Whole Genome Shotgun WGS Sequencing, Clone, Genomic Library, Viruses, Bacteria, Archaea, Fruitfly, Craig Venter, Celera Genomics

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Home >> Biotechnology and Genomics >> Methods and Uses of Genomics and Proteomics Research >> Whole Genome Shotgun (WGS) Sequencing

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Whole-genome shotgun (WGS) sequencing.

Whole-genome shotgun sequencing involves shearing or cleavage (partial digestion) of genomic DNA followed by cloning, to produce a genomic library. This is followed by sequencing of cloned DNA-fragments at random, followed by assembly of the fragment sequences into larger units on the basis of their overlaps. The techniques is described s shotgun assembly and is illustrated. This approach does not require any or physical maps of the genome for whole genome sequencing. In the past, this approach was used for small genomics, each having very little or no repetitive sequences (e.g. viruses, bacteria, archaea, fruitfly, etc) but later (during 1998-2001), this approach was extensively utilized by Craig Venter at a private company named Celera Genomics in USA (Rockville, Maryland) not only for sequencing Drosophila genome, but also for sequencing the human genome.

However, while using WGS sequencing approach for human genome sequencing, Craig Venter also made use of publicly available hierarchical shotgun DNA sequence data generated by the International Human Genome Sequencing Consortium (IHGSC). In this WGS approach, 8-10 fold sequencing of the whole genome is needed for coverage of the whole genome. Much less amount of sequencing (4-5 fold) is needed is hierarchical shotgun approach (consult next section), although in this latter approach, considerable work is involved in preparing the whole genome physical map using BACs, and PACs; this work has been completely dispensed with in the WGS sequencing

Different Steps Involved in Whole Genome Sequencing (WGS)

Different Steps involved in whole genome sequencing (WGS) technique used for sequencing the genome of Haemophilus influenzae

Using WGS approach, genome of the bacterium, Haemophilus influenzae was the first genome that was sequenced and the results published in 1995. The sequences were initially obtained in the form of 140 sequenced contigs, each contig, consisting of 2-20 overlapping clones and representing different non-overlapping portions of the genome (a contig is a set of contiguous overlapping clones, each contig having two to more than 25 clones and a singleton is a clone not incorporated into any contig).

The gaps between these contigs were filled later. For this purpose, the genomic library was searched for singletons, whose end sequences may match those of the ends of two different contigs. If such a clone (singleton) is available, its sequence will fill the gap between two contigs. As many as 99 gaps were filled in this manner, while sequencing the genome of H. influenzae.

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The remaining gaps were filled by preparing fresh phage lambda genomic library, and probing it with oligonucleotides, each carrying the sequence that is found at one of the two ends of an unlinked contig (on either side of a gap). This allowed identification of lambda clones. Each hybridizing with two oligonucleotides, so that the sequencing of this clone will bridge this gap between two unlinked contigs. Another strategy selected on the basis of Southern hybridization pairs of oligonucleotides (lambda clones) as primers in PCR with genomic DNA as template so that if a PCR product is obtianed, this could be sequenced to fill the gap

The above WGS approach was extensively utilized during 1990s to sequence dozens of microbial genomes. The strength of this approach was that is could generate sequences faster and that it did not need any physical or genetic map as a prerequisite for sequencing. However, the weakness of the WGS approach is the complexity and limitation of analysing much more sequence data than is needed in “map first-sequence later” approach, discussed

A technique that was used for filling the gaps for assembly of the complete sequence of the genome of Haemophilus Infuenzae

Closing a Sequence gap

Different contigs (1-9); the two ends of each contig are used to derive two oligonucleotides

Closing a Physical Gap; probe a second clone library with oligonucleotides

Complete sequence of the genome

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