Sanger in October 1954, Crick began developing a theory which argued that the arrangement of nucleotides in DNA determined the sequence of amino acids in proteins, which in turn helped determine the function of a protein. He published this theory in 1958.

RNA sequencing was one of the earliest forms of nucleotide sequencing. The major landmark of RNA sequencing is the sequence of the first complete gene and the complete genome of Bacteriophage MS2, identified and published by Walter Fiers and his coworkers at the University of Ghent (Ghent, Belgium), in 1972 and 1976. Traditional RNA sequencing methods require the creation of a cDNA molecule which must be sequenced.

The first method for determining DNA sequences involved a location specific primer extension strategy established by Ray Wu at Cornell University in 1970. DNA polymerase catalysis and specific nucleotide labeling, both of which figure prominently in current sequencing schemes, were used to sequence the cohesive ends of lambda phage DNA. Between 1970 and 1973, Wu, R Padmanabhan and colleagues demonstrated that this method can be employed to determine any DNA sequence using synthetic location specific primers. Frederick Sanger then adopted this primer extension strategy to develop more rapid DNA sequencing methods at the MRC Centre, Cambridge, UK and published a method for "DNA sequencing with chain terminating inhibitors" in 1977. In 1973, Gilbert and Maxam reported the sequence of 24 basepairs using a method known as wandering spot analysis. Advancements in sequencing were aided by the concurrent development of recombinant DNA technology, allowing DNA samples to be isolated from sources other than viruses.

The first full DNA genome to be sequenced was that of bacteriophage φX174 in 1977. Medical Research Council scientists deciphered the complete DNA sequence of the Epstein Barr virus in 1984, finding it contained 172,282 nucleotides. Completion of the sequence marked a significant turning point in DNA sequencing because it was achieved with no prior genetic profile knowledge of the virus. Followed by the commercialization of the DNA sequencer "Direct Blotting Electrophoresis System GATC 1500" by GATC Biotech, which was intensively used in the framework of the EU genome sequencing programme, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II. This was followed by Applied Biosystems' marketing of the first fully automated sequencing machine, the ABI 370, in 1987 and by Dupont's Genesis 2000 which used a novel fluorescent labeling technique enabling all four dideoxynucleotides to be identified in a single lane. By 1990, the U. S. National Institutes of Health (NIH) had begun large scale sequencing trials on Mycoplasma capricolum, Escherichia coli, Caenorhabditis elegans, and Saccharomyces cerevisiae at a cost of US$0.75 per base. Meanwhile, sequencing of human cDNA sequences called expressed sequence tags began in Craig Venter's lab, an attempt to capture the coding fraction of the human genome. In 1995, Venter, Hamilton Smith, and colleagues at The Institute for Genomic Research (TIGR) published the first complete genome of a free living organism, the bacterium Haemophilus influenzae. The circular chromosome contains 1,830,137 bases and its publication in the journal Science marked the first published use of whole genome shotgun sequencing, eliminating the need for initial mapping efforts. By 2001, shotgun sequencing methods had been used to produce a draft sequence of the human genome.]] Several new methods for DNA sequencing were developed in the mid to late 1990s and were implemented in commercial DNA sequencers by 2000. Together these were called the "next generation" or "second generation" sequencing (NGS) methods, in order to distinguish them from the earlier methods, including Sanger sequencing. In contrast to the first generation of sequencing, NGS technology is typically characterized by being highly scalable, allowing the entire genome to be sequenced at once. Usually, this is accomplished by fragmenting the genome into small pieces, randomly sampling for a fragment, and sequencing it using one of a variety of technologies, such as those described below. An entire genome is possible because multiple fragments are sequenced at once (giving it the name "massively parallel" sequencing) in an automated process. NGS technology has tremendously empowered researchers to look for insights into health, anthropologists to investigate human origins, and is catalyzing the "Personalized Medicine" movement. However, it has also opened the door to more room for error. There are many software tools to carry out the computational analysis of NGS data, often compiled at online platforms such as CSI NGS Portal, each with its own algorithm. Even the parameters within one software package can change the outcome of the analysis. In addition, the large quantities of data produced by DNA sequencing have also required development of new methods and programs for sequence analysis. Several efforts to develop standards in the NGS field have been attempted to address these challenges, most of which have been small scale efforts arising from individual labs. Most recently, a large, organized, FDA funded effort has culminated in the BioCompute standard. On 26 October 1990, Roger Tsien, Pepi Ross, Margaret Fahnestock and Allan J Johnston filed a patent describing stepwise ("base by base") sequencing with removable 3' blockers on DNA arrays (blots and single DNA molecules). In 1996, Pål Nyrén and his student Mostafa Ronaghi at the Royal Institute of Technology in Stockholm published their method of pyrosequencing. On 1 April 1997, Pascal Mayer and Laurent Farinelli submitted patents to the World Intellectual Property Organization describing DNA colony sequencing. The DNA sample preparation and random surface polymerase chain reaction (PCR) arraying methods described in this patent, coupled to Roger Tsien et al. 's "base by base" sequencing method, is now implemented in Illumina's Hi Seq genome sequencers. In 1998, Phil Green and Brent Ewing of the University of Washington described their phred quality score for sequencer data analysis, a landmark analysis technique that gained widespread adoption, and which is still the most common metric for assessing the accuracy of a sequencing platform. Lynx Therapeutics published and marketed massively parallel signature sequencing (MPSS), in 2000. This method incorporated a parallelized, adapter/ligation mediated, bead based sequencing technology and served as the first commercially available "next generation" sequencing method, though no DNA sequencers were sold to independent laboratories.

Allan Maxam and Walter Gilbert published a DNA sequencing method in 1977 based on chemical modification of DNA and subsequent cleavage at specific bases. Also known as chemical sequencing, this method allowed purified samples of double stranded DNA to be used without further cloning. This method's use of radioactive labeling and its technical complexity discouraged extensive use after refinements in the Sanger methods had been made. Maxam Gilbert sequencing requires radioactive labeling at one 5' end of the DNA and purification of the DNA fragment to be sequenced. Chemical treatment then generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule. The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation. To visualize the fragments, the gel is exposed to X ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred.

The chain termination method developed by Frederick Sanger and coworkers in 1977 soon became the method of choice, owing to its relative ease and reliability. When invented, the chain terminator method used fewer toxic chemicals and lower amounts of radioactivity than the Maxam and Gilbert method. Because of its comparative ease, the Sanger method was soon automated and was the method used in the first generation of DNA sequencers. Sanger sequencing is the method which prevailed from the 1980s until the mid 2000s. Over that period, great advances were made in the technique, such as fluorescent labelling, capillary electrophoresis, and general automation. These developments allowed much more efficient sequencing, leading to lower costs. The Sanger method, in mass production form, is the technology which produced the first human genome in 2001, ushering in the age of genomics. However, later in the decade, radically different approaches reached the market, bringing the cost per genome down from $100 million in 2001 to $10,000 in 2011.

The objective for sequential sequencing by synthesis (SBS) is to determine the sequencing of a DNA sample by detecting the incorporation of a nucleotide by a DNA polymerase. An engineered polymerase is used to synthesize a copy of a single strand of DNA and the incorporation of each nucleotide is monitored. The principle of real time sequencing by synthesis was first described in 1993 with improvements published some years later. The key parts are highly similar for all embodiments of SBS and includes (1) amplification of DNA (to enhance the subsequent signal) and attach the DNA to be sequenced to a solid support, (2) generation of single stranded DNA on the solid support, (3) incorporation of nucleotides using an engineered polymerase and (4) real time detection of the incorporation of nucleotide The steps 3 4 are repeated and the sequence is assembled from the signals obtained in step 4. This principle of real time sequencing by synthesis has been used for almost all massive parallel sequencing instruments, including 454, PacBio, IonTorrent, Illumina and MGI.

Large scale sequencing often aims at sequencing very long DNA pieces, such as whole chromosomes, although large scale sequencing can also be used to generate very large numbers of short sequences, such as found in phage display. For longer targets such as chromosomes, common approaches consist of cutting (with restriction enzymes) or shearing (with mechanical forces) large DNA fragments into shorter DNA fragments. The fragmented DNA may then be cloned into a DNA vector and amplified in a bacterial host such as Escherichia coli. Short DNA fragments purified from individual bacterial colonies are individually sequenced and assembled electronically into one long, contiguous sequence. Studies have shown that adding a size selection step to collect DNA fragments of uniform size can improve sequencing efficiency and accuracy of the genome assembly. In these studies, automated sizing has proven to be more reproducible and precise than manual gel sizing. The term "de novo sequencing" specifically refers to methods used to determine the sequence of DNA with no previously known sequence. De novo translates from Latin as "from the beginning". Gaps in the assembled sequence may be filled by primer walking. The different strategies have different tradeoffs in speed and accuracy; shotgun methods are often used for sequencing large genomes, but its assembly is complex and difficult, particularly with sequence repeats often causing gaps in genome assembly. Most sequencing approaches use an in vitro cloning step to amplify individual DNA molecules, because their molecular detection methods are not sensitive enough for single molecule sequencing. Emulsion PCR isolates individual DNA molecules along with primer coated beads in aqueous droplets within an oil phase. A polymerase chain reaction (PCR) then coats each bead with clonal copies of the DNA molecule followed by immobilization for later sequencing. Emulsion PCR is used in the methods developed by Marguilis et al. (commercialized by 454 Life Sciences), Shendure and Porreca et al. (also known as "polony sequencing") and SOLiD sequencing, (developed by Agencourt, later Applied Biosystems, now Life Technologies). Emulsion PCR is also used in the GemCode and Chromium platforms developed by 10x Genomics.

Shotgun sequencing is a sequencing method designed for analysis of DNA sequences longer than 1000 base pairs, up to and including entire chromosomes. This method requires the target DNA to be broken into random fragments. After sequencing individual fragments using the chain termination method, the sequences can be reassembled on the basis of their overlapping regions.

SMRT sequencing is based on the sequencing by synthesis approach. The DNA is synthesized in zero mode wave guides (ZMWs) – small well like containers with the capturing tools located at the bottom of the well. The sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labelled nucleotides flowing freely in the solution. The wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected. The fluorescent label is detached from the nucleotide upon its incorporation into the DNA strand, leaving an unmodified DNA strand. According to Pacific Biosciences (PacBio), the SMRT technology developer, this methodology allows detection of nucleotide modifications (such as cytosine methylation). This happens through the observation of polymerase kinetics. This approach allows reads of 20,000 nucleotides or more, with average read lengths of 5 kilobases. In 2015, Pacific Biosciences announced the launch of a new sequencing instrument called the Sequel System, with 1 million ZMWs compared to 150,000 ZMWs in the PacBio RS II instrument. SMRT sequencing is referred to as "third generation" or "long read" sequencing.

The DNA passing through the nanopore changes its ion current. This change is dependent on the shape, size and length of the DNA sequence. Each type of the nucleotide blocks the ion flow through the pore for a different period of time. The method does not require modified nucleotides and is performed in real time. Nanopore sequencing is referred to as "third generation" or "long read" sequencing, along with SMRT sequencing. Early industrial research into this method was based on a technique called 'exonuclease sequencing', where the readout of electrical signals occurred as nucleotides passed by alpha(α) hemolysin pores covalently bound with cyclodextrin. However the subsequent commercial method, 'strand sequencing', sequenced DNA bases in an intact strand. Two main areas of nanopore sequencing in development are solid state nanopore sequencing, and protein based nanopore sequencing. Protein nanopore sequencing utilizes membrane protein complexes such as α hemolysin, MspA (Mycobacterium smegmatis Porin A) or CssG, which show great promise given their ability to distinguish between individual and groups of nucleotides. In contrast, solid state nanopore sequencing utilizes synthetic materials such as silicon nitride and aluminum oxide and it is preferred for its superior mechanical ability and thermal and chemical stability. The fabrication method is essential for this type of sequencing given that the nanopore array can contain hundreds of pores with diameters smaller than eight nanometers.

The first of the high throughput sequencing technologies, massively parallel signature sequencing (or MPSS, also called next generation sequencing), was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner and Sam Eletr. MPSS was a bead based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides. This method made it susceptible to sequence specific bias or loss of specific sequences. Because the technology was so complex, MPSS was only performed 'in house' by Lynx Therapeutics and no DNA sequencing machines were sold to independent laboratories. Lynx Therapeutics merged with Solexa (later acquired by Illumina) in 2004, leading to the development of sequencing by synthesis, a simpler approach acquired from Manteia Predictive Medicine, which rendered MPSS obsolete. However, the essential properties of the MPSS output were typical of later high throughput data types, including hundreds of thousands of short DNA sequences. In the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels. It combined an in vitro paired tag library with emulsion PCR, an automated microscope, and ligation based sequencing chemistry to sequence an E. coli genome at an accuracy of >99.9999% and a cost approximately 1/9 that of Sanger sequencing. It was developed internally at Solexa by those named on the relevant patents. In 2004, Solexa acquired the company Manteia Predictive Medicine in order to gain a massively parallel sequencing technology invented in 1997 by Pascal Mayer and Laurent Farinelli.

This method is an upgraded modification to combinatorial probe anchor ligation technology (cPAL) described by Complete Genomics which has since become part of Chinese genomics company BGI in 2013. The two companies have refined the technology to allow for longer read lengths, reaction time reductions and faster time to results. In addition, data are now generated as contiguous full length reads in the standard FASTQ file format and can be used as is in most short read based bioinformatics analysis pipelines. The two technologies that form the basis for this high throughput sequencing technology are DNA nanoballs (DNB) and patterned arrays for nanoball attachment to a solid surface. The result is sequences of quantities and lengths comparable to Illumina sequencing.

DNA nanoball sequencing is a type of high throughput sequencing technology used to determine the entire genomic sequence of an organism. The company Complete Genomics uses this technology to sequence samples submitted by independent researchers. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. Unchained sequencing by ligation is then used to determine the nucleotide sequence. However, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult. The reads are short, averaging 35 bp. What made this technology especially novel was that it was the first of its class to sequence non amplified DNA, thus preventing any read errors associated with amplification steps. In 2009 a human genome was sequenced using the Heliscope, however in 2012 the company went bankrupt.

There are two main microfluidic systems that are used to sequence DNA; droplet based microfluidics and digital microfluidics. Microfluidic devices solve many of the current limitations of current sequencing arrays. Abate et al. studied the use of droplet based microfluidic devices for DNA sequencing. These devices have the ability to form and process picoliter sized droplets at the rate of thousands per second. The devices were created from polydimethylsiloxane (PDMS) and used Forster resonance energy transfer, FRET assays to read the sequences of DNA encompassed in the droplets. Each position on the array tested for a specific 15 base sequence. Significant advantages include the portability of the device, reagent volume, speed of analysis, mass manufacturing abilities, and high throughput. This study provided a proof of concept showing that digital devices can be used for pyrosequencing; the study included using synthesis, which involves the extension of the enzymes and addition of labeled nucleotides. They used an electro wetting device to create, mix, and split droplets. The sequencing uses a three enzyme protocol and DNA templates anchored with magnetic beads. The device was tested using two protocols and resulted in 100% accuracy based on raw pyrogram levels. The advantages of these digital microfluidic devices include size, cost, and achievable levels of functional integration. This shows that many of these DNA sequencing techniques will be able to be applied further and be used to understand more about genomes and transcriptomes.

DNA sequencing methods currently under development include reading the sequence as a DNA strand transits through nanopores (a method that is now commercial but subsequent generations such as solid state nanopores are still in development), and microscopy based techniques, such as atomic force microscopy or transmission electron microscopy that are used to identify the positions of individual nucleotides within long DNA fragments (>5,000 bp) by nucleotide labeling with heavier elements (e. g., halogens) for visual detection and recording. Third generation technologies aim to increase throughput and decrease the time to result and cost by eliminating the need for excessive reagents and harnessing the processivity of DNA polymerase.

Another approach uses measurements of the electrical tunnelling currents across single strand DNA as it moves through a channel. Depending on its electronic structure, each base affects the tunnelling current differently, allowing differentiation between different bases. The use of tunnelling currents has the potential to sequence orders of magnitude faster than ionic current methods and the sequencing of several DNA oligomers and micro RNA has already been achieved.

Sequencing by hybridization is a non enzymatic method that uses a DNA microarray. A single pool of DNA whose sequence is to be determined is fluorescently labeled and hybridized to an array containing known sequences. Strong hybridization signals from a given spot on the array identifies its sequence in the DNA being sequenced. This method of sequencing utilizes binding characteristics of a library of short single stranded DNA molecules (oligonucleotides), also called DNA probes, to reconstruct a target DNA sequence. Non specific hybrids are removed by washing and the target DNA is eluted. Hybrids are re arranged such that the DNA sequence can be reconstructed. The benefit of this sequencing type is its ability to capture a large number of targets with a homogenous coverage. A large number of chemicals and starting DNA is usually required. However, with the advent of solution based hybridization, much less equipment and chemicals are necessary. The higher resolution of DNA fragments permitted by MS based methods is of special interest to researchers in forensic science, as they may wish to find single nucleotide polymorphisms in human DNA samples to identify individuals. These samples may be highly degraded so forensic researchers often prefer mitochondrial DNA for its higher stability and applications for lineage studies. MS based sequencing methods have been used to compare the sequences of human mitochondrial DNA from samples in a Federal Bureau of Investigation database and from bones found in mass graves of World War I soldiers. Early chain termination and TOF MS methods demonstrated read lengths of up to 100 base pairs. Researchers have been unable to exceed this average read size; like chain termination sequencing alone, MS based DNA sequencing may not be suitable for large de novo sequencing projects. Even so, a recent study did use the short sequence reads and mass spectroscopy to compare single nucleotide polymorphisms in pathogenic Streptococcus strains.

In microfluidic Sanger sequencing the entire thermocycling amplification of DNA fragments as well as their separation by electrophoresis is done on a single glass wafer (approximately 10 cm in diameter) thus reducing the reagent usage as well as cost. In some instances researchers have shown that they can increase the throughput of conventional sequencing through the use of microchips. Research will still need to be done in order to make this use of technology effective.

This approach directly visualizes the sequence of DNA molecules using electron microscopy. The first identification of DNA base pairs within intact DNA molecules by enzymatically incorporating modified bases, which contain atoms of increased atomic number, direct visualization and identification of individually labeled bases within a synthetic 3,272 base pair DNA molecule and a 7,249 base pair viral genome has been demonstrated.

This method is based on use of RNA polymerase (RNAP), which is attached to a polystyrene bead. One end of DNA to be sequenced is attached to another bead, with both beads being placed in optical traps. RNAP motion during transcription brings the beads in closer and their relative distance changes, which can then be recorded at a single nucleotide resolution. The sequence is deduced based on the four readouts with lowered concentrations of each of the four nucleotide types, similarly to the Sanger method. A comparison is made between regions and sequence information is deduced by comparing the known sequence regions to the unknown sequence regions.

A method has been developed to analyze full sets of protein interactions using a combination of 454 pyrosequencing and an in vitro virus mRNA display method. Specifically, this method covalently links proteins of interest to the mRNAs encoding them, then detects the mRNA pieces using reverse transcription PCRs. The mRNA may then be amplified and sequenced. The combined method was titled IVV HiTSeq and can be performed under cell free conditions, though its results may not be representative of in vivo conditions.

While there are many different ways to sequence DNA, only a few dominate the market. In 2022, Illumina had about 80% of the market ; the rest of the market is taken by only a few players (PacBio, Oxford, 454, MGI)

The success of any DNA sequencing protocol relies upon the DNA or RNA sample extraction and preparation from the biological material of interest. A successful DNA extraction will yield a DNA sample with long, non degraded strands. A successful RNA extraction will yield a RNA sample that should be converted to complementary DNA (cDNA) using reverse transcriptase—a DNA polymerase that synthesizes a complementary DNA based on existing strands of RNA in a PCR like manner. Complementary DNA can then be processed the same way as genomic DNA. After DNA or RNA extraction, samples may require further preparation depending on the sequencing method. For Sanger sequencing, either cloning procedures or PCR are required prior to sequencing. In the case of next generation sequencing methods, library preparation is required before processing. Assessing the quality and quantity of nucleic acids both after extraction and after library preparation identifies degraded, fragmented, and low purity samples and yields high quality sequencing data. The high throughput nature of current DNA/RNA sequencing technologies has posed a challenge for sample preparation method to scale up. Several liquid handling instruments are being used for the preparation of higher numbers of samples with a lower total hands on time:
companyLiquid handlers / Automationlower mark USDupper mark USDlanding urlOpentronsOpenTrons OT 2$6,500$20,000https://www. opentrons. com/GilsonGilson Pipetmax$20,000$40,000https://gb. gilson. com/GBSV/system pipetmax. htmlNeotecNeotec EzMate$25,000$45,000http://neotec. co. il/pipetting device/FormulatrixFormulatrix Mantis$40,000$60,000https://formulatrix. com/liquid handling systems/mantis liquid handler/Hudson RoboticsHudson Robotics SOLO$40,000$50,000https://hudsonrobotics. com/products/applications/automated solutions next generation sequencing ngs/HamiltonHamilton Microlab NIMBUS$40,000$80,000https://www. hamiltoncompany. com/automated liquid handling/platforms/microlab nimbus#specificationsTTP LabtechTTP Labtech Mosquito HV Genomics$45,000$80,000https://www. sptlabtech. com/products/liquid handling/mosquito hv genomics/Beckman CoulterBiomek 4000$50,000$65,000https://www. mybeckman. uk/liquid handlers/biomek 4000/b22640HamiltonHamilton Genomic STARlet$50,000$100,000https://www. hamiltoncompany. com/automated liquid handling/assay ready workstations/genomic starletEppendorfEppendorf epMotion 5075t$95,000$110,000https://www. eppendorf. com/epmotion/Beckman CoulterBeckman Coulter Biomek i5$100,000$150,000https://www. beckman. com/liquid handlers/biomek i5HamiltonHamilton NGS STAR$100,000$200,000http://www. hamiltonrobotics. com/PerkinElmerPerkinElmer Sciclone G3 NGS and NGSx Workstation$150,000$220,000https://www. perkinelmer. com/uk/product/sciclone g3 ngs workstation cls145321AgilentAgilent Bravo NGS$170,000$290,000https://www. agilent. com/en/products/automated liquid handling/automated liquid handling applications/bravo ngsBeckman CoulterBeckman Coulter Biomek i7$200,000$250,000https://www. beckman. com/liquid handlers/biomek i7Labcyte Echo 525Beckman Coulter Labcyte Echo 525$260,000$300,000https://www. labcyte. com/products/liquid handling/echo 525 liquid handlerTecanTecan NGS$270,000$350,000https://lifesciences. tecan. com/ngs sample preparation

In October 2006, the X Prize Foundation established an initiative to promote the development of full genome sequencing technologies, called the Archon X Prize, intending to award $10 million to "the first Team that can build a device and use it to sequence 100 human genomes within 10 days or less, with an accuracy of no more than one error in every 100,000 bases sequenced, with sequences accurately covering at least 98% of the genome, and at a recurring cost of no more than $10,000 (US) per genome." Each year the National Human Genome Research Institute, or NHGRI, promotes grants for new research and developments in genomics. 2010 grants and 2011 candidates include continuing work in microfluidic, polony and base heavy sequencing methodologies.

The sequencing technologies described here produce raw data that needs to be assembled into longer sequences such as complete genomes (sequence assembly). There are many computational challenges to achieve this, such as the evaluation of the raw sequence data which is done by programs and algorithms such as Phred and Phrap. Other challenges have to deal with repetitive sequences that often prevent complete genome assemblies because they occur in many places of the genome. As a consequence, many sequences may not be assigned to particular chromosomes. The production of raw sequence data is only the beginning of its detailed bioinformatical analysis. Yet new methods for sequencing and correcting sequencing errors were developed.

Human genetics have been included within the field of bioethics since the early 1970s and the growth in the use of DNA sequencing (particularly high throughput sequencing) has introduced a number of ethical issues. One key issue is the ownership of an individual's DNA and the data produced when that DNA is sequenced. Regarding the DNA molecule itself, the leading legal case on this topic, Moore v. Regents of the University of California (1990) ruled that individuals have no property rights to discarded cells or any profits made using these cells (for instance, as a patented cell line). However, individuals have a right to informed consent regarding removal and use of cells. Regarding the data produced through DNA sequencing, Moore gives the individual no rights to the information derived from their DNA. For instance, one concern is that insurers may use an individual's genomic data to modify their quote, depending on the perceived future health of the individual based on their DNA. In May 2008, the Genetic Information Nondiscrimination Act (GINA) was signed in the United States, prohibiting discrimination on the basis of genetic information with respect to health insurance and employment. In 2012, the US Presidential Commission for the Study of Bioethical Issues reported that existing privacy legislation for DNA sequencing data such as GINA and the Health Insurance Portability and Accountability Act were insufficient, noting that whole genome sequencing data was particularly sensitive, as it could be used to identify not only the individual from which the data was created, but also their relatives. In most of the United States, DNA that is "abandoned", such as that found on a licked stamp or envelope, coffee cup, cigarette, chewing gum, household trash, or hair that has fallen on a public sidewalk, may legally be collected and sequenced by anyone, including the police, private investigators, political opponents, or people involved in paternity disputes. As of 2013, eleven states have laws that can be interpreted to prohibit "DNA theft". Ethical issues have also been raised by the increasing use of genetic variation screening, both in newborns, and in adults by companies such as 23andMe. It has been asserted that screening for genetic variations can be harmful, increasing anxiety in individuals who have been found to have an increased risk of disease. For example, in one case noted in Time, doctors screening an ill baby for genetic variants chose not to inform the parents of an unrelated variant linked to dementia due to the harm it would cause to the parents. However, a 2011 study in The New England Journal of Medicine has shown that individuals undergoing disease risk profiling did not show increased levels of anxiety.