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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). https://doi.org/10.1186/1479-5876-9-222