Ion torrent

Next Generation Sequencing Platform

1. Ion Torrent Sequencing Aishwarya Babu

2. Introduction Continuation of the original pyrosequencing technique. The current Ion Torrent system detects H +  ions, another product released during DNA polymerization. The basic principle, the detection of H +  ions, has been licensed from DNA Electronics in London.

3. Technology combines semiconductor sequencing technology with biochemistry, enabling the direct translation of chemical information into digital sequence data. Eliminates the need for expensive optics, lasers, and complex sequencing chemistries with fluorescently labelled nucleotides. More affordable and cost-effective, faster, scalable, and relatively simple to operate.

4. Principle Detection of hydrogen ion release during incorporation of new nucleotides into the growing DNA template. In nature, when a nucleotide is incorporated into a strand of DNA by a polymerase, a hydrogen ion is released as a by-product. 4

5. Ion Torrent, with its Ion Personal Genome Machine (PGM™) sequencer Uses a high-density array of micro-machined wells to perform nucleotide incorporation in a massively parallel manner. Each well holds a different DNA template. Beneath the wells is an ion-sensitive layer followed by a proprietary ion-sensor. The ion changes the pH of the solution, which is detected by an ion sensor.

6. 6 If there are two identical bases on the DNA strand, the output voltage is doubled, and the chip records two identical bases. Instead of detecting light as in 454 pyrosequencing, Ion Torrent technology creates a direct connection between the chemical and digital events.

7. 7 Hydrogen ions are detected on ion-semiconductor sequencing chips. These ion semiconductor chips are designed and manufactured like any other semiconductor chips used in electronic devices. These are cut in the form of wafers from a silicon boule. The transistors and circuits are then pattern-transferred and subsequently etched onto the wafers using photolithography. This process is repeated 20 times or more creating a multi-layer system of circuits.

8. Instrumentation Ion Torrent’s first instrument, the Ion Personal Genome Machine (PGM) - least expensive next generation sequencer on the market. Run up to 5.5 million reads, with an output reaching 2 Gb, a reading length up to 400 bases, and a run time between 2 and 7 h. Targeted towards smaller genomes and targeted sequencing. It uses disposable chips which come in three varieties of increasing output.

9. Ion Proton : Allows for larger chips with higher densities needed for exome and whole genome sequencing. Substantially more expensive. Capable of generating much larger outputs. The first chip, the PI, is able to generate ~10Gb per run. The number of reads is up to 80 million, the read length is 200 bases, and the run time is 2–4 h.

10. Technology Description The workflow consists of four major steps: library construction, template prep, sequencing and analysis.

11. 1. Library Construction The first step in the workflow is library construction. Involves taking DNA (or RNA converted to DNA). Fragmenting it to a uniform size (generally 200-400b). Then adding sequencing adapters.

12. 2. Template Prep/Amplification The fragments generated during the library prep are attached to beads and amplified using emulsion PCR ( emPCR ). 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.

13. Clonal amplification of each fragment is then performed within the microdroplets. Following amplification the emulsion is ‘broken’ - by organic extraction and centrifugation. The amplified beads are enriched in a glycerol gradient with unamplified beads pelleting at the bottom. While the emulsion PCR process is effective, it is slow and complicated.

14. 3. Sequencing Based on the standard pyrosequencing chemistry, a form of ‘sequencing by synthesis’. Individual bases are introduced one at a time and incorporated by DNA polymerase. Ion Torrent system measures the direct release of H+ from the reaction. Relatively inexpensive instruments coupled with disposable chips, which essentially act as pH meters. Sequencing reactions are relatively fast, with 200b reads taking about 2 hours. System can use unmodified nucleotides, which are cheaper and better tolerated by DNA polymerase.

15. Each bead is placed into a single well of a slide. Like 454, the slide is flooded with a single species of dNTP, along with buffers and polymerase, one NTP at a time. The pH is detected in each of the wells, as each H +  ion released will decrease the pH. The changes in pH allow us to determine if that base, and how many thereof, was added to the sequence read. The dNTPs are washed away, and the process is repeated cycling through the different dNTP species.

16. The pH change, if any, is used to determine how many bases (if any) were added with each cycle.

17. 4. Data analysis Generate standard output files like FASTQ, data analysis is generally straightforward. Ion Torrent offers the ‘Torrent Browser’ software, which acts as the primary interface for a number of basic functions.

18. Technique Features 1. Simple: Simple sequencing chemistry based on semi-conductor technology, without optical detection. Low sample requirement. 2. Fast: Sequencing within 2-3 hours, rapid turnaround time (~2 days) from sample to DNA sequences. 3. Flexible: Flexible scaled chips on PGM and Proton systems for different throughput needs. 4. High Accuracy: 99.97%.

19. Applications 1. Small genome sequencing ( eg : Microbial de-novo & resequencing; Mitochondrial sequencing) 2. Amplicon sequencing ( eg : 16S meta genome sequencing) 3. Targeted resequencing 4. Transcriptome and whole exon sequencing (Ion Proton)

20. Limitations : The read length of 200 bp lies in between short and long read length NGS technologies. Whereas short read technologies are facilitated by huge data generation, Ion lags behind in total data output. In this way, Ion Torrent has to prove itself as a standalone sequencing technique for  de novo sequencing projects of big genomes. 21

21. Improvements on the previous technology The four main advantages of NGS over classical Sanger sequencing are:

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