Ion semiconductor sequencing
Encyclopedia
Ion Semiconductor Sequencing is a method of DNA sequencing
based on the detection of hydrogen ion
s that are released during the polymerization
of DNA
. This is a method of "sequencing by synthesis", during which a complementary
strand is built based on the sequence of a template stand.
A microwell containing a template DNA strand to be sequenced is flooded with a single species of deoxyribonucleotide
(dNTP). If the introduced dNTP is complementary
to the leading template nucleotide, it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, which indicates that a reaction has occurred. If homopolymer repeats are present in the template sequence, multiple dNTP molecules will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.
This technology differs from other sequencing
technologies in that no modified nucleotides or optics
are used. Ion semiconductor sequencing may also be referred to as ion torrent sequencing, pH-mediated sequencing, silicon sequencing, or semiconductor sequencing. It was developed by Ion Torrent Systems Inc. and was released in February 2010. Ion Torrent have marketed their machine as a rapid, compact and economical sequencer that can be utilized in a large number of laboratories as a bench top machine.
(dNTP) into a growing DNA strand involves the formation of a covalent bond
and the release of pyrophosphate
and a positively charged hydrogen ion
. A dNTP will only be incorporated if it is complementary
to the leading unpaired template nucleotide. Ion semiconductor sequencing exploits these facts by determining if a hydrogen ion is released upon providing a single species of dNTP to the reaction.
Microwells on a semiconductor chip that each contain one single-stranded template DNA molecule to be sequenced and one DNA polymerase
are sequentially flooded with unmodified A, C, G or T
dNTP. If an introduced dNTP is complementary to the next unpaired nucleotide on the template strand it is incorporated into the growing complementary strand by the DNA polymerase. If the introduced dNTP is not complementary there is no incorporation and no biochemical reaction. The hydrogen ion that is released in the reaction changes the pH
of the solution, which is detected by a hypersensitive ion sensor. The unattached dNTP molecules are washed out before the next cycle when a different dNTP species is introduced.
ion sensor. All layers are contained within a CMOS
semiconductor chip, similar to that used in the electronics industry.
Each released hydrogen ion triggers the ISFET
ion sensor. The series of electrical pulses transmitted from the chip to a computer is translated into a DNA sequence, with no intermediate signal conversion required. Each chip contains an array of microwells with corresponding ISFET detectors. Because nucleotide incorporation events are measured directly by electronics, the use of labeled nucleotides and optical measurements are avoided.
reads, with 100 Mb per run. The read-length as of February 2011 was 100 base pairs. The accuracy for homopolymer repeats of 5 repeats in length was 98%.. It should be noted that these figures have not yet been independently verified outside of the company.
Because the system records natural polymerase-mediated nucleotide incorporation events, sequencing can occur in real-time. In reality, the sequencing rate is limited by the cycling of substrate nucleotides through the system. Ion Torrent Systems Inc., the developer of the technology, claims that each incorporation measurement takes 4 seconds and each run takes about one hour, during which 100-200 nucleotides are sequenced. If the semiconductor chips are improved (as predicted by Moore’s law), the number of reads per chip (and therefore per run) should increase.
The cost of acquiring a pH-mediated sequencer from Ion Torrent Systems Inc. at time of launch was priced at around $50,000 USD, excluding sample preparation equipment and a server for data analysis. The cost per run is also thought to be significantly lower than that of alternative automated sequencing methods.
. Signals generated from a high repeat number are difficult to differentiate from repeats of a similar but different number (e.g. 7 from 8 homorepeats).
Another limitation of this system is a short read length compared to other sequencing methods such as Sanger sequencing or pyrosequencing
. Longer read lengths are beneficial for de novo
genome assembly. The read length achieved by Ion Torrent Systems Inc. is currently 200 base pairs per run. The throughput is currently lower than that of other high-throughput sequencing technologies, although the developers hope to change this by increasing the density of the chip.
Table 1. Comparing metrics and performance of next-generation DNA sequencers.
Due to the ability of alternative sequencing methods
to achieve a greater read length (and therefore being more suited to whole genome analysis) this technology may be best suited to small scale applications such as microbial genome sequencing, microbial transcriptome
sequencing, targeted sequencing, amplicon sequencing, or for quality testing of sequencing libraries.
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
based on the detection of hydrogen ion
Hydrogen ion
Hydrogen ion is recommended by IUPAC as a general term for all ions of hydrogen and its isotopes.Depending on the charge of the ion, two different classes can be distinguished: positively charged ions and negatively charged ions....
s that are released during the polymerization
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
. This is a method of "sequencing by synthesis", during which a complementary
Complementarity (molecular biology)
In molecular biology, complementarity is a property of double-stranded nucleic acids such as DNA, as well as DNA:RNA duplexes. Each strand is complementary to the other in that the base pairs between them are non-covalently connected via two or three hydrogen bonds...
strand is built based on the sequence of a template stand.
A microwell containing a template DNA strand to be sequenced is flooded with a single species of deoxyribonucleotide
Deoxyribonucleotide
A deoxyribonucleotide is the monomer, or single unit, of DNA, or deoxyribonucleic acid. Each deoxyribonucleotide comprises three parts: a nitrogenous base, a deoxyribose sugar, and one phosphate group. The nitrogenous base is always bonded to the 1' carbon of the deoxyribose, which is distinguished...
(dNTP). If the introduced dNTP is complementary
Complementarity (molecular biology)
In molecular biology, complementarity is a property of double-stranded nucleic acids such as DNA, as well as DNA:RNA duplexes. Each strand is complementary to the other in that the base pairs between them are non-covalently connected via two or three hydrogen bonds...
to the leading template nucleotide, it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, which indicates that a reaction has occurred. If homopolymer repeats are present in the template sequence, multiple dNTP molecules will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.
This technology differs from other sequencing
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
technologies in that no modified nucleotides or optics
Optics
Optics is the branch of physics which involves the behavior and properties of light, including its interactions with matter and the construction of instruments that use or detect it. Optics usually describes the behavior of visible, ultraviolet, and infrared light...
are used. Ion semiconductor sequencing may also be referred to as ion torrent sequencing, pH-mediated sequencing, silicon sequencing, or semiconductor sequencing. It was developed by Ion Torrent Systems Inc. and was released in February 2010. Ion Torrent have marketed their machine as a rapid, compact and economical sequencer that can be utilized in a large number of laboratories as a bench top machine.
Technology
Sequencing Chemistry
In nature, the incorporation of a deoxyribonucleotideDeoxyribonucleotide
A deoxyribonucleotide is the monomer, or single unit, of DNA, or deoxyribonucleic acid. Each deoxyribonucleotide comprises three parts: a nitrogenous base, a deoxyribose sugar, and one phosphate group. The nitrogenous base is always bonded to the 1' carbon of the deoxyribose, which is distinguished...
(dNTP) into a growing DNA strand involves the formation of a covalent bond
Covalent bond
A covalent bond is a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms. The stable balance of attractive and repulsive forces between atoms when they share electrons is known as covalent bonding....
and the release of pyrophosphate
Pyrophosphate
In chemistry, the anion, the salts, and the esters of pyrophosphoric acid are called pyrophosphates. Any salt or ester containing two phosphate groups is called a diphosphate. As a food additive, diphosphates are known as E450.- Chemistry :...
and a positively charged hydrogen ion
Hydrogen ion
Hydrogen ion is recommended by IUPAC as a general term for all ions of hydrogen and its isotopes.Depending on the charge of the ion, two different classes can be distinguished: positively charged ions and negatively charged ions....
. A dNTP will only be incorporated if it is complementary
Complementarity (molecular biology)
In molecular biology, complementarity is a property of double-stranded nucleic acids such as DNA, as well as DNA:RNA duplexes. Each strand is complementary to the other in that the base pairs between them are non-covalently connected via two or three hydrogen bonds...
to the leading unpaired template nucleotide. Ion semiconductor sequencing exploits these facts by determining if a hydrogen ion is released upon providing a single species of dNTP to the reaction.
Microwells on a semiconductor chip that each contain one single-stranded template DNA molecule to be sequenced and one DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
are sequentially flooded with unmodified A, C, G or T
Nucleic acid notation
The nucleic acid notation currently in use was first formalized by the International Union of Pure and Applied Chemistry in 1970. This universally accepted notation uses the Roman characters G, C, A, and T, to represent the four nucleotides commonly found in deoxyribonucleic acids...
dNTP. If an introduced dNTP is complementary to the next unpaired nucleotide on the template strand it is incorporated into the growing complementary strand by the DNA polymerase. If the introduced dNTP is not complementary there is no incorporation and no biochemical reaction. The hydrogen ion that is released in the reaction changes the pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
of the solution, which is detected by a hypersensitive ion sensor. The unattached dNTP molecules are washed out before the next cycle when a different dNTP species is introduced.
Signal Detection
Beneath the layer of microwells is an ion sensitive layer, below which is a hypersensitive ISFETISFET
ISFET pH electrode also redirects here.An ISFET is an ion-sensitive field-effect transistor used for measuring ion concentrations in solution; when the ion concentration changes, the current through the transistor will change accordingly. Here, the solution is used as the gate electrode...
ion sensor. All layers are contained within a CMOS
CMOS
Complementary metal–oxide–semiconductor is a technology for constructing integrated circuits. CMOS technology is used in microprocessors, microcontrollers, static RAM, and other digital logic circuits...
semiconductor chip, similar to that used in the electronics industry.
Each released hydrogen ion triggers the ISFET
ISFET
ISFET pH electrode also redirects here.An ISFET is an ion-sensitive field-effect transistor used for measuring ion concentrations in solution; when the ion concentration changes, the current through the transistor will change accordingly. Here, the solution is used as the gate electrode...
ion sensor. The series of electrical pulses transmitted from the chip to a computer is translated into a DNA sequence, with no intermediate signal conversion required. Each chip contains an array of microwells with corresponding ISFET detectors. Because nucleotide incorporation events are measured directly by electronics, the use of labeled nucleotides and optical measurements are avoided.
Sequencing Characteristics
The per base accuracy achieved in house by Ion Torrent on the Ion Torrent ion semiconductor sequencer as of February 2011 was 99.6% based on 50 baseBase pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...
reads, with 100 Mb per run. The read-length as of February 2011 was 100 base pairs. The accuracy for homopolymer repeats of 5 repeats in length was 98%.. It should be noted that these figures have not yet been independently verified outside of the company.
Strengths
The major benefits of ion semiconductor sequencing are rapid sequencing speed and low upfront and operating costs. This has been enabled by the avoidance of modified nucleotides and optical measurements.Because the system records natural polymerase-mediated nucleotide incorporation events, sequencing can occur in real-time. In reality, the sequencing rate is limited by the cycling of substrate nucleotides through the system. Ion Torrent Systems Inc., the developer of the technology, claims that each incorporation measurement takes 4 seconds and each run takes about one hour, during which 100-200 nucleotides are sequenced. If the semiconductor chips are improved (as predicted by Moore’s law), the number of reads per chip (and therefore per run) should increase.
The cost of acquiring a pH-mediated sequencer from Ion Torrent Systems Inc. at time of launch was priced at around $50,000 USD, excluding sample preparation equipment and a server for data analysis. The cost per run is also thought to be significantly lower than that of alternative automated sequencing methods.
Limitations
If homopolymer repeats of the same nucleotide (e.g. GGGGG) are present on the template strand (strand to be sequenced) multiple introduced nucleotides are incorporated and more hydrogen ions are released in a single cycle. This results in a greater pH change and a proportionally greater electronic signal. This is a major limitation of the system in that it is difficult to enumerate long repeats. This is shared by other techniques that detect single nucleotide additions such as pyrosequencingPyrosequencing
Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides...
. Signals generated from a high repeat number are difficult to differentiate from repeats of a similar but different number (e.g. 7 from 8 homorepeats).
Another limitation of this system is a short read length compared to other sequencing methods such as Sanger sequencing or pyrosequencing
Pyrosequencing
Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides...
. Longer read lengths are beneficial for de novo
De novo
In general usage, de novo is a Latin expression meaning "from the beginning," "afresh," "anew," "beginning again." It is used in:* De novo transcriptome assembly, the method of creating a transcriptome without a reference genome...
genome assembly. The read length achieved by Ion Torrent Systems Inc. is currently 200 base pairs per run. The throughput is currently lower than that of other high-throughput sequencing technologies, although the developers hope to change this by increasing the density of the chip.
Comparison to other sequencing methods
| Ion Torrent | 454 Sequencing | Illumina | SOLiD | |
|---|---|---|---|---|
| Sequencing Chemistry | Ion semiconductor sequencing | Pyrosequencing Pyrosequencing Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides... |
Polymerase-based sequence-by-synthesis | Ligation-based sequencing Sequencing by ligation Sequencing by ligation is a DNA sequencing method that uses the enzyme DNA ligase to identify the nucleotide present at a given position in a DNA sequence. Unlike most currently popular DNA sequencing methods, this method does not use a DNA polymerase to create a second strand... |
| Amplification approach | Emulsion PCR | Emulsion PCR | Bridge amplification | Emulsion PCR |
| Mb per run | 100 | 100 | 600,000 | 170,000 |
| Time per run | 1.5 hours | 7 hours | 9 days | 9 days |
| Read length | 200 bp | 400 bp | 2x100 bp | 35x75 bp |
| Cost per run | $ 350 USD | $ 8,438 USD | $ 20,000 USD | $ 4,000 USD |
| Cost per Mb | $ 5.00 USD | $ 84.39 USD | $ 0.03 USD | $ 0.04 USD |
| Cost per instrument | $ 50,000 USD | $ 500,000 USD | $ 600,000 USD | $ 595,000 USD |
Table 1. Comparing metrics and performance of next-generation DNA sequencers.
Application
The developers of ion semiconductor sequencing have marketed it as a rapid, compact and economical sequencer that can be utilized in a large number of laboratories as a bench top machine. The company hopes that their system will take sequencing outside of specialized centers and into the reach of hospitals and smaller laboratories. A January 2011 New York Times article, "Taking DNA Sequencing to the Masses", underlines these ambitions.Due to the ability of alternative sequencing methods
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
to achieve a greater read length (and therefore being more suited to whole genome analysis) this technology may be best suited to small scale applications such as microbial genome sequencing, microbial transcriptome
Transcriptome
The transcriptome is the set of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA produced in one or a population of cells.-Scope:...
sequencing, targeted sequencing, amplicon sequencing, or for quality testing of sequencing libraries.