半导体测序技术

Ion Semiconductor Sequencing is a method of DNA sequencing based on the detection of hydrogen ions 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, silicone sequencing, or semiconductor sequencing. It was developed by Ion Torrent Systems Inc. and was released in February 2010.1 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.2TechnologyThe incorporation of deoxyribonucleotide into a growing DNA strand causes the release of hydrogen and pyrophosphate.The release of hydrogen ions indicate if zero, one or more nucleotides were incorporated.Released hydrogens ions are detected by an ion sensor. Multiple incorporations lead to a corresponding number of released hydrogens and intensity of signal.Sequencing ChemistryIn nature, the incorporation of a deoxyribonucleotide (dNTP) into a growing DNA strand involves the formation of a covalent bond and the release of pyrophosphate and a positively charged hydrogen ion.1 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.134 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.5 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.13 The unattached dNTP molecules are washed out before the next cycle when a different dNTP species is introduced.3Signal DetectionBeneath the layer of microwells is an ion sensitive layer, below which is a hypersensitive ISFET ion sensor.2 All layers are contained within a CMOS semiconductor chip, similar to that used in the electronics industry.26Each 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.37 Each chip contains an array of microwells with corresponding ISFET detectors.3 Because nucleotide incorporation events are measured directly by electronics, the use of labeled nucleotides and optical measurements are avoided.26Sequencing CharacteristicsThe 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 base reads, with 100 Mb per run.8 The read-length as of February 2011 was 100 base pairs.8 The accuracy for homopolymer repeats of 5 repeats in length was 98%.8. It should be noted that these figures have not yet been independently verified outside of the company.StrengthsThe major benefits of ion semiconductor sequencing are rapid sequencing speed and low upfront and operating costs.47 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.9 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.710 If the semiconductor chips are improved (as predicted by Moores law), the number of reads per chip (and therefore per run) should increase.7The cost of acquiring a pH-mediated sequencer from Ion Torrent Systems Inc. at time of launch was priced at around $50,000 USD, excluding sample preparation equipment and a server for data analysis.4710 The cost per run is also thought to be significantly lower than that of alternative automated sequencing methods.48LimitationsIf homopolymer repeats of the same nucleotide (e.g. GGGGG) are present on the template strand (strand to be sequenced) multiple introduced nucleotides are incorporated and more hydrogen ions are released in a single cycle. This results in a greater pH change and a proportionally greater electronic signal.7 This is a major limitation of the system in that it is difficult to enumerate long repeats. This is shared by other techniques that detect single nucleotide additions such as pyrosequencing.11 Signals generated from a high repeat number are difficult to differentiate from repeats of a similar but different number (e.g. 7 from 8 homorepeats).Another limitation of this system is a short read length compared to other sequencing methods such as sanger sequencing or pyrosequencing. Longer read lengths are beneficial for de novo genome assembly. The read length achieved by Ion Torrent Systems Inc. is currently 100 base pairs per run.14 The throughput is currently lower than that of other high-throughput sequencing technologies, although the developers hope to change this by increasing the density of the chip.1Comparison to other sequencing methodsIon Torrent 1012454 Sequencing 13Illumina 14SOLiD 15Sequencing ChemistryIon semiconductor sequencingPyrosequencingPolymerase-based sequence-by-synthesisLigation-based sequencingAmplification approachEmulsion PCREmulsion PCRBridge amplificationEmulsion PCRMb per run100 Mb100 Mb600 Gb3000 MbTime per run2 hours7 hours9 days5 daysRead length100 bp400 bp2x100 bp35-50 bpCost per run$ 500 USD$ 8,438 USD$ 20,000 USD$ 17,447 USDCost per Mb$ 5.00 USD$ 84.39 USD$ 0.03 USD$ 5.81 USDCost per instrument$ 50,000 USD$ 500,000 USD$ 600,000 USD$ 591,000 USDTable 1. Comparing metrics and performance of next-generation DNA sequencers. 16ApplicationThe 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.12 The company hopes that their system will take sequencing outside of specialized centers and into the reach of hospitals and smaller laboratories.17 A January 2011 New York Times article, Taking DNA Sequencing to the Masses, underlines these ambitions.17Due 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.14References1. a b c d e f g h Rusk, N. (2011). Torrents of sequence. Nat Meth 8(1): 44-44.2. a b c d e Ion Torrent Official Webpage.3. a b c d e Pennisi, E. (2010). Semiconductors inspire new sequencing technologies. Science 327(5970): 1190.4. a b c d e f Perkel, J., Making contact with sequencings fourth generation. Biotechniques, 2011.5. Alberts B, Molecular Biology of the Cell. 5th Edition ed. 2008, New York: Garland Science.6. a b Karow, J. (2009) Ion Torrent Patent App Suggests Sequencing Tech Using Chemical-Sensitive Field-Effect Transistors. In Sequence.7. a b c d e f Bio-IT World, Davi