Ion Torrent Next-Generation Sequencing

Overview

• A type of next-generation sequencing (NGS) that involves DNA synthesis.
• Exploits the release of hydrogen ions during DNA polymerization.
• Detects pH changes using a semiconductor chip.

Process Steps

1. Library Preparation

   • Fragment DNA via sonication or nebulization.
   • Add specific adapters to both the 5' and 3' ends of the DNA.

2. Emulsion PCR

   • Incubate DNA with a bead coated with complimentary oligonucleotides.
   • Dilute the mixture to ensure one template strand per bead.
   • Create an emulsion with oil to form microvesicles containing beads with DNA.
   • Each microvesicle acts as a microreactor for PCR, refining the DNA strands.
   • Amplifies DNA through cycles (30-60 times) to produce thousands of copies.

3. Loading onto Ion Chip

   • Break the emulsion and transfer beads onto chips with millions of micro wells.
   • Each well can hold a single bead.

4. Signal Processing

   • Flood the micro wells with nucleotides, one type at a time.
   • Use an ion-sensitive field effect transistor (ISFET) to detect hydrogen ions released during incorporation of nucleotides.
   • No voltage detected if nucleotide does not match; voltage change indicates incorporation of a complementary nucleotide.
   • Release of hydrogen ions allows for detection of multiple bases at once, resulting in higher voltage signals.

Key Components

• Ion-sensitive Field Effect Transistor (ISFET): Measures ion concentrations and detects signal changes.
• Electrical Pulses: Generated for each hydrogen ion released during nucleotide incorporation, translating into a DNA sequence.
• Software Processing: Assembles the sequence without needing fluorescently labeled nucleotides or optics.

Advantages

• Fast, efficient, and adaptable compared to other sequencing methods.