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2. The shotgun strategies
2.1 Whole genome shotgun sequencing
J.Craig Venter recognized the labor and time saving possibilities of the whole genome shotgun method, and he and his team developed an assembly algorithm that can handle large volumes of sequencing data produced by shotgun sequencing of large scale genomes such as the human genome.
The whole genome shotgun method is a strategy that skips the laborious and time-consuming mapping process of the hierarchical shotgun approach by reassembling random fragments taken from the genome as a whole using computer algorithms (Figure 3.). The strategy seems very simple and straightforward, but when it is applied to the sequencing of large genomes such as the human genome, it is necessary to produce millions of sequence fragments and reassemble them in a limited time. The strategy requires high-throughput DNA sequencers that enable the sequencing of hundreds of thousands of bases per day, and a computer algorithm that permits the assembly of millions of random fragments. Venter founded The Institute for Genome Research (TIGR), where an algorithm was developed for the assembly of millions of cDNA fragments synthesized with the human messenger RNA(13). Using this algorithm, Venter and his coworkers at TIGR completed the sequencing of the Haemophilus influenza genome, which contains 1.8 million bases(14). Although sequencing of a megabase-scale genome was possible, most genomic research scientists thought it would not work for the human genome, which contains millions of repetitive DNA sequences.
2.2 Hierarchical shotgun sequencing
The sequencing of the human genome can lead to an understanding of human evolution, underlying molecular mechanisms of disease, and the interplay between the environment and heredity in defining the human condition. The sequencing of the human genome was started as an international project, the Human Genome Project (HGP), and in the early phase of the sequencing of the human genome by HGP, efforts were concentrated on making genetic, physical and sequence map. Later, a hierarchical shotgun approach (Figure 3.) to sequencing the human genome was adopted.
In the hierarchical shotgun approach, the entire genome is shredded into large fragments of several hundred thousands bases, and these large fragments are then positioned on the genome chromosomes by looking for distinctive marker sequences, whose locations have already been pinpointed (mapping process). The large fragments are then shattered into tiny fragments and each fragment is sequenced, and computer algorithms that recognize matching sequence information from overlapping fragments are used to reconstruct the complete sequence of the large fragments. The mapping process turned out to be laborious and time-consuming and the sequencing speed was not fast enough due to the limited productivity of the DNA sequencers available in those days. It was obvious that both methodological and technological innovations were necessary to complete the human genome sequencing on schedule.
2.3 The development of the whole genome shotgun strategy and sequencing of the human genome
In January 1998, the emergence of the fully automated high throughput DNA sequencer, the PRISM3700, and the development of the whole genome shotgun sequencing algorithm, changed the situation that confronted human genome sequencing. Right before launching the sequencer on the market, Hunkapiller invited Venter to see the new sequencer(15) and made a bold proposal to found a private company whose business would involve sequencing the human genome using the newly developed DNA sequencers, building up a database for the human genome, and selling it to customers. Hunkapiller and Venter discussed the feasibility of the plan and evaluated the strategies for completing the sequencing of the human genome.
They set up a strategy to apply the whole genome shotgun method to sequencing the human genome. The strategy (the whole genome shotgun strategy) involves the use of a modular sequencing system from sample preparation to assembly of sequencing data(16) (Figure 4.). Four principal modules were designed to operate independently: (i) library transformation, plating, and colony picking; (ii) DNA template preparation; (iii) dideoxy sequencing reaction set-up and purification; and (iv) sequence determination with the ABI PRISM3700 DNA sequencer. Also an automated trace-processing pipeline was developed to process each sequence file. In order to assure the quality of the processes, procedural controls were established to maintain the validity of the sequencing results as the sequencing reactions proceeded through the process.
In May 1998, they founded a publicly-traded company, Celera Genomics, with three hundred automated high-throughput PRISM3700 DNA sequencers, and large scale computers to sequence the human genome. They declared that they would complete the sequencing of the human genome in three years using the whole genome shotgun method(17).
Before challenging the sequencing of the human genome, they tested the whole genome strategy by sequencing the Drosophila genome which contains 120 million bases, and completed the sequencing in only 4 months(18).
Armed with these developments at Celera Genomics and referring to the publicly available data, Hunkapiller, Venter, and coworkers completed the draft sequence of the human genome in 9 months and proved the validity of their strategy(16). Without Hunkapiller and Venter, the acceleration of human genome sequencing would not have been occurred.
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