Sequencing_Whole_Genomes - Sequencing Whole Genomes...

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Sequencing Whole Genomes Hierarchical Shotgun Sequencing v. Shotgun Sequencing How do you sequence a whole genome? There are two general strategies for sequencing a complete genome. The method preferred by the Human Genome Project is the hierarchical shotgun sequencing method. 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. The BAC inserts are isolated and mapped to determine the order of each cloned 150 Mb fragment. This is referred to as the Golden Tiling Path . Each BAC fragment in the Golden Path is fragmented randomly into smaller pieces and each piece is cloned into a plasmid and sequenced on both strands. These sequences are aligned so that identical sequences are overlapping. These contiguous pieces are then assembled into finished sequence once each strand has been sequenced about 4 times to produce 8X coverage of high quality data. Figure 1. Schematic diagram of sequencing strategy used by the publicly funded Human Genome Project. The DNA was cut into 150 Mb fragments and arranged into overlapping contiguous fragments. These contigs were cut into smaller pieces and sequenced completely.
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The method developed and preferred by Celera is simply called shotgun sequencing . This approach was developed and perfected on prokaryotic genomes which are smaller in size and contain less repetitive DNA. Shotgun sequencing randomly shears genomic DNA into small pieces which are cloned into plasmids and sequenced on both strands, thus eliminating the BAC step from the HGP's approach. Once the sequences are obtained, they are aligned and assembled into finished sequence. Figure 2. Schematic diagram of sequencing strategy used by Celera. The DNA was cut into small pieces and sequenced completely. These fragments were organized into contigs based on overlapping sequences. The advantage to the hierarchical approach is sequencers are less likely to make
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Sequencing_Whole_Genomes - Sequencing Whole Genomes...

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