Break genome into many long fragments ii. emPCR The Water-in-Oil-Emulsion. Shotgun Sequencing. Clone-by-clone or hierarchical sequencing 2. Shotgun sequencing— sequencing of random clones followed by sequence assembly based on the overlaps—was suggested by … Next-gen sequencing: shotgun library preparation. 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. These methods came into immediate use. It is named by analogy with the rapidly expanding, quasi-random shot grouping of a shotgun.. Online version of (1) – Walking rice genome i. sequence could be read off immediately, going up the four lanes by size to infer the order of bases. … ... • Used to sequence whole genomes • Steps: – DNA is broken up randomly into smaller fragments – Dideoxy method produces reads – Look for overlap of reads . Massively Parallel Sequencing. 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. Shotgun sequencing. 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. Those ordered arrays were known as physical maps, and they served to break large … 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! However, the method required more chemical steps than a chain termination method which makes it more complex. 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.) How do you sequence a whole genome? 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. Sequence each fragment with shotgun 2. In genetics, shotgun sequencing is a method used for sequencing random DNA strands. 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). There are two general strategies for sequencing a complete genome. Pyrosequencing. 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 Strategies for whole‐genome sequencing 1. All the small fragments are then cloned and sequenced. Computers analyze the sequence data for overlapping regions and assemble the sequences into several large contigs. Map each long fragment onto the genome iii. ... 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