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06. Semiconductor Sequencing pH-mediated sequencing, silicon sequencing or semiconductor sequencing

Semiconductor sequencing is another sequencing by synthesis method that is based on detection of H+ ions released during the polymerization of DNA. With this technique, Life Technologies released the Personal Genome Machine in 2011 as, "a rapid, compact and economical bench top machine."

One great thing about this technology is that there is no need for a modified nucleotide, or oligonucleotides as we have seen in reversible chain terminators and Sanger sequencing.

Procedure

1) Emulsion PCR

Emulsion PCR allows for enriched beads to be placed in microwells (see micro-machined well). Just underneath these microwells are pH sensors that are able to detect the most miniscule changes in pH.

Microwell containing the DNA template with an ISFET ion sensor.
Microwell containing the DNA template with an ISFET ion sensor. Each microwell is loaded up with a bead from emulsion PCR.

pH

Remember that pH is just a logarithmic scale that measures the amount of hydrogen ions (H+) in a solution. The lower the pH, the more hydrogen ions there are.

2) dNTP's flooded

A particular dNTP is released. If the growing sequence requires that particular dNTP, then a H+ ion and pyrophosphate group is released.

Hydrogen ion being released with DNA polymerization.
A H+ ion is released when a corresponding dNTP is incorporated. Adapted from David Tack.

3) Signal detection

The signal is picked up by the ISFET sensor and translated into a base call. Any homopolymers (multiple of the same base) result in a strong signal.

4) Wash and repeat

Unattached dNTP molecules are washed out, and the cycle repeats with a new dNTP.

Analysis

Ion Proton System.
The Ion Proton System was release from Life Technologies in 2012.
Ion Torrent Personal Genome Machine Ion Proton System
Bases per run 1 Gb Up to 10Gb
Read Length 35-400 bp 200 bp
Run time 4.5 hours 2-4 hours

Pros

  • Rapid sequencing speed.
  • Low upfront and operating costs.
  • Real-time sequencing.
  • No need for modified nucleotides or special enzymes.
  • No need for expensive optical equipment.

Cons

  • Difficult to capture homopolymer regions such as CCCCCC. This results in multipled hydrogen ions going off at once, and a greater pH change. However, there is a loss of resolution as the number of repeated elements increases.
  • Short read lengths compared to Sanger sequencing and pyrosequencing.
  • Rate limited by dNTP flow.

Videos

Watch how the Ion Torrent system works.

A more detailed look into the Ion Proteon Sequencer.

References

  1. Wikipedia - Ion semiconductor sequencing.
  2. Life Technologies - Ion Torrent
07. Bridge PCR
05. 454 Roche Pyrosequencing

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