Overview Edit

Shotgun sequencing is a method used to sequence large strands of DNA unable to be sequenced using standard standard methods, such as Sanger sequencing, through breaking up the strands randomly and deriving the full sequence based on where pieces overlap.[1] This is achieved through two different methods known as whole genome shotgun sequencing and hierarchical shotgun sequencing. [2]

Methods Edit

Whole genome shotgun sequencing involves the random fragmentation of the entire genome prior to sequencing. This is achieved through shearing DNA into undefined segments and cloning into vectors for sequence analysis. The obtained sequence fragments are then used to computationally obtain the full sequence of the desired DNA by scanning for overlapping sections

WholeGenomeShotgun (1)

A graphic representation of whole genome shotgun sequencing Image Source

and assembling the DNA based on those with high consensus.[1] Hierarchical shotgun sequencing differs from whole genome shotgun sequencing in that the initial step prior to random fragmentation is a separate, more ordered fragmentation of DNA sequences. This is achieved through the use of partial restriction enzyme digest and cloning into Bacterial Artificial Chromosome (BAC) vectors. These fragments are then amplified, fragmented, and sequenced in the shotgun method. Through the use of this method, larger sequences can be determined than those that the whole genome method allows. [3]

Both methods also have drawbacks. The whole genome approach is more prone to error due to the entire genome being fragmented and the potential for overlap sequences to be attributed to the wrong position.The hierarchical approach is a more time consuming method due to the specific fragmenting and the extra precautions that need to be taken to make sure that initial fragments are isolated. In addition to individual drawbacks, both methods are prone to incomplete covarage. [2]

Applications Edit

Shotgun sequencing is incredibly important for both genomics and metagenomics alike. The power of the technique is put on display most prominently in its metagenomic use. This is because the amount of genomes in a given community is so large that it would be impossibly time consuming to make an attempt at sequencing the DNA in full strands with the drawback that it may not be as sensitive as one would like. This drawback is made up for by the fact that it has been successfully used to sequence incredibly varied communities of bacteria and viruses. One such prominent example is the characterization of the microbiota of human saliva, in which it was successfully shown that humans carry a large number of benign bacteria as well as some common infectious bacteria in their saliva at all times.[4] However it can also be applied to genomics as well, seeing as any single genome would also take a substantial amount of time to sequence, and has revolutionized the way that sequencing occurs.

Sources Edit

  1. 1.0 1.1 R. Staden (June 1979). A strategy of DNA sequencing employing computer programs. Nucleic Acids Res. 6(7): 2601–2610. PubMed
  2. 2.0 2.1 J. Commins, C. Toft, and M. A. Fares (2009). Computational Biology Methods and Their Application to the Comparative Genomics of Endocellular Symbiotic Bacteria of Insects. Biol Proced Online. 11: 52–78.PubMed
  3. Chial, H. (2008) DNA sequencing technologies key to the Human Genome Project. Nature Education 1(1):219 Link
  4. Hasan, N. A. et al. (2014) Microbial community profiling of human saliva using shotgun metagenomic sequencing. PLoS One. 9(5):e97699 PubMed