Ion Torrent Sequencing: Principle, Steps, Method, Uses microbiologystudy

Ion Torrent Sequencing is a next-generation sequencing method that uses a semiconductor chip to directly detect the hydrogen ions (H⁺) released during DNA synthesis. Ion Torrent technology introduced the first commercially available semiconductor chip capable of directly converting chemical signals into digital data.

Ion Torrent sequencing is distinct because it does not rely on traditional optical detection methods. While highly accurate, optics-based technologies tend to be very expensive. Instead, Ion Torrent involves direct detection without the need for scanning, cameras, or light so the sequence is instantly captured. Ion Torrent sequencing is known for its fast, cost-effective, and highly scalable sequencing capabilities. Due to its speed, low cost, and simplicity, Ion Torrent sequencing has found wide applications in different fields including genomics, oncology, microbiology, and personalized medicine.

Unlike traditional approaches that rely on complex imaging, fluorescence detection, cameras, or scanners, Ion Torrent uses the direct measurement of pH changes during the sequencing process. As the DNA polymerase incorporates nucleotides, the release of hydrogen ions results in pH changes, which are detected electronically. This speeds up the sequencing process and also simplifies the overall workflow.

Principle of Ion Torrent Sequencing

The principle of Ion Torrent sequencing is based on detecting protons (H⁺) released when nucleotides are added to a growing DNA strand during DNA synthesis. As DNA polymerase incorporates a nucleotide into the complementary strand, a proton is released which lowers the pH in the surrounding environment. This change is detected by the ion sensor in the semiconductor chip which is converted into a voltage signal. This signal is used to identify the sequence of the DNA template. Unlike other sequencing technologies like Illumina which uses light to detect labeled nucleotides, Ion Torrent technology uses pH-based detection. 

Ion Torrent sequencing uses semiconductor chips called complementary metal-oxide semiconductors (CMOS). These chips are similar to those found in digital cameras and mobile phones. It uses an electrochemical detection system known as ion-sensitive field-effect transistors (ISFETs), which can detect ions released during DNA synthesis. The release of protons leads to a slight shift in pH which is detected by the sensors. The sensors consist of wells filled with dNTPs and beads containing a DNA template. Beneath each well lies a metal oxide sensing layer. This setup allows the electronic information from pH changes to be transmitted to the semiconductor for analysis.

Video on Ion Torrent Sequencing

Steps of Ion Torrent Sequencing

1. DNA Extraction and Library Preparation

• The first step is creating a sequencing library which consists of DNA fragments that are ready for sequencing. 
• The process begins with fragmenting the DNA using methods like sonication or enzymatic methods. This step is necessary for generating small and manageable DNA pieces that can be sequenced efficiently.
• After fragmentation, specific adapters are attached to both ends of the DNA fragments. These adapters help in immobilizing the DNA on beads for further amplification.
• Then the DNA fragments are selected based on the specific size requirements for the library using gel electrophoresis. For short-read sequencers like Ion Torrent, it is best to use shorter fragments. The final DNA fragment size must be uniform to ensure efficient downstream sequencing.
• The prepared library is then quantified and checked for quality to ensure accurate sequencing. 

2. Amplification

• After library preparation, the DNA fragments are bound to the surface of tiny beads called Ion Sphere Particles (ISPs) and amplified using emulsion PCR (emPCR). Each bead is loaded with a single DNA fragment. 
• The beads are emulsified in oil, creating tiny water-in-oil droplets that act as microreactors. This allows the amplification of DNA fragments on individual beads and results in a collection of beads containing clonally amplified target molecules.
• After emPCR, the beads containing successfully amplified DNA are enriched by centrifugation, ensuring that only beads with amplified DNA proceed to the next steps.

3. Sequencing

• The amplified beads are then loaded into microwells on the semiconductor chip. Each well holds a single bead. 
• During the sequencing process, nucleotides are sequentially flooded over the chip and the incorporation of the nucleotide is detected by the ion sensor on the chip.
• If a nucleotide matches the DNA template, it releases hydrogen ions which change the pH of the solution. The ion sensor on the chip detects the change in pH as an electrical signal. These signals are used to determine nucleotides incorporated during the sequencing process. If there is no match, no signal is detected.

4. Data analysis

• Once the sequencing run is complete, the generated data are transferred to the server where the signal is processed and base calling algorithms are used to determine the DNA sequence of each fragment.
• The data from the server can be viewed through a web interface and can be downloaded in formats that are compatible with various next-generation sequencing analysis tools.
• Different software tools are used to assemble sequences, align them to the reference genomes, identify variants, and annotate the data.

Advantages of Ion Torrent Sequencing

• Ion Torrent sequencing is faster compared to many other sequencing technologies which is ideal for time-sensitive applications such as clinical research or diagnostics. 
• Ion Torrent sequencing does not rely on optical signals to detect nucleotide incorporation. It eliminates the need for cameras, light sources, or scanners. Instead, it uses a direct detection method based on pH changes which directly converts nucleotide incorporation into voltage signals, speeding up the sequencing process.
• It is cost-effective as it offers a relatively low cost per run making it more accessible to smaller labs. 
• The workflow of Ion Torrent sequencing is simple. It involves fewer complex steps.
• It can be used for a wide range of applications. It supports multiple sequencing approaches such as RNA sequencing, targeted DNA sequencing, and whole-exome sequencing.

Applications of Ion Torrent Sequencing

• Ion Torrent sequencing plays an important role in cancer research, allowing the study of various biomarkers that can be used to develop cancer drugs and study the immune system’s response to cancer. It can be used to perform targeted sequencing for oncology research.
• Ion Torrent sequencing has applications in microbial research. It helps in studying microbial diversity and tracks pathogen outbreaks. This can also be used to detect antibiotic-resistance mutations. It has applications in de novo microbial sequencing, bacterial typing to identify bacterial strains, viral typing, and metagenomics studies to study complex microbial communities. 
• Ion Torrent is also useful for studying complex diseases like autoimmune disorders and neurodegenerative conditions. It can be used in gene expression profiling to understand how genes behave. It can be used in targeted DNA and RNA sequencing to focus on disease-causing regions in the genome. 
• It is used in plant and animal genotyping to identify traits that enhance agriculture. This supports innovations in crop and livestock breeding and helps address food security challenges.
• Ion Torrent sequencing has applications in targeted DNA sequencing which studies specific regions of interest.
• Ion Torrent sequencing can be used in forensic DNA analysis to identify individuals from DNA samples. This helps with genetic profiling in crime investigations.
• It has applications in clinical diagnostics to detect genetic mutations linked to different diseases. It is also an important tool in personalized medicine and allows treatments based on the genetic profile of a patient.

Limitations of Ion Torrent Sequencing

• Ion Torrent sequencing struggles with homopolymeric regions which are sequences of repeating nucleotides. When multiple nucleotides are incorporated simultaneously, a large amount of H⁺ ions are released which generates more signal. It has difficulties in accurately sequencing homopolymers and can lead to errors in base calling.
• This technology has a relatively short read length compared to other sequencing methods like Sanger sequencing. This limits certain types of analysis that require longer stretches of continuous DNA sequence. 
• The process of emPCR used to amplify DNA can introduce errors and bias or loss of certain DNA sequences.
• Due to its shorter read lengths, lower throughput, and challenges with assembling complex genomes, it is less suitable for large-scale genome sequencing projects.

References

1. EMBL-EBI. (n.d.). Ion Torrent: Proton / PGM sequencing | Functional genomics II. Retrieved from https://www.ebi.ac.uk/training/online/courses/functional-genomics-ii-common-technologies-and-data-analysis-methods/next-generation-sequencing/ion-torrent-proton-pgm-sequencing/
2. Ion Torrent | Thermo Fisher Scientific – NP. (n.d.). Retrieved from https://www.thermofisher.com/np/en/home/brands/ion-torrent.html
3. Ion TorrentTM next-gen sequencing technology. (n.d.). Retrieved from https://www.thermofisher.com/np/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-technology.html
4. Pereira, R., Oliveira, J., & Sousa, M. (2020). Bioinformatics and computational tools for Next-Generation sequencing analysis in clinical genetics. Journal of Clinical Medicine, 9(1), 132. https://doi.org/10.3390/jcm9010132
5. Porterfield, A. (2020, April 2). How the Ion Torrent Sequencer works. Retrieved from https://bitesizebio.com/13547/how-the-ion-torrent-sequencer-works/
6. Saadat, A. (2020, August 30). Ion Torrent sequencing. Retrieved from https://biobinge.pubpub.org/pub/its/release/1
7. Slatko, B. E., Gardner, A. F., & Ausubel, F. M. (2018). Overview of Next‐Generation Sequencing Technologies. Current Protocols in Molecular Biology, 122(1). https://doi.org/10.1002/cpmb.59