Ion torrent sequencing

Ion torrent sequencing

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  Ion Torrent Semi-conductor sequencing AshiniSilva, Dilmi Croos, Hafsa Thaha, Vindyani Peiris, Kanishka Pathmakumara and Sureshni Fernando
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  INTRODUCTION First generation sequencing –Sanger sequencing: based on the chain termination method developed by Sanger and Coulson in 1975. – Maxam Gilbert sequencing Next generation sequencing – novel techniques of DNA sequencing directly from DNA fragments without the need of cloning vectors. – massively parallel sequencing / 2nd generation sequencing / deep sequencing.
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  ION TORRENT • Ionsemiconductor sequencing / Ion torrent sequencing / pH mediated sequencing / semiconductor sequencing. • No modified nucleotides or optics are used. • Detects the pH change caused due to the release of H+ during the addition of dNTP to the DNA. • Ion Torrent has marketed their machine as a rapid compact economical sequencer that can be utilized in a large number of laboratories as a bench top machine.
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  PRINCIPLE • When acomplementary nucleotide is incorporated to a strand of DNA, a phosphodiester bond is formed releasing a H+ ion which changes the pH of the medium. • The ion sensor converts the chemical information into digital electronic information. Electric signal
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  PROCEDURE 1. Fragmentation ofunknown DNA strand 2. Ligation of adaptor and unknown DNA strand 3. Denaturation 4. Amplification through emulsion PCR 5. Placement of beads into the wells of the chip 6. Flooding of the chip with dNTPs 7. pH detection
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  Fragmentation Ligation Denaturation Amplification dNTP Unknown DNAsequence Adaptor
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  pH DETECTION • Complementarymetal oxide semiconductor (CMOS) chip contains more than 1 million wells. • The base of the chip contains Ion-sensitive field- effect transistor (ISFET) that can detect the pH changes G G T A C
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  APPLICATIONS • Genomic sequencing •Drug resistant testing • Targeted sequencing in cancer studies • Forensic and criminal studies • Microbial characterization
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  ADVANTAGES • Faster sequencing •Smaller instrument size • Significantly lower operating cost
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  • ThermoFisher hasan Ion PGM machine which contains Ion AmpliSeq panels, • Custom design fits the demands from the clinical research market • Very easy to use and it is very important for technicians who do not have a lot of NGS experience.
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  INSTRUMENT ADVANTAGES DISADVANTAGES IonTorrent – PGM -Low cost instrument -Disposable -Very simple machine with few moving parts -Three chips available with varying numbers of reads -More hands-on time and fewer reads at higher cost per Mb relative to MiSeq; -Smaller user community -Libraries made for 400 base reads don’t work well on Proton Ion Torrent – Proton Moderate cost -More hands on time -Fewer overall bases of data than Illumina -Higher cost per MB of data than most Illumina instruments -Smaller user community Ion Torrent – S5/S5 xl -Cost-competitive -Brightest future among Ion Torrent instruments -User community not yet established for molecular ecology -Fewer data analysis tools & pipelines
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  Challenges High cost compared to Illumina sequencing Runfor each nucleotide at a time High complexity of workflow and result Low number of total reads Informatic challenges for analysis and reporting