Ion Torrent Sequencing
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The document summarizes Ion Torrent next generation sequencing. It detects chemiluminescence given off when a proton is released during sequencing. DNA fragments are attached to beads and undergo emulsion PCR amplification. Sequencing is based on standard pyrosequencing and measures light from chemiluminescent reagents released during the reaction. The output is in FASTQ format.
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Ion Torrent Sequencing
- 1. Next Generation Sequencing Ion Torrent Method
- 2. Overview • Similar idea as SMRT • Detects the chemiluminescence given off when a proton (H+) is released • Use ion chip
- 3. Library Construction • If RNA, converted into cDNA • DNA fragmentation • Adapter ligated to DNA • Denaturation Image provided by http://www.thermoscientificbio.com/ngs-library-preparation-kits/
- 4. Template Prep/ePCR Amplification • Fragments are attached to beads • ePCR
- 5. Sequencing • Based on standard pyrosequencing • Measures light from chemiliuminescent reagents by measuring direct release of H+ (protons) from the sequencing reaction
- 6. Data Analysis • Output FASTQ Image provided by http://mendel.iontorrent.com/ion-docs/On-Dataflow.html
- 7. Work Cited • Ambry Genetics. Making Sense of NextGent Sequencing. Kelly Gonzalez, MS, CGC, and Senior Manager of Clinical Genomics. Available at http://www.ambrygen.com/sites/default/files/pdfs/NERRG_4-10- 12_Making_Sense_of_NetGen_Sequencing_KG(3).pdf. Access verified May 21, 2014. • Omixon. Allen Van Deynze, 2010; Solanaceae Coordinated Agricultural Project. Next Generation Sequencing. Available at http://www.omixon.com/the-basics- of-next-generation-sequencing/. Access verified May 21, 2014. • AllSeq. Life Technologies – Ion Torrent. http://allseq.com/knowledgebank/sequencing-platforms/life-technologies-ion- torrent Access verified Aug. 1, 2014.
- 8. Recommended Video See what you've been missing: Explore RNA-Seq for Gene Expression research https://www.youtube.com/user/ionto rrent
Editor's Notes
- This video gives an overview on Ion Torrent Sequencing, another type of Next Generational Sequencing.
- The Ion Torrent method uses the same idea as the Small Molecule Real Time sequencing where they sequence at enzymatic rate using the DNA polymerase. Instead of having a fluorescent they recognized that when a base is added to a DNA sequence a hydrogen ion is released. It is that ion that the computer is able to detect and differentiate which base had been added to give off the ion. The whole basis is this computer chip where the sequencing is going on. While this technology uses a sequencing-by-synthesis method and emulsion PCR (emPCR) similar to other platforms, it differs in that it doesn’t use fluorescence or chemiluminescence. Instead, it measures the H+ ions released during base incorporation. The lack of any optics has allowed Ion Torrent to rapidly expand the output from their systems by approximately 10-fold every six months. This rapid pace of improvement, along with fast runs of about 2 hours and inexpensive consumables has made Ion Torrent a popular new platform.
- The first step in the workflow is library construction. While there are a number of options available, the process is fairly standard and generally involves taking DNA (or RNA converted to DNA), fragmenting it to a uniform size (generally 200-400b) and then adding sequencing adapters. Ion Torrent has specific kits for a variety of applications, including DNA fragmentation for small genomes, total RNA-Seq and cancer-specific targeted sequencing.
- The fragments generated during the library prep are attached to beads and amplified using emulsion PCR (ePCR). Beads coated with complementary primers are mixed with a dilute aqueous solution containing the fragments to be sequenced along with the necessary PCR reagents. This solution is then mixed with oil to form an emulsion of microdroplets. The concentration of beads and fragments is kept low enough such that each microdroplet contains only one of each (or possibly none, but almost never more than one). Clonal amplification of each fragment is then performed within the microdroplets. Following amplification the emulsion is ‘broken’ (generally by organic extraction and centrifugation) and the amplified beads are enriched in a glycerol gradient (with unamplified beads pelleting at the bottom).
- What really differentiates Ion Torrent’s systems is the sequencing technology. It is based on the standard pyrosequencing chemistry, a form of ‘sequencing by synthesis’ whereby individual bases are introduced one at a time and incorporated by DNA polymerase. However, unlike other platforms based on pyrosequencing, rather than measuring light released from chemiluminescent reagents, the Ion Torrent system measures the direct release of H+ (protons) from the reaction. Because optics aren’t required, they’re able to make relatively inexpensive instruments coupled with disposable chips, which essentially act as pH meters. The lack of optics also means they don’t have to contend with slow image scans, so the sequencing reactions are relatively fast, with 200b reads taking about 2 hours. Finally, the lack of fluorescence or chemiluminescence means that the system can use unmodified nucleotides, which are cheaper and better tolerated by DNA polymerase. While the error rates are generally pretty good (~1%), pyrosequencing chemistry has trouble with long homopolymers (stretches of the same base, e.g., AAAAAA). Because the chemistry doesn’t pause after each base incorporation, stretches of the same base will result in a single, stronger, signal. While short stretches can be differentiated, it becomes increasingly difficult with longer stretches.
- As the Ion Torrent systems generate standard output files like FASTQ, data analysis is generally straightforward. In addition to a variety of available third party analysis solutions, Ion Torrent offers the ‘Torrent Browser’ software, which acts as the primary interface for a number of basic functions. Later this year, they will be launching a cloud-based solution called ‘Ion Reporter’ that will serve as a front-end for a variety of open source analysis solutions.
- This is the Work Cited for this video.
- This is a recommended video in order to further understand how Ion Torrent sequencing works. See what you've been missing: Explore RNA-Seq for Gene Expression research https://www.youtube.com/user/iontorrent This is the last Next Generational Sequencing video, thank you for watching.