The demonstration and experimental operation is performed mostly in the Liping Wei laboratory in the National Institute of Biological Sciences
in the National Institute of Biological Sciences (NIBS), Beijing .
The first step is the library preparation.
The input sample for the Ion Torrent PGM system can be the purified DNA after fragmentation, the cDNA generated by reverse transcription, or
mixed DNA fragments (e.g. fragments with the PCR amplicon)
To construct the library for Ion Torrent PGM system, we can blunt the ends of purified DNA samples and ligate the adapters. A 5-7 cycles of
enrichment will then be applied to ensure that each fragment of the library for constructing templates is flanked by two adapters
simultaneously.
As the video shows, this step can be done by general PCR machines in the laboratory.
The next step is to prepare the template for sequencing.
This step is automatically performed primarily in the Ion OneTouch 2 and Ion OneTouch ES instruments.
This is the Ion OneTouch 2.
This step is to amplify the library fragments on ISPs by emulsion PCR and enrich those ISP that can be used as templates.
The emulsion PCR amplifies the PCR system in tiny aqueous phase droplets, which are enclosed by oil phase to avoid cross-contamination.
Note that the templates are the library DNA fragments ligated with adapters; for the primer, one of its ends is bound to the ISP, and the other is in the
PCR system.
Now as you can see, the research staff is putting the emulsion PCR system into the Ion OneTouch instrument.
The Ion OneTouch can automatically perform the generation of the emulsion, PCR amplification, and ISP centrifugal recovery.
After getting the precipitated ISPs, we will use Ion OneTouch Enrichment System (ES) for automatic enrichment.
This is the instrument.
The instrument uses the streptavidin magnespheres for enrichment of specific sequences to recycle the ISPs that can be used
as templates.
After the enrichment, we add to them the sequencing primers, the polymerase, and the control ISPs, and mix thoroughly.
We finally get the mixture in the tube as the video shows.
Then we can add the sequencing sample into a semiconductor chip.
This is the semiconductor chip. It consists of millions of wells and each chip well is expected to have inside a Ion Sphere particle (ISP).
Itâs not so unimaginable to add the sequencing sample into the chip. There is a small hole on the chip. You can add slowly the sequencing
sample through this hole by rotating the pipetteâs volume control knob at a speed of about 1 uL/s.
Now we can see the experimenter is loading sample into the chip.
After that, we can start the sequencing run.
Here is the Ion Torrent PGM in our laboratory.
Before sequencing, we need to initialize the instrument.
As mentioned before, the sequencer identify which base is added by detecting changes in pH. So the initialization process is actually cleaning
the entire sequencing system and calibrating the pH value.
Then we can see the chip is being installed into the Ion Torrent PGM sequencer . We can run the sequencing after that.