Single-molecule real-time sequencing

Definition

Single-molecule real-time sequencing (SMRT sequencing) is a cutting-edge DNA sequencing technology that allows for the observation of DNA synthesis in real-time at the single-molecule level. This approach enables the direct measurement of nucleotide incorporation and can produce long reads of DNA, making it particularly valuable for sequencing complex genomes and detecting structural variants.

5 Must Know Facts For Your Next Test

1. SMRT sequencing utilizes zero-mode waveguides (ZMWs) to allow for real-time observation of DNA polymerase activity as it incorporates nucleotides.
2. One significant advantage of SMRT sequencing is its ability to generate long reads, often exceeding 10,000 base pairs, which improves assembly of complex genomes.
3. The technology can detect various types of modifications on DNA bases, providing insights into epigenetic changes and transcriptional regulation.
4. SMRT sequencing has applications in diverse fields such as metagenomics, transcriptomics, and cancer genomics due to its high accuracy and ability to handle repetitive regions.
5. Error rates in SMRT sequencing are typically lower than traditional short-read methods when dealing with homopolymeric regions, making it preferable for certain applications.

Review Questions

• How does single-molecule real-time sequencing differ from traditional sequencing methods in terms of data acquisition?
  • Single-molecule real-time sequencing offers a distinct advantage over traditional methods by allowing the observation of DNA synthesis at the single-molecule level. In contrast to methods like Sanger or Illumina sequencing that rely on amplification and produce shorter reads, SMRT captures real-time data on nucleotide incorporation directly from a single DNA molecule. This unique approach enables researchers to obtain longer contiguous sequences and provides more information about structural variants in genomes.
• What are the technological innovations that enable single-molecule real-time sequencing to produce long reads, and why are these important for genomic research?
  • The key technological innovation behind single-molecule real-time sequencing is the use of zero-mode waveguides (ZMWs), which allow for the observation of individual DNA polymerase molecules as they incorporate nucleotides in real time. This setup not only permits the generation of long reads but also enhances the resolution of complex genomic regions, facilitating accurate genome assembly and detection of structural variations. Long reads are particularly important for genomic research as they help resolve repetitive sequences that can be challenging for short-read technologies.
• Evaluate the implications of using single-molecule real-time sequencing in understanding complex genomic features compared to conventional methods.
  • The use of single-molecule real-time sequencing significantly enhances our understanding of complex genomic features by providing longer read lengths and direct observation of nucleotide incorporation. This capability allows researchers to detect structural variants and epigenetic modifications that conventional short-read methods might miss due to their reliance on amplification and shorter sequences. By improving our understanding of these features, SMRT technology plays a crucial role in advancing fields such as cancer genomics and metagenomics, enabling more comprehensive analyses of genetic diversity and regulation.

Related terms

Polymerase: An enzyme that synthesizes DNA by adding nucleotides one at a time during the replication process.

Long-read sequencing: A sequencing method that produces longer contiguous sequences of DNA, which helps in resolving complex genomic regions.

Nanopore sequencing: A technology that involves passing a single strand of DNA through a nanopore and measuring changes in electrical conductivity to determine the sequence.