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- Introduction
- Real-time PCR
- Microarrays
- RNA sequencing
- Biological interpretation of gene expression data
- Genotyping, epigenetic and DNA/RNA-protein interaction methods
- DNA/RNA-protein interactions
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Third-generation sequencing
The first paper deploying TGS sequencing was published in 2009. However, TGS gained momentum only in the later 2010s; this is mostly due to third-sequencing technologies initially presenting lower accuracy than second-generation ones, an issue that HiFi sequencing from PacBio has recently overcome.
The distinguishing feature of TGS is that it is a single-molecule sequencing technology, i.e. the DNA amplification step essential for the previous generations is not required. TGS ‘reads’ the DNA molecule, allowing for faster sequencing and longer reads generation (in the range of kilobases).
This is particularly relevant when sequencing and de novo assembly, both for genomic and transcriptomics studies.
TGS currently consists of two main technologies:
- Nanopore Sequencing, which is based on membrane current alterations generated by nucleotides of a DNA strand as they pass through a membrane nanopore: each nucleotide produces a specific change in current, allowing for the DNA molecule to be ‘read’. This is the sequencing method used by Oxford Nanopore.
- SMRT (Single Molecule Real Time) sequencing, which takes advantage of zero-mode waveguide technology to perform sequencing by synthesis of single DNA molecules; this is the sequencing method used by HiFi PacBio.
On the following two pages, you will discover more about these two sequencing methodologies. If you are interested in diving deeper into TGS and its applications, you can read this review.