In addition, in genetics, shotgun sequencing, known as shotgun cloning, is a method used for sequencing long DNA strand. Expanding rapidly, it is named by analogy with the accidental firing model gun almost. In order to obtain the entire sequence to be able to use the (100 to 1000 base pairs) relatively short-chain, the longer sequence, it must be divided into smaller fragments, chain termination method of DNA, then reassembly. The entire chain, and fast, little by little, but it is a more complex process, by running through the shotgun sequencing, to use the fragment random chromosome walking: the main two ways, used for this purpose are.
In order to obtain a value read at random shotgun sequencing in DNA, using the chain termination method, and is divided into smaller multiple segments that are connected in series. Overlapping reading of the target DNA Multiple obtained by performing several rounds of sequencing fragments and this. Is read in order to use an end portion overlapping and computer programs, and different, and assemble them into a continuous sequence. Shotgun sequencing, is a precursor technology was responsible for the complete genome.
Shotgun sequence is a method of including the entire chromosome and the maximum is designed for the analysis of DNA sequences of length 1000 base pairs. This method requires the target DNA to be split into random fragments. After sequencing fragments, sequences can be assembled on the basis of the overlapping region. In this very simple example, it is not read can be assembled in the original sequence to turn the length of the original sequence of the whole, none of the read or a duplicate for the end to unite covers 4. In fact, I am using a large amount of information that this process is full of sequencing error and ambiguity. The assembly of complex genomes, which means that it can come from different parts of the complete series, by the great abundance of repetitive sequences, short lead such more complicated. Many overlap is required for reading each segment of the DNA of the original, to overcome the sequence assembly and accurate these difficulties. For example, to complete the Human Genome Project, that is, each base end sequence is a sequence of coverage of 12x there are more than 12 average hit most of the human genome. However, in these methods, I was not able to produce or isolate sequences of reliability of (chromatin) human genome about 1%.
Sequencing whole genome shotgun of the genome of the small (4000-7000 base) is used in 1979 already. Couple end sequence known colloquially as shotgun shotgun sequencing, wide application benefited. Sequencing project as began to take complex DNA longer, several groups have begun to realize can be obtained by sequencing the ends of DNA fragments useful information. Sequence of both ends of the monitoring data fragments and the same is spaced the length of the fragment, heavier than the one end of the sequence of the two fragments of knowledge two arrays that are oriented in opposite directions on it is connected But other is beneficial to recover the sequence of a fragment of the original object.
End limited to close the gap of the implementation of the approach shotgun sequence traditional but, at first, as part of the place HGPRT human, use the pair announced the description of the use of both ends of the paired in 1990 . Assuming a fragment of a certain length, theoretical explanation of the first clean bed end sequencing strategy was 1991 it. The optimal fragment length for a couple end sequence, there is a community consensus that it is three times read the length of the sequence at that time. 1995, and Roach. introduces the innovation of using fragments of different sizes, it indicates that it is possible to target the end of pure strategy pair array is large. Institute for Genomic Research (TIGR), then, strategy, adopted by the human genome and sequence from Celera Genomics sequence of Drosophila melanogaster genome genome then Haemophilus influenzae bacteria in 1995 to 2000 then.
In order to implement a strategy fragmented randomly fragmented high molecular weight DNA, size and (usually 2,10,50,150 KB) selected and cloned into an appropriate vector. Clones were sequenced from both ends using the chain termination method to obtain a short two sequences. Each sequence is called a read or read-end, two people is Meitopea to read from the same branch. Usually, at all, read the long 500-1000 bases as chain termination method, branch Meitopea The minimum can be generated only do almost overlap.
Original sequence was reconstructed from read using the sequence assembly software. First, overlay view is collecting the sequence of many ingredients called configuration. Configuration can be linked together in a skeleton following the link between Meitopea. The average fragment libraries are known, if it has a narrow window of deviation, it is possible to derive from the position Mattopea the distance between configurations. It may be used to according to the size of the space between the assembly, various techniques find gaps in order. The difference, if it is a (5-20 kilobytes) than after the use of PCR to amplify the desired region followed by sequencing. If the difference is large (> 20 kilobytes), the large fragment was cloned into the vector of specific BAC array of vector followed (such as bacterial artificial chromosome).
Proponents of this approach, suggesting that it is a whole-genome sequencing sequencer is made of conventional approaches much more efficient, simultaneous use of large amounts is possible. Critics have claimed a large region sequence, for the genome with the local repetitive in particular, that there is a doubt the ability to connect these areas properly as soon as the technology of DNA. Capacity to be cheaper assembly sequence program becomes complicated, it is possible to overcome this limitation.