There is particular interest to researchers crimes higher resolution to resolve DNA fragments by the method of MS-based, they can in order to identify an individual, and decides to search the nucleotide polymorphism in a sample of human DNA possible. The forensic investigator, you might want to prefer the mitochondrial DNA of continuity applications and research higher stability than often, greatly reduced these samples. Sequencing method based MS were used to compare the sequence of the mitochondrial DNA samples from human bones found in mass graves soldiers WWII database and FBI.

Method chain and early termination of TOF MS showed reads of base pairs up to 100. As sequencing alone chain termination, not able to exceed the average read size and DNA sequencing of the MS-based researchers may not be suitable for projects Denoboshiken large. The Even so, a recent study, short sequence reads, and has benefited from mass spectrometry in order to compare the single nucleotide polymorphism in the pathogenic strain of streptococcus.

Mass spectrometry (MS) is an analytical technique that generated the (single array) Mass spectrum of the molecule, including samples of materials. Is used to determine the elemental composition of the sample mass and molecular particles, spectra were elucidation of the chemical structure of the compound and other molecules, such as peptides. Mass spectrometry operates by chemical compound that generates ions by measuring the mass-to-charge ratios and their molecular fragments or charged molecules.

In a typical procedure, the MS, the sample may be solid, liquid or a gas is ionized. Ions are separated according to their mass to cost ratio. Ions are detected by the detection mechanism of charged particles. Result of the signal processing is shown as spectrum of the relative amount of ions as a function of the mass ratio of the cost. It is possible to identify the combined masses of the characteristic fragmentation table or the identified molecules or atoms. Detector ion source and mass analyzer: mass spectrometer comprises three components.

I will convert a portion of the sample to the ion in the ionizer. A technique of ion species ways depending (solid, liquid, gaseous) phase in the efficiency and sample various mechanisms ionization unknown. Evacuation system removes ions from the sample was trajected through the detector and mass analyzer on the next. The difference of the mass of the fragments allows mass analysis to sort the mass ion their to cost ratio. By measuring the value of the index number, the detector provides data for the calculation of the abundance of each ion present. Further detector Some provide spatial information, such as multi-channel plate.

Mass spectrometry, has a goal of both qualitative and quantitative. That it determines the identification of unknown compounds, the isotopic composition of elements in the molecule and to determine the structure of the compounds according to the picture contains them. Other applications, contains the (neutral in a vacuum and the chemical nature of the ions) to study the basis of gas-phase ion chemistry or to determine the amount of the compound in the sample quantitative. MS is now using a very common in research laboratories biological properties physical, chemical or various compounds.

Mass analyzer, an electric field is created between the bars 4 parallel Alternatively, oscillating erratically, or stabilize the trajectory of ions passing through a radio frequency (RF) a quadrupole field quadrupole uses. Only ions passed through the system at any point within a certain range the mass / charge ratio, but a wide range of values ​​of M / Z of the sample quickly to individual hop series change in the potential of the bar continuously or made possible. Mass analyzer, a point called acts as a mass selective filter, ion trap quadrupole closely, instead of passing the ions are not trapped, it is because it is designed to collect those trapped quadrupole as a transmission quadrupole associated with the ion trap quadrupole linear especially Except.

In a general embodiment of the transmission, it is a quadrupole of triple quadrupole mass spectrometer. The first four acting as a filter for transmitting the table four “three”, the input of a specific collision compositions possible successive stages 3 and the ion, ions are divided into fragments electrode Fourth Weight It is composed of a quadrupole. It acts as a mass filter to transmit the fragment ion detector quadrupole third particular. If you have been made to repeat cycle quickly through a set of configuration of the mass filter quadrupole, you can record the full spectrum. Similarly, it is possible to be executed by the type of different scanning properties of tandem mass spectrometry of four triple 4

In order to determine the order in which they appear in the DNA strand nucleotide, nanopore sequencing is the method of development from 1995. Nanopore is a small hole in the order of 1 nanometer inner diameter slightly. Done (tens of nm), by etching a slightly larger hole in the piece of silicon also leads to much more features and porous transmembrane cell protein some, such as Nanopor Nanopor, filled using the modified method of long ion then gradually small hole diameter of the nanopores – being. Further graphene, and are considered as synthetic substrate for a solid Nanopor. By (voltage) is applied to the above theory behind nanopore array be observed current due to conduction of ions through the nanopore when the nanopore is immersed in the fluid and it has the potential possible. The amount of current is very sensitive to the size and shape of the nanopore. If (base) single nucleotide, chains of molecules of DNA or other is near the nanopore or pass, this will allow you to create a characteristic change in the amount of current passing through the nanopore.

DNA may be transferred via the nanopore for various reasons. For example, it is possible to pass through it possibly to attract the electrophoresis of DNA for nanopore and can. Or enzyme that is associated with the nano-pores, can lead to DNA nanopore. Like thread through the eye of a needle more, the size of the nanopores means that the base of one, is forced from the hole as long string at a time is the DNA. As it did, each nucleotide of the DNA molecule, can prevent the nanopores Features degree of another. Thus, unlike the nanopore issued by whether they are blocked, the amount of current that can at any time through the nanopore varies DNA of the current through the nanopore by the DNA molecule through the nanopore direct reading of T C, G, or sequencing. This approach is demonstrated by Hagan Bailey Oxford nanopores Technology professor – Alternatively, the nanopore can be used for them to identify the DNA bases individual passing through the nanopore in the correct order.

Potential, is that it is possible without the need for identifying or labeling substances chemical labeling step or one molecule intervention PCR amplification step of the DNA, the need for optical instruments, and sequenced directly using the nanopore . As of July 2010, the information is publicly available, nanopore sequencing, shows that data backup and laboratory-based, it is still being developed some of the various components of the method You. Despite these improvements, sequencing of the nanopores is not useful enough to counter the “next generation sequencing” methods are currently available parallelism also routineized,. The DNA analysis technology of nanopore-based, through the R & D solid sequencing and internal (protein Nanopor, in order to establish a chain sequencing and direct exo sequencing in collaboration with academic institutions Oxford nanopore technology in industry (Nanopor is used in combination with fluorescent labels NobleGen and Nanopor samples were developed using the library (DNA NabSys, these probes, a) to detect when it is hybridized to single-stranded DNA,) to). The research project IBM, some projects solid nanopores, characterized by computer simulations of the transfer of DNA strands to identify the DNA bases in any direction.

nanopores of the bacteria that cause the dissolution α-hemolysin (αHL), red blood cells, has been studied for more than 15 years. By this time, studies have shown that it is possible to identify four bases all using the ion current between the αHL pores. To identify a specific base movement through the holes is preferably ΑHL structure. The αHL pores, it is long in the 10 Nm ~ 5 nM in in two distinct sections. The upper example, it may be made of (R1, R2, R3) capable of recognizing three sites consisting of large structure, such as lower entrance and to distinguish each of the base to another. has been developed through the structure variation and basic research of sequence with the αHL, is moving toward the consistency of a very long read. Protein mutation αHL has improved the detection capability of the pore. The next step to be associated with exonuclease αHL pores are planned. Enzyme will cleave itself periodically to allow to identify the base pores continuous. It is possible to slow down the transfer of DNA holes connecting the exonuclease organic pores and improve the accuracy of the data collection. Recent studies, I have demonstrated the ability of αHL to detect the nucleotide at the site of two separate lower half of the pores. Create a measurable ion current value 16 different four instead, while moving through the hole, the site of the R2 and R1, you can each site occurs twice. This method can improve the single nanopore read by doubling the site that sequence is read nanopores.

To determine the order the building blocks of DNA in the gene encoding the individual called DNA (nucleotides), Genetics learning proceeds, it is one of the methods used for testing of genetic diseases. A new technology that allows rapid sequencing of DNA mass have been developed. Sequence original technology is called breakthrough consisting of (named after the development scientist game, the Frederick Sanger) Sanger sequencing, and help scientists to determine the genetic code of humans, but this is time-consuming and it is expensive is. Sanger method, which is automated in order to more quickly, with short work laboratory sequence DNA, are still in use today, (also known as the human genome) man years which the DNA of all in order take. While reducing development costs more recently (for example, several weeks, a few days, for sequencing of the human genome) several techniques are called (sequencing or next-generation) to accelerate generation sequencing process are.

The next sequence, it is possible to DNA sequences large amount of current, for example, it is all part of the DNA of the individual to provide a procedure for the preparation of the protein. These pieces called exons are considered to constitute one percent of the human genome. Together, all exons of the genome is known, exome sequence method is known as exome sequencing in its entirety. In this method, in order to identify genes also are a few selected Rather, it allows the variation of the protein coding region of any gene. Mutations most known cause disease because occurs in exon exome entire sequence is considered to be an effective method for the identification of possible pathogenic mutations.

However, exon outer DNA mutants, researchers have discovered that it is possible to influence leads to deformation of the complete or genetic disease, an abbreviated sequence exome, the activity of production genes and proteins. Another method, called the whole genome sequence, can determine the order of the total nucleotides in the DNA of an individual will be determined by each variation of the genome. Genetic changes of many rather than identified by whole-genome sequencing and whole exome, you can choose the gene sequences, but the significance of much of this information is unknown. Since there is no genetic changes all health, determining mutants identified whether is contained in the state of interest is difficult. And is associated with genetic diseases various identification examples are not yet been diagnosed occasionally. As well as being used in clinical, exome whole, the total genome sequence method is valuable for researchers. Continuing with the study of the genome and exome, it may help to determine whether genetic variation new associated with the health state help diagnose disease in the future.

A first method for DNA sequencing have been developed in the mid 1970s. At that point, it is possible to base pairs of an array or only some years, researchers not enough for all of the sequence of a single gene much less of the entire human genome. The HGP, by the time it began in 1990, only a few laboratories were able to make a 100 000 house only a sequence, the cost of sequencing is still very high. Since then, to reduce the cost to the point where it can be increased and automation technology improvement, the speed, individual genes are sequenced daily basis, several laboratories, the bases of more than 100 million per year You may order.

Beginning in the late 1990s, the scientific community, witnessed the culmination of significant achievements related to DNA sequencing. Is created in the genome of the important model organisms, including some (Rattus norvegicus) mouse now (Mus musculus), rat, (D. pseudoobscura and Drosophila melanogaster) of two fruits, two in addition to history Thing of the genomic sequence of human (C. briggsae and (Caenorhabditis elegans) nematode) nematode One, the (intestinal and C Ciona intestinalis savignyi) fungi some other, malaria (Plasmodium falciparum), 2 Hoya and yeast (Saccharomyces cerevisiae), (Anopheles) mosquito that carries the malaria parasite that causes; (including rice) list mustard grass long (Arabidopsis thaliana), rice, and micro-organisms, some plants. Work of the sequence is in progress bee (honeybee), or just the beginning, when you start immediately chimpanzee (chimpanzee), bovine (cow), the dog (Canis familiaris) chicken (Gallus gallus) This is expected. It becomes genetic simplicity relative, and terrain ideal for technology development in the future of those many of these model organisms.

The synthesis technique has been developed (also known as SMRT) by the Pacific Ocean Biosciences Inc., is parallelized a single molecule DNA sequencing single molecule real-time sequence. Use zero-mode wave, which was developed in the laboratory of Harold G. and (ZMW), Watt W. Webb of Cornell University, and single-molecule real-time sequencing Craighead. The enzyme, DNA polymerase is located at the bottom of the ZMW the DNA molecule as a template. Is a DNA polymerase having the structure illuminated observation volume small enough is created to observe only one nucleotide (also known as base) in the DNA ZMW. Each of the 4 DNA bases are attached to one of the different fluorescent dyes four. If it is incorporated by DNA polymerase, cut a fluorescent marker nucleotides are expanded surveillance area ZMW whose fluorescence is not observed. Detecting the fluorescent signal of the nucleotide incorporation, the base, the detector is a call by fluorescence corresponding dyes. Sequence data generated from a single molecule real time sequencing, was published in January 2009 in the journal Science first.

The DNA sequencing, I went a chip that contains the ZMWs many. In each ZMW, through may be created through the imaging camera capable of light to track the activity of DNA polymerases at the molecular level, DNA polymerase and a single single-stranded DNA template molecule, the bottom is fixed. Signal from the incorporated nucleotide phosphate binders is detected go to DNA synthesis, as results in DNA sequencing in real time by a DNA polymerase. The nucleotide bases each detector has a fluorescent dye for each of the four in order to allows to determine on the basis of the from being taken by the DNA polymerase DNA synthesis to occur. And is attached to the phosphate of the nucleotide chains of the fluorescent dye molecule. Nucleotides, when it is incorporated by DNA polymerase, when establishing a phosphodiester bond to the DNA chain elongation, as part of the natural process of DNA synthesis, the fluorescent dye is split and a phosphoric acid chains. Fluorescent dye molecules which are separated and then the fluorescent signal becomes not detected, the spray from the detection of sound. It is a nano-photonic birth structure is composed of a circular hole in the lining of aluminum foil silica was deposited (ZMW) on a clear substrate in wave mode of zero. In diameter ~ 70nm in ZMW hole, there is about 100 Nm depth. It is due to the behavior of light passing through the small hole of the optical field profile decays exponentially into the chamber. ~ 20 zeptoliters observables in ZMW is illuminated (10-21 × 20 liters). This volume inside, it is possible to easily detect the activity of DNA polymerase for single nucleotide.

In other words, real-time single molecule sequence to be applied to a variety of genomic research. Please refer to the length of the molecule from a single real-time sequence greater than that of the method sequence based on the Sanger dideoxy chain termination or equivalent: genome sequence new de. I want to enable the genome assembly and easy de novo of long read length genome. The SMRT sequencing, in the New England Journal of Medicine, in 2011, publication of Denobogenomu was demonstrated for analyzing the occurrence of E. coli in Germany in the cholera epidemic in Haiti in 2010. The scientists also, in order to balance the short read sequence data and read sequence data long-term, I use a single molecule real-time sequence of de novo genome hybrid meeting. In 2012, publication of peer review of some proven, the genome of complete bacteria that are automated including a newspaper that updates the assembler Celera genome finish pipe using the SMRT sequencing long read was released.

DNA molecules, and using an enzyme-shift circuit for separating the DNA strand newly synthesized from the template, it is possible to create a circular DNA template, and re-sequencing is independently: rearranged. SMRT is other Pacific Ocean Biosciences, using this approach round consensus with the sequencing to prove the validity of the activating internal tandem duplication mutation in FLT3 as a therapeutic target in acute myeloid leukemia, and the University of California scientists from institutions. These results, it is published in Nature magazine in April 2012. August 2012, scientists of the Broad Institute, announced the evaluation of SNP calls for SMRT sequencing. Methylation Detection: to indicate base whether it is methylated, and the dynamics of the polymerase. In order to detect changes and other methylation based on peer-reviewed articles in a series in 2011, scientists, shows the use of real-time sequencing of single molecules. In 2012, for the team of scientists SMRT used to generate a full methylomes, sequencing the six bacteria said the findings in the study of nucleic acids. November 2012, scientists have published a report on the methylation of genome-wide epidemic strain of E. coli.

And work together as an integrated system, genetic Helicos assay system is made up of several components. DNA molecules for sequencing target hybridizes to the site of a single stream of glass cell. The sample with the probe Helicos loader temperature can be adjusted optimal hybridization, it is loaded into the flow cell. Each of the 25 channels, it can be considered separately in other steps necessary to sample was added to prepare a sample standard cells flow. The sequencing system together synthetic that is inserted in the sample HeliScope When loaded properly when the flow cell, and all reagents needed for sequencing by image. Was the sequence for as long as necessary, the image is processed in real time Helicsope engine analysis sequencing system. It processes the image from the physical location of any analysis engine read from these pictures the construction sequence. After execution, and full-field imaging process is transferred to the cluster data to calculate the reference group or aligned properly.

Single molecule sequencing (SMS), and provides information about both series quantitative accurate, Helicos , provides a unique view of genome biology through direct sequencing of cellular nucleic acids in an equitable way. To avoid size variations seen in other techniques and GC content, sample preparation is not required PCR amplification or ligation. Subsequently poly simply, DNA was hybridized to the surface of the flow cell containing oligo dT for sequencing-by-synthesis and molecular parallel shear, the several billion. This process will require much less material compared to other technologies. The measured gene expression is carried out by direct quantitative method based on the first strand cDNA or by the (RNA-seq) so that it is possible to use a new approach that enables direct sequence hybridization and cellular RNA I can.

The successful diversification of applications, chip array using picogram weight DNA copy number variation study of both of FFPE tissue samples and tumor tissue fresh, sequence of DNA was degraded and ancient, RNA, a small study, that is the exact mutation This leads to the identification of a new class of RNA have been made, including the genome sequence for detection and capture an array of RNA from 250 cells only cell mass directly. Most of the next generation technology, since it requires, it is not possible range of sequence-specific amplification and size of the target DNA molecules that do not meet these criteria sequencing reliably. Obtaining is unnecessary, it is possible to use as a template directly the degraded molecules or have changed, the sequence of the single molecule is not subject to these limitations it. ® explaining the principles and methods for using a genetic analysis system Helicos.

Generally, samples for sequencing, poly (A) tail was prepared to be longer than (dT) 50 oligonucleotide on the surface of the flow cell. Residues that are not paired are needed to fill the treatment, to lock Thing avoided. After hybridization, 37 ◦ C, and then in the dGTP and dATP the lowered virtual terminator nucleotides corresponding dTTP, and dCTP, was added together with DNA polymerase temperature. Contains an extra base and due to the chemical structure attached to the nucleotide, the virtual terminator nucleotides prevent more involved. Accordingly, TTP is filled with all of the unpaired DASS present in poly (A) tail. Hybridizing molecules are locked into place when it detects the insertion and non-residue of the first nucleotide virtual polymerase terminator. Color image application will include all molecules incorporation of a nucleotide is possible because there is a need to DNA molecules each have now. Further, since the label can be assigned to any base, it does not give sequence information at this stage. Thus, for the majority of the molecule sequence, starting from the base of the second of the original molecule.

The DNA sample, it is necessary to generate a nucleic acid that is compatible with the purpose of the hybridization of these surfaces normally I was hybridized primers immobilized to the flow cell on for sequencing. Target sequence attached to the surface of the flow cell may have a sequence that can be synthesized in theory, in practice, oligo flow cell standard commercial is (dT) 50. To be compatible oligo surface on the flow cell (dT) primer and 50, it is necessary to generate a 50 NT least the 3 ‘end of the molecule poly tail to be (DA) sequencing. When you lock the step and filling, so will fill, is that rather than on the surface oligo (dT) as long as, T excess, it is desirable at least tail it. Generation poly (DA) tail 3 ‘can be carried out using a polymerase or ligase variety. If the average duration DNA, and sufficient for the mass measurement, it is possible to determine the exact amount of dATP, but added to produce a tail poly (DA) from 90 to 200 nucleotides it is possible to. To generate the tail of this length, is in the sample there, it is the first to determine the 3 ‘end of how to use the terminal transferase in the tail of the range of optimal size, mix of the DNA of the right, the dATP There is a need.

DNA sequencing nanoballs, has a high-throughput sequencing technology used for the determination of the entire genome sequence of an organism. Way is to use a rolling circle replication to amplify small fragments of genomic DNA to the DNA nanoballs. Associated with additional DNA, and then fluorescence probe is connected to the relevant anchor sequence with known sequences in the DNA template. The main purpose, is determined by the associated probe fluorescence and tyrant. The method can be sequenced to run at a lower cost than the drug generation sequencing platforms other DNA nanoballs many DNA. However, limitations of this method is to produce a short sequence of only DNA has become a challenge to move the reading of the reference genome. The company has used the DNA sequence to sequence nano ball samples that were submitted from the complete genome researchers.

The ligated fragments circularizing fragments and adapter sequence, the fragment (BP) cleavage of a small 400-500 base pairs, DNA sequences nanoballs involves the isolation of DNA to be sequenced. As a result of the single-stranded copies of many of the fragments, fragment of circular, is copied by a rolling circle replication. Head, and I pushed into the DNA nanoballs to tail in a thread long DNA copy associate. In nanoballs, and adsorption to the input cell sequence. Unlinked sequencing reactions, contact the site of the nucleotide specific nanoballs by connecting a fluorescent probe DNA. Color fluorescence in each interrogation position is detected by the high-resolution camera. Is used to analyze the fluorescence data, the production of each call, as well as for applying the 35-BP mate pair, bioinformatics, reads the reference genome. Genome is assembled, were identified polymorphisms all present in the sequence.

The lysed cells were extracted from the cell lysate DNA. High molecular weight DNA, often for long, megabase pairs some destroyed sonicated double-stranded DNA at random intervals. DNA sample is most effective if it contains a narrow range of length reads the mapping sequencing already. Therefore, sequencing of DNA fragments and selection of fragment length ideal, are separated polyacrylamide gel electrophoresis, size by (PAGE). Was purified by gel extraction as a result of DNA having (400-500 base pairs, typically) length of a narrow range, the DNA of the appropriate size range.

Adapter DNA sequence must be applied to DNA fragments of the DNA sequences of unknown unknown DNA clearance, as is known. I was PCR amplified the DNA and, attached to the left and right sides of the DNA fragments in the first round of (AD1) adapter ligation of the left and right adapters. Left AD1 is modified to create a single-stranded termini additional are connected to one another so as to form a circular DNA. Were cut 13 BP right right adapter DNA, restriction enzymes, are added. This results in double-stranded DNA linear. Are connected to the ends of (Ad 2) linear DNA in the left and right sides of the adapter sequence, the product is amplified using PCR. It is modified so that after a sequence of Ad 2, connect to each other to form a circular DNA. Restriction enzyme is used again to cleave circular DNA left BP AD1 13. The results, linear DNA fragments are (Figure 3). The lateral coupling (AD3) adapter sequence to the left and right of the linear DNA, was amplified by PCR product. The adapter is fixed to couple to each other because they form a circular DNA. Cutting the DNA of 26 bp to the left of the AD3, 26 bp to the right ad 2 depending on the type III restriction EcoP15, were added. In this step, to remove most of the DNA, and the DNA linearized again. Was amplified by PCR to combine the left and right adapters (AD4) DNA, the product, AD4 sequence is modified such that they bind to each other. The result is a complete circular DNA template.

Ion semiconductor array is a method for DNA sequencing based on the detection of the hydrogen ions released during the polymerization of DNA. While the complementary strand is built based on the sequence of the template strand, which is a method of “sequencing-by-synthesis”. Chain DNA template consisting of microplate was determined the nucleotide sequence of one deoxyribonucleotide triphosphates of (a dNTP) is flooded. If it is incorporated into the complementary strand template nucleotides leading complementary dNTP was introduced is growing. This leads to the release of hydrogen ions which indicates that the reaction has occurred to trigger the ISFET ion sensor. If present in the template sequence repeat homopolymer, dNTP molecule contains more than one cycle. This results in a corresponding number of hydrogen atoms, which has announced a higher proportional electrical signal.

This technology is different from the other techniques for sequencing and to use nucleotide optical system, has not changed. Further ionic semiconductor sequences mediated sequence SEQ silicon ion flux, the pH, or it may be a sequence in a semiconductor. DNA Electronics Co., Ltd., which was developed by Ion Torrent Systems, has been licensed from the Corporation, the technology, was released in February 2010. Can be as a desktop machine, to be used in many laboratories, commercial ion Trent machine sequencer as economical high-speed, compact. The Roche 454 Life Sciences DNA Electronics partner reading for a long time, in the development of high-density semiconductor sequencing platform that uses this technology.

In nature, (dNTP a) include deoxyribonucleotide triphosphates with growing chain of DNA involves the formation of hydrogen release positively charged ions with pyrophosphate and covalent bond. It contains dNTP when it is complementary to the tip template nucleotide pair. In order to establish hydrogen ions whether the release delivery of dNTP kind of reaction, ion semiconductor sequencing, uses these facts. Be full unmodified sequentially microplate DNA polymerase and a semiconductor chip that contains a single-stranded DNA template molecule to be sequenced, respectively, with the dNTP T C, or G,. Complementary pairs of nucleotides template strand, is incorporated into the growing complementary strand of DNA polymerase if you enter the next dNTP. Of dNTP complementary was introduced included, if it is not biochemical reaction. The pH detected by the ISFET, hydrogen ions are released in the reaction of the solution changes. DNTP from molecules that are not attached is cleaned before the next cycle of dNTP different types is introduced.

I have an ion-sensitive layer beneath is a ISFET ion sensor under the well layer. CMOS semiconductor chips similar to those used in the electronics industry, all layers are included within. Each chip contains a number of wells and ISFET of the detector. Then, hydrogen ions are released each leads to ISFET ion sensor. The events associated with signal for incorporation of nucleotides, the sequence of electrical pulses sent from computer chips, optical measurement and nucleotides consists of DNA sequence without intermediate conversion was measured directly the electronic device, it is labeled use of is avoided. It can be implemented in software assembly and DNA signal processing. Precision reached base House Trent Trent ion ion ion semiconductor sequencer, in February 2011, was 99.6%, based on the 50 reading 100 MB of each execution. The length of the reading of February 2011 was 100 base pairs. Accuracy was 98% of the repeating homopolymer length of five replicates. It should be noted that the data is still independent, it is not validated external

It is also commercially available in 2008 developed two base encoding called solid (sequence detection oligonucleotides and ligation) by Applied Biosystems, and next-generation sequencing techniques. These techniques generate a read hundreds of thousands of small sequence. Well-known example of such a method can be found in the system and stable (introduced in 2006) Solexa DNA 454 pyrosequencing of the system. Price decreases in 2006 to nearly $ 0.0001/base of 2004 from $ 0.01/base years, these methods, sequence capacity of one million base machine day in 2004 to base / machine more than 100 million 2006 has increased / day. 2-based encoding based on the array than ligation sequencing-by-synthesis rather. However, fluorescent labels 8 amount can be changed for using a fluorescence-labeled 9-mer probe 6 bases differ only in place, two base encoding, like, but to distinguish the three bases most basic two How Macevicz over 6BP view thereby utilizing the probe body can be obtained. Without having to run twice at work 2-based encoding, you can twice, read each base.

1988, and Whiteley. I show you how to use the fluorescence-labeled oligonucleotide ligation detection of DNA variants. In order to detect changes in DNA, 1995 Macevicz showed that coupling the plurality of oligonucleotides adjacent to each other. In 2003, for example, Dressman. It shows the use of emulsion PCR that can be repeatedly executed to generate one million clones amplification analysis beads ligation thereof. Such as 2005 Shendure. Complete the steps of sequencing that combines Dressman technique ligation and Whiteley 8 ‘9 probe fluorescently labeled “degenerate base to distinguish the evidence differently depending on the modification and the basis labeled probe. This process, using the same primer was repeated, but I read the labeled probe and 5 that have been identified on the basis of degenerate 6BP another array – read 7BP at three and three-way> -> 5 direction.

In fact, the literal translation of the color reading as it could lead to a shift of the frame-based call one time that occurs in the color error is so named, it is not recommended basically. It is recommended that you convert the nucleotide sequence your reference color space to best use of property “error correction” of the base two encodings. Clear conversion of the reference nucleotide sequence in the color space is present, but a possible implementation of the four color strings main strings. I think the amino acid translation. There is a clear base of amino acid translation, but there are many solutions for the transfer of amino acid to the base.

Mapping color space, reads the color space can be used the basic encoding rules of two adjacent difference in color may represent the polymorphic base true only appropriate standards. If you want to translate the color to read and decipher the database directly, without any knowledge of the other, you will not be able to do this effectively. In particular, this method, error correction, not a tool for error conversion equipment. Color space, converts the error the most common mode of your frequency to another in the form of DNA changes most commonly. Base change single these affect the colors adjacent to each other in the color space. There are logical rules to correct the error of error adjacent to the “invalid” and “valid” in the neighborhood. It is possible to measure the probability of receiving an error on the 50-BP reading two adjacent. There are 49 make changes 50 continuous string in (50-BP reading). There are 1225 to make changes to the string you do not have 50 consecutive. The error of 49 1225, simple, assuming full random error is a candidate for SNP only (usually, a high frequency at the end of the read these). Furthermore, in order to provide error 16 May 1225 by third only candidates for EPP errors only in the neighborhood and can be effective according to the bug labeling known samples. It reduces the false positive results low coverage, such as Smith, which is particularly useful for detecting low SNP of the coating.

Part of the now Solexa, Illumina, a method for technology reversible dye terminator sequencing-based, it has developed the engineering of polymerases have developed internally. Solexa Solexa system design was invented by Klennerman and Balasubramanian of the Chemistry Department at the University of Cambridge in was developed internally [49] wound chemistry. The company has acquired the Solexa Manteia prediction drugs that massivelly get the parallel sequencing technology based on “DNA cluster” containing the cloned DNA amplification on the surface in 2004. Cluster technology was acquired in the treatment of joint California Lynx. Solexa, Inc. merged with Solexa’s form of Lynx later

In this method, primers and DNA molecules, are applied to the first slide, DNA of the local clone colonies were amplified by polymerase minted later “DNA cluster” to be formed. The sequenced, four kinds of bases reversible terminator (RT-based) was added, and nucleotides unincorporated is eluted. Camera is removed from the DNA that makes it possible to get the image of nucleotides that are fluorescently labeled, and starts the next cycle chemically dyes with terminal 3 ‘blocker thereafter. Unlike pyrosequencing, DNA strand extending one nucleotide and image acquisition time may be performed in time slow allows large arrays very DNA colonies should be covered by Continuous shooting from a camera.

Separation and shooting enzyme reaction allows unlimited capacity Thing in theory and optimal performance. The optimum configuration of an analog in this manner eventually – determining the amount of the chamber multiplied by the number of cameras in only digital conversion pixel of DNA colonies required (approximately 10 pixels for optimum viewing thereof colonies reach divided by the number, the performance of the instrument). In 2012, after working in enzymatics optics, and fluid camera is available with A / D conversion speed MHz, more than 10, is a multiple of one million nucleotides  corresponds to 1X to about one human genome equivalent bandwidth coating of the human genome sequence at the time and again on one instrument (camera) equipment the day it is possible.

The basic steps for sequencing Illumina dye response is as follows. DNA molecules is attached to the primer first slide, I was amplified colonies local to be formed. This technology 1998 Laurent Farine Li and Geneva Medical Glaxo Wellcome Institute Dr. Pascal Mayer Dr. cluster generation was invented, has been developed, was presented to the public for the first time in the O end of 1996. Labeled with a fluorescent color different termination bases (adenine, cytosine, guanine, and thymine) reversibly four types, is added associated with the blocking group. Then, the four bases were then compete for binding sites of the template DNA for removing molecules sequenced, are not captured. Each laser after fusion is applied as a result of the probe and the removal of the terminal 3 ‘blocking groups. Then, to enable the start of the next cycle and identification sequence, the identification of detectable fluorescent color for one of the four bases were observed. Until sequencing the complete DNA molecule, the process is repeated.

This technique provides a number of advantages over traditional methods of sequencing by Sanger sequencing. I get a real data series quickly and can be due to the nature of the sequence Illumina dye that is automated, to thread multiple sequences at the same time. In addition, in contrast to the enzyme very expensive and required by sequencing other, this method uses a DNA polymerase only.

454 Thing use the possible scale parallel pyrosequencing system is about 400-600 Mega-based sequencing of the DNA of 10 hours per run of GS FLX Titanium series reagents FLX genome sequencer. The system relies on determining were sprayed DNA capture bead small water-in-oil, a DNA fragment was ligated adapter. I was amplified by PCR DNA was fixed to these beads. To 29 meters – and I was placed PicoTiterPlate, the optical on-chip DNA binding strip any. It is a mixture of enzymes such as are packaged in the well DNA polymerase, ATP sulfurylase, luciferase and such. I will be placed in the GS FLX system sequence PicoTiterPlate then. The acquisition by the release of GS20 sequencing machine and Roche Diagnostics in 2005, next-generation DNA sequencing market since the first 454 has experienced rapid growth. 454 sequence run-GS FLX Titanium series reagents for use in genome sequencer FLX device capable of 400 to 600 million base pairs sequence of motion and 400-500 base pair 2008 reads a length. Sequencing, the company plans to release a kit that can read up to a length of 1000 bps to 2010. The end of 2009, 454 Life Sciences, announced the desktop version GS Junior system, the Genome Sequencer FLX system.

454 array can be analyzed as a FASTA file and bioinformatics standard tools, but the information can not be used in a pure nucleotide sequence flow value is included. Thus, the use of the flow makes a direct value algorithm several groups proposed. Are called to operate in, “flowspace” “Unlike nucleotide space”, this approach is to suppress the loss of information. For example, the set of flowgrams, the method using a maximum likelihood PyroNoise to determine whether there is a possibility that lead to sequence biological basic one or more separate. In a similar manner, the use of Bayesian statistics PyroBayes method, the number of base that is specified the flux values ​​are observed to determine the length of each run as perhaps homo. If the likelihood of the base exceeds a predetermined threshold, it is added to the execution of the homopolymer than the base. This is the number of the insertion error will be increased, as is unique to 454 pyrosequencing that may occur only for very undercalls substituted error consistently, it deletion and it is possible to reduce the number of replacement. To call centers more homopolymers, in order to allow higher recognition rates SNP, this trend will work.

For discovery mapping, a small RNA that flowgrams the target genome is an effective way directly been proven. It is also possible to achieve speeds higher accuracy of the nucleotide sequence of the assembly building flowspace consensus sequence of the data that is oversampled very much. Metagenomics is another area where the quality of the 454-pyrosequenced data has been attracting attention. The study for various purposes, and have a major impact on the processing of the data in a specific way that has not been used in sensitive characteristics of pyrosequencing data, but there is indicated the value of the direct current artifacts some are. For example, you can collapse the homo all sequences of length 1 of pyrosequencing that may introduce errors in homopolymer length when matching the index approach of the 454 series. For a long homo, also errors of many, there is work to improve the 454 series on the side of the chemical in particular, distribution of overlap is caused by a signal that leads to ambiguous base calls and wide. In addition to determining the exact length of the homopolymer, the base or the calling party (i.e., the noise flow rate) is particularly important low optical signal. There is a possibility to come from the most basic of conversation, but flow value of 0.49 is treated as noise from the 454 caller based it.