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Shotgun sequencing method

Shotgun DNA sequencing : Topic index : 1) principle 2) procedure a) DNA fragmentation b) Agarose gel electrophoresis c) purification and modification d) cloning e) sequencing f) overlapping 3) Advantage 4) Limitations Fragmentation: Fragmentation of the DNA can be done by using restriction enzymes, physically by breaking it into small pieces (usually 2, 10, 50, and 150 kb ) by passing it through a narrow gauge syringe or sonicating it, which is the way of breaking the sample using sound waves, by mechanical method, shearing. It is a random process, so the sequences of the fragments will have some overlap between them. Fragments of about 150Mb are obtained. Cloning: Next step is joining of DNA fragments with a vector which is a carrier DNA. This method is known as cloning. And a sequence library is created. Sequencing of entire genome will be created by using this library. Sequencing: Sequencing of each clone in library is done. Individual fragments are sequenced individually using the chain termination method to obtain reads. Multiple overlapping reads are obtained for the target DNA by performing several rounds of this fragmentation and sequencing. Advantages and Disadvantages of Shotgun Sequencing Shotgun sequencing had a number of important advantages over previous methods: 1)Faster because the mapping process was eliminated 2)Uses less DNA than other methods 3)Less expensive than approaches requiring a map Some disadvantages of shotgun sequencing include: 1) Requires computer processing power beyond what an ordinary laboratory would possess 2) Can introduce errors in the assembly process 3) Requires a reference genome 4) May not be able to assemble repetitive sequences