Shotgun sequencing

Shotgun sequencing is a method used in genomics to sequence long DNA strands. It was first proposed by Frederick Sanger, a British biochemist and a two-time Nobel laureate. The method involves breaking the DNA into random fragments, which are then sequenced to obtain reads. These reads are then assembled to yield the complete sequence.

History

Shotgun sequencing was first used in the 1970s by Frederick Sanger to sequence the genome of the bacteriophage ΦX174. The method was later improved and used to sequence the first complete genome of a free-living organism, the bacterium Haemophilus influenzae, in 1995.

Method

Shotgun sequencing involves several steps:

1. Fragmentation: The DNA is broken into random fragments using either physical methods (such as sonication) or enzymes (such as restriction enzymes).
2. Cloning: The fragments are inserted into a vector, such as a plasmid, to create a library of DNA fragments.
3. Sequencing: Each fragment in the library is sequenced using a method such as Sanger sequencing or next-generation sequencing.
4. Assembly: The sequences (reads) are assembled into the complete sequence using bioinformatics software.

Applications

Shotgun sequencing has been used to sequence the genomes of many organisms, including humans. It has also been used in metagenomics, a field that studies the collective genomes of microbial communities.

Limitations

Despite its wide use, shotgun sequencing has several limitations. For example, it can be difficult to assemble the reads if the DNA contains many repetitive sequences. Also, the method requires a large amount of DNA, which can be a limitation when sequencing rare or precious samples.

See also

• Whole genome sequencing
• Next-generation sequencing
• Sanger sequencing
• Metagenomics