An Introduction to Shotgun Sequencing: Fire in the Hole
In the midst of all the cool new sequencing techniques and technologies out there today, you may have overlooked the tried and true method of Shotgun Sequencing.
What is Shotgun Sequencing Anyway?
Shotgun sequencing gets its name from the concept that a large sequence is essentially broken up in to many, many smaller pieces, similar to how a shotgun shell breaks apart when fired.
You essentially put in your entire sequence (the fire in the hole bit) and break it up into fragments. These fragments, called “reads”, are then pieced together by looking for overlap in sequences. Reads can be anywhere between 2-300 kilobases in length (yourgenome.com). This process is repeated a few times to create more and more reads. More reads equals better coverage of your sequence, which leads to a more accurate result.
With the help of computer software, the reads are put in relative order by looking for unique areas of sequence where the reads may overlap. The overlapping reads are then put into longer reads called “contigs”. With this process, you’re basically reordering a jumbled up sentence. Think of nouns and verbs as unique sections of sequence that generally only show up once and articles (like the, a, and an) are the non-unique portions that can show up anywhere. This diagram illustrates shotgun sequencing well.
Once contigs are built, the matching process is essentially repeated to build larger sections, or “scaffolds” and framework, which are then mapped to a reference genome.
Why Is This Method Useful?
Shotgun sequencing works well for large pieces of DNA sequence. Using a next gen sequencing method on large pieces of DNA can be incredibly expensive, while shotgun sequencing is generally cheaper and faster (when a reference sequence is available). Because you are using a reference sequence, you also do not need to waste time mapping clones like you would in clone-by-clone sequencing. This can save you a lot of time and energy, particularly when you are interested in those larger sequences.
Shotgun sequencing also needs much less DNA than other methods of sequencing. This means that you can make your DNA sample go farther, which can be especially important if it is irreplaceable!
The drawbacks of shotgun sequencing are that you need a reference sequence to compare your sequence of interest to. This means you have a sequence from an ideal or similar organism that the software (or you!) can compare to. This helps fill in the gaps with best guesses for parts where reads just don’t overlap.
You are sacrificing accuracy for speed with this technique, but the use of a reference can help minimize errors or, at least, make them easier to resolve!
Another drawback is that shotgun sequencing needs many unique areas in the sequence. Sequences with lots of repeating sections are tough or impossible for this method because the software can’t map those sections.
What do you think of shotgun sequencing? Would you use it? Comment below!
- Adams, J. (2008) Complex genomes: Shotgun sequencing. Nature Education 1(1):186
- Wellcome Genome Campus: Public Engagement, Your Genome, 2016.
- Howard Hughes Medical Institute, 2016
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