Third-generation sequencing

Third-generation Sequencing

Third-generation sequencing (TGS) refers to a class of DNA sequencing technologies that enable the direct sequencing of single DNA molecules without the need for amplification. These technologies have revolutionized genomics by providing longer read lengths, faster sequencing times, and the ability to detect epigenetic modifications.

History

The development of third-generation sequencing technologies began in the early 2000s as researchers sought to overcome the limitations of second-generation sequencing technologies, such as short read lengths and the need for PCR amplification. The first commercially available third-generation sequencer was released by Pacific Biosciences in 2011.

Technologies

Several third-generation sequencing technologies have been developed, each with unique mechanisms and advantages.

Single-Molecule Real-Time (SMRT) Sequencing

Single-Molecule Real-Time Sequencing is a technology developed by Pacific Biosciences. It uses zero-mode waveguides (ZMWs) to observe the synthesis of DNA in real-time. This method allows for the detection of long reads, often exceeding 10,000 base pairs, and can also identify epigenetic modifications such as DNA methylation.

Nanopore Sequencing

Nanopore sequencing is a technology developed by Oxford Nanopore Technologies. It involves passing a single DNA molecule through a nanopore and measuring changes in electrical current to determine the sequence of bases. This technology is notable for its ability to produce ultra-long reads, sometimes over 100,000 base pairs, and for its portability, as devices like the MinION are small and easy to use in the field.

Applications

Third-generation sequencing has a wide range of applications in various fields of biology and medicine.

Genomics

In genomics, third-generation sequencing is used for whole genome sequencing, particularly for complex genomes with repetitive regions that are difficult to resolve with short-read technologies. It is also used for de novo genome assembly and for sequencing highly heterozygous genomes.

Transcriptomics

In transcriptomics, third-generation sequencing allows for the sequencing of full-length mRNA transcripts, providing a more complete view of the transcriptome. This is particularly useful for identifying alternative splicing events and for characterizing isoforms.

Epigenomics

Third-generation sequencing technologies can directly detect epigenetic modifications such as DNA methylation and histone modifications, providing insights into the regulation of gene expression and the role of epigenetics in disease.

Clinical Applications

In the clinical setting, third-generation sequencing is used for cancer genomics, where it helps identify structural variants and mutations that drive cancer. It is also used in infectious disease diagnostics, allowing for rapid identification of pathogens and their resistance genes.

Advantages and Limitations

Advantages

- Long Read Lengths: Third-generation sequencing provides much longer read lengths compared to second-generation technologies, which is beneficial for resolving complex genomic regions. - Real-Time Sequencing: The ability to sequence in real-time allows for faster data acquisition and analysis. - Direct Detection of Modifications: These technologies can directly detect epigenetic modifications without the need for additional chemical treatments.

Limitations

- Error Rates: Third-generation sequencing technologies generally have higher error rates compared to second-generation sequencing, although this is being improved with advances in technology and bioinformatics. - Cost: The cost per base is higher than that of second-generation sequencing, although this is decreasing over time.

Future Directions

The future of third-generation sequencing is promising, with ongoing developments aimed at improving accuracy, reducing costs, and expanding applications. Integration with other technologies, such as CRISPR-Cas9 for targeted sequencing, is also being explored.

See Also

- Second-generation sequencing - Genome sequencing - Epigenetics

External Links

• [Pacific Biosciences](https://www.pacb.com/)
• [Oxford Nanopore Technologies](https://nanoporetech.com/)

Third-generation sequencing

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  MinION Portable Gene Sequencer

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  Hybrid Assembly

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  Third-generation sequencing