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shotgun sequncing
- 1. Shotgun Genome Sequencing Saif Saleh Mohsen Ali Dept of Biochemistry Genetics & Genomics
- 2. • Genome is all genetic material of an organism. It consists of DNA (or RNA in RNA viruses). The genome includes both the genes (the coding regions) and the noncoding DNA, as well as mitochondrial DNA and chloroplast DNA. The study of the genome is called genomics. • What is the genome sequencing? • Genome sequencing is the technique that allows researchers to read the genetic information found in the DNA of anything from bacteria to plants to animals • Sequencing involves determining the order of bases, the nucleotide subunits- Adenine(A), Guanine(G), Cytosine(C) and Thymine(T), found in DNA What is the genome
- 3. Determining the Sequence of DNA Methods: 1) Maxam and Gilbert chemical degradation method. 2) Chain termination or Dideoxy method • Fredrick Sanger . 3) Genome sequencing method: • Shotgun sequencing • Clone contig approach 4) Second generation sequencing methods: • Pyrosequencing • Nanopore sequencing • Illumina sequencing • Solid sequencing
- 4. Factors that determine sequencing strategy are :- • a) Genome size • b) Chromosomal structure • c) Repeat content and character • d) Desired end product
- 5. What is shotgun sequencing? • Shotgun sequencing involves randomly breaking up DNA sequences into lots of small pieces and then reassembling the sequence by looking for regions of overlap. • Shotgun sequencing was originally used by Fred Sanger and his colleagues to sequence small genomes such as those of viruses and bacteria . • In whole genome shotgun sequencing the entire genome is broken up into small fragments of DNA for sequencing.
- 6. Shotgun Genome Sequencing •Two different methods used :- 1) Hierarchical shotgun sequencing Useful for sequencing genomes of higher vertebrates that contain repetitive sequences 2) Whole genome shotgun sequencing Useful for smaller genome
- 8. History • Shotgun sequencing has been developed for sequencing of large fragments of DNA in 1979. • The first genome sequenced by shotgun sequencing was that of cauliflower mosaic virus, published in 1981. • Shotgun sequencing was the initiative for full genome sequencing.
- 9. The principal of shotgun sequencing • The chain termination method of DNA sequencing can only be used for fairly short strands of 100 to 1000 base pairs. •Longer sequences are subdivided into smaller fragments that can be sequenced separately, and subsequently they are re- assembled to give the overall sequence.
- 10. Steps of shotgun genome sequencing • Break genome into smaller fragments. • Sequence those smaller pieces. • Piece the sequences of the short fragments together .
- 12. Example for Shotgun Sequencing
- 13. Advantages of shotgun sequencing: • By removing the mapping stages, whole genome shotgun sequencing is a much faster process than clone-by-clone sequencing. • Whole genome shotgun sequencing uses a fraction of the DNA that clone-by-clone sequencing needs. • Whole genome shotgun sequencing is particularly efficient if there is an existing reference sequence. • It is much easier to assemble the genome sequence by aligning it to an existing reference genome . • Shotgun sequencing is much faster and less expensive than methods requiring a genetic map.
- 14. Disadvantages of shotgun sequencing: • Vast amounts of computing power and sophisticated software are required to assemble shotgun sequences together. To sequence the genome from a mammal (billions of bases long), you need about 60 million individual DNA sequence reads. • Errors in assembly are more likely to be made because a genetic map is not used. However these errors are generally easier to resolve than in other methods and minimised if a reference genome can be used. • Whole genome shotgun sequencing can only really be carried out if a reference genome is already available, otherwise assembly is very difficult without an existing genome to match it to. • Whole genome shotgun sequencing can also lead to errors which need to be resolved by other, more labour-intensive types of sequencing, such as clone-by-clone sequencing. • Repetitive genomes and sequences can be more difficult to assemble. •