15.13: Genome Sequencing II - Next-Gen Methods
The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features. Next-generation sequencing allows for the parallel sequencing of millions of fragments of DNA as opposed to the traditional sequencing methods. The pure genomic DNA is first fragmented into smaller fragments to make a sequencing library. This DNA library is then amplified for use in the actual sequencing reactions. While the reversible terminator sequencing method uses fluorescent dNTPs with a reversible terminator as a critical ingredient in the sequencing reaction, pyrosequencing utilizes the pyrophosphate released after the addition of each nucleotide. This pyrophosphate is appropriated for a light-generating reaction by the firefly luciferase enzyme, which can then be detected. Hence, both these methods work on the principle of ‘sequencing by synthesis.’ On the other hand, ‘sequencing by ligation’ methods rely on the specificity and sensitivity of DNA ligases towards mismatch base-pairing to decipher the nucleotide sequence of a DNA fragment.
Application of Next-Generation Sequencing
Next-generation sequencing methods are not solely applied to whole-genome sequencing. They are often used in the field of clinical diagnostics, epigenetics, metagenomics, epidemiology, and transcriptomics. Next-generation sequencing technologies also have the potential to be applied in personalized medicine to accelerate early detection and intervention of some disorders, including cancer.