Semiconductor Sequencing: Principle, Steps, Uses microbiologystudy

Semiconductor sequencing is a technology that uses electronic sensors fixed in microchips to sequence nucleic acids by directly measuring electrical signals caused by proton release during DNA synthesis.

This sequencing method merges semiconductor technology with DNA sequencing by combining complementary metal-oxide semiconductor (CMOS) chips with sequencing by synthesis (SBS) chemistry.  

DNA sequencing was dominated by the chain-termination method for decades and most next-generation sequencing methods have relied on detecting light signals. However, sequencing technologies have advanced to develop innovative methods like semiconductor sequencing. Semiconductor sequencing technology is a powerful alternative to traditional DNA sequencing technologies. Instead of relying on optical detection which involves complex and costly equipment, semiconductor sequencing systems use electrical detection technologies to detect DNA sequences. This makes sequencing faster, more scalable, and more cost-effective.

The first commercial use of semiconductor sequencing technology was Ion Torrent sequencing. It was introduced in 2010 by Jonathan Rothberg, who also developed the Roche 454 sequencing platform. This semiconductor-based sequencing technology was developed and commercialized by Ion Torrent Systems which was later acquired by Life Technologies and is now part of Thermo Fisher Scientific.

Principle of Semiconductor Sequencing

Semiconductor sequencing works on the principle of sequencing by synthesis (SBS) and uses ion-sensitive field-effect transistors (ISFETs) arranged in CMOS chips to detect changes in electric potential during nucleotide incorporation.

This sequencing platform is based on detecting the release of hydrogen ions (H⁺) during the polymerization of DNA. The chips contain millions of tiny wells and pH sensors to allow massively parallel sequencing.

The process involves amplifying adapter-ligated DNA fragments using emulsion PCR (emPCR) where the fragments are encapsulated with small beads along with PCR reagents in an oil-based emulsion. Beads carrying amplified DNA are enriched from the emulsion and loaded into sequencing chips. During sequencing, nucleotides are sequentially added into the reaction wells. When a nucleotide matches the complementary base on the DNA template, it is incorporated into the newly synthesized strand. Each nucleotide addition results in the release of a hydrogen ion as a byproduct of the polymerization process. The released H⁺ ions change the pH of the solution and cause a change in electric potential in the well. These changes are detected by the ISFET sensor at the bottom of each well. The electrical signals generated by these changes are processed and converted into data to identify nucleotides.

CMOS Chips and ISFETs in Semiconductor Sequencing

• The development of semiconductor sequencing technology is built on decades of advancements in semiconductor sensor devices and DNA sequencing techniques. 
• Semiconductor sequencing uses biosensors which combine ISFET sensors with CMOS technology.
• CMOS chips are the basis of semiconductor sequencing technology. It was originally developed in the 1970s and has been widely used in modern electronic devices like computers, smartphones, and digital cameras. 
• In semiconductor sequencing, CMOS chips allow millions of sequencing reactions to occur directly on the chip. This removes the need for traditional optical detection systems by using electronic sensors, simplifying sequencing and reducing costs. 
• ISFET is an electrochemical sensor that is sensitive enough to measure even the smallest changes in ion concentration. 
• ISFETs are essential to semiconductor sequencing systems because they are compatible with standard CMOS technology which allows millions of sensors to be integrated onto a single chip.
• The use of CMOS-based sensors in sequencing has advanced sequencing technologies. They have the advantage of being small and compatible with large-scale integration at low cost.

Process of Semiconductor Sequencing

1. Library Preparation

• The process begins with library preparation similar to other sequencing methods.
• First, genomic DNA is extracted and fragmented. Then, adapter sequences are attached to the ends of each DNA fragment to facilitate sequencing.

2. Template Amplification

• The prepared DNA fragments are amplified using emulsion PCR to create millions of copies of each fragment which are then loaded onto the sequencing chip.
• DNA fragments are first attached to beads called Ion Sphere Particles (ISPs) through the adapter sequences.
• The ISPs are suspended in a water-oil emulsion, creating microreactors where clonal amplification occurs.
• Only beads with successfully amplified DNA are isolated and purified for sequencing.

3. Sequencing

• The enriched beads are loaded onto the semiconductor sequencing chip. The chip contains millions of microwells each designed to fit a single ISP.
• Semiconductor sequencing uses sequencing-by-synthesis approach.
• The DNA polymerase enzyme adds nucleotide to the DNA template in the presence of sequencing reagents.
• When a nucleotide is incorporated, a hydrogen ion is released which changes the pH of the solution in the well.
• The ISFET sensor at the bottom of the microwell detects this pH change and converts the chemical signal into an electrical signal.
• The electrical signal is processed into voltage output that represents nucleotide incorporation. 

4. Data Analysis

• The electrical signals recorded during sequencing are translated into nucleotide sequences.
• The raw sequencing data undergoes quality control and alignment to reference genomes, generating meaningful biological information.
• Advanced bioinformatics tools analyze the data to reconstruct the DNA sequence.

Advantages of Semiconductor Sequencing

• Semiconductor sequencing is more affordable. The use of CMOS technology reduces manufacturing and operational expenses. Silicon-based pH sensors are easier and less expensive to manufacture compared to advanced optical systems.
• This technology eliminates the need for complex optics, simplifying the sequencing workflow. 
• It uses natural, unmodified nucleotides instead of chemically modified or fluorescently labeled bases.
• The real-time nature of ion detection reduces sequencing time and produces rapid results. This makes them ideal for applications such as clinical diagnostics and pathogen detection. 
• Semiconductor sequencers are smaller and easier to handle compared to optical-based systems. It eliminates bulky optical equipment leading to smaller and more portable sequencing systems. The compact setup makes it suitable for laboratories with limited space.
• It is easier to use and requires minimal training compared to other platforms. 

Limitations of Semiconductor Sequencing

• Semiconductor sequencing produces shorter read lengths compared to some other methods.
• It requires advanced data analysis software and expertise to interpret the results accurately.
• Semiconductor sequencing struggles with accurately reading homopolymer regions. This can lead to inaccurate base calling, making it less suitable for genomes with repetitive sequences. 
• Changes in environmental conditions and buffer systems can affect the precision of pH detection. Inconsistent pH measurements can lead to errors in base calling.
• Although it is faster and simpler, its overall error rates may be higher than other technologies, particularly in repetitive and GC-rich regions.

Applications of Semiconductor Sequencing

• Semiconductor sequencing has applications in clinical diagnostics and research. It can be used in cancer research to identify cancer-specific mutations and study tumor genetics. It is also useful in the diagnosis of infectious diseases by identifying pathogens. 
• It is useful in microbiome studies to study gut microbiome and understand microbial communities in the environment.
• It has applications in agricultural genomics by facilitating crop improvement and animal breeding.
• Semiconductor sequencing is well-suited for targeted DNA and RNA sequencing. 
• It can also be used for exome sequencing to identify mutations associated with genetic diseases.
• Semiconductor sequencing can be used in chromatin immunoprecipitation sequencing (ChIP-Seq) to map protein-DNA interactions and study epigenetic modifications.

References

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