Sugar … All the small fragments are then cloned and sequenced. In genetics, shotgun sequencing is a method used for sequencing random DNA strands. Sequence each fragment with shotgun 2. How do you sequence a whole genome? Strategies for whole‐genome sequencing 1. … Shotgun Sequencing Sequencing reactions are performed with a universal primer on a random selection of the clones in the shotgun library. Massively Parallel Sequencing. Shotgun Sequencing. However, the method required more chemical steps than a chain termination method which makes it more complex. sequence could be read off immediately, going up the four lanes by size to infer the order of bases. Pyrosequencing. Computers analyze the sequence data for overlapping regions and assemble the sequences into several large contigs. These methods came into immediate use. emPCR The Water-in-Oil-Emulsion. Clone-by-clone or hierarchical sequencing 2. Methods in DNA Sequencing Big Picture Large-scale sequencing requires DNA to be broken into fragments Cutting (with enzymes) Shearing (with mechanical forces) DNA is duplicated into a vector Individually sequenced Assembled electronically Shotgun sequencing Brief Bio Background Nucleotides Components in DNA, consists of 3 portions: Nitrogenous base (Adenine, Guanine, etc.) Break genome into many long fragments ii. In this approach, genomic DNA is cut into pieces of about 150 Mb and inserted into BAC vectors, transformed into E. coli where they are replicated and stored. Other articles where Hierarchical shotgun sequencing is discussed: whole genome sequencing: Sequencing methods: from genes to genomes: …that sequenced large genomes involved hierarchical shotgun sequencing, in which segments of genomic DNA were cloned (copied) and arranged into ordered arrays. Shotgun sequencing. There are two general strategies for sequencing a complete genome. Furthermore, the read length is too short as compared with the automated sequencing. The method preferred by the Human Genome Project is the hierarchical shotgun sequencing method. 454: Data Processing Image Processing Base- Whole-genome shotgun sequencing Used both whole-genome assembly and regional chromosome assembly Incorporated data from the public project Raised ethical concerns about the ownership of the human genome and patentability of genes Whole-genome shotgun sequencing: Yet another modification of the Sanger’s chain termination method is the whole-genome shotgun sequencing. Next-gen sequencing: shotgun library preparation. Shotgun Sequencing Celera Started in Sept 1999, goal was to do in $300M and 3 years what the public project was doing for $3B and 15 years! emPCR Emulsion PCR is a method of clonal amplification which allows for millions of unique PCRs to be performed at once through the generation of micro-reactors. These sequencing reads are assembled in to contigs, identifying gaps (where there is no sequence available) and single-stranded regions (where there is sequence for only one strand). The first step in shotgun sequencing an entire genome is to digest the genome into a large number of small fragments suitable for sequencing. Online version of (1) – Walking rice genome i. Hierarchical – Clone‐by‐clone yeast, worm, human i. Map each long fragment onto the genome iii. Those ordered arrays were known as physical maps, and they served to break large … Shotgun vs. Hierarchical Method Celera vs. Human Genome Project Hierarchical (top-down) assembly: – The genome is carefully mapped – “Shotgun” into large chunks of 150kb • Exact location of each chunk is known – Each piece is again “shotgun” into 2kb and sequenced ... • Used to sequence whole genomes • Steps: – DNA is broken up randomly into smaller fragments – Dideoxy method produces reads – Look for overlap of reads . 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