What is Sequencing?   Sequencing is a set of laboratory techniques used to determine the sequence of nucleotides in a sample of genetic material. Examples of genetic material that can be sequenced include the whole genome, exons, and transcriptomes.

Applications of Sequencing:

  • Detecting mutations in specific diseases.
  • Identifying microorganisms such as specific bacterial species for diagnosis of infections.
  • Identifying polymorphisms involved in disease development and potential therapeutic targets.
  • Pharmacogenomic studies to investigate the effects of drugs on patients.

What are the Different Types of Sequencing?

First-generation or direct sequencing (Maxam-Gilbert and Sanger sequencing) are high-accuracy sequencing methods that use gel electrophoresis. These methods are not used as frequently because of high cost and low throughput. First-generation sequencing is used for:

  • Moderate length molecules
  • Detection of small variations in sequencing
  • Samples in which PCR amplification is not required.
  • Validation of next-generation sequencing data.

Second-generation sequencing (Roche 454, Illumina GA, and ABI SOLiD) are next-generation sequencing methods that also provide highly accurate results, but have been developed to offset the high cost-to-throughput ratio encountered in first-generation sequencing through parallel sequencing. Second-generation sequencing methods require PCR amplification and are used for:

  • Short molecules
  • Sequencing mRNA molecules, copy number variants, and single nucleotide polymorphisms.

Third-generation sequencing is the newest addition to gene sequencing and is still under active development. Unlike first- and second-generation technologies, the third generation has been found to have a lower read accuracy. However, it still has several advantages and potential applications:

  • Ability to produce long-read sequences of over 1000 base pairs.
  • Substantially quicker results.
  • May be used for structure variant calling, which is more forgiving to higher error rates.
  • Identification of epigenetic markers.

Source: Ilkhanoff, L., Mouli, S., & Lin, S. (2011, December). Genomic sequencing in clinical trials. Journal of Translational Medicine, 9(1).