in this video we're going to cover a type of next generation sequencing called ion torrent in the process of DNA synthesis incorporating a new nucleotide involves forming a new covalent bond here pausing the release of these two phosphate molecules called a pyrophosphate and a positively charged hydrogen ion ion torrent sequencing exploits this by sequencing DNA through the detection of these hydrogen ions which are released during DNA polymerization in ion torrent sequencing lots of hydrogen ions in O L will cause the pH to change and will change the conductivity detected by a semiconductor chip which we loaded our DNA onto we can then process these signals to determine the DNA sequence and it does this in four steps library preparation emulsion PCR loading onto ion chip and signal processing so first we need to prepare the DNA for sequencing we need to fragment it which we can do by sonication or a nebulization then we add specific adapters to both ends of the DNA both the five prime and the three prime ends then we need to amplify the DNA through emulsion PCR which is a variation of PCR that some next-generation technologies used to replicate DNA sequences to perform emulsion PCR we incubate the DNA with a microscopic bead which is bound all around with complimentary ala goes to our adapters that we stuck on to the ends of the template DNA earlier this allows each single-stranded DNA to stick to the DNA capture bead and we dilute the mixture to ensure that each bead only has one template strand attached then we add oil after all this is an emulsion which means that we have two liquids that aren't miscible and this creates microvesicles so small that they can only hold one beat per vesicle like you see here then inside these drops is not only one bead bound to one template DNA but the components you need in a typical PCR reaction like polymerase dntps primer and buffer so if you think about it these micro vesicles act like a micro reactor for PCR to occur and amplify the DNA strands so that we have thousands of copies all of these micro vesicles floating around the mixed are simultaneously amplifying DNA so we end up with millions and millions of copies PCR makes a complimentary strand of the DNA so in each micro vesicle we have a bead and one fragment anneals to its complimentary adapter site on the bead the polymerase which was added to the PCR mix amplifies the strand from the bead so that it creates another copy then the original strand teenagers but the strand which was just created is connected to the bead by the sugar phosphate backbone so it's forced to stick around the Strand which dissociates will float away and anneal onto another ala go on the same bead and the cycle repeats itself 30 to 60 more times until we end up with several thousands of the same DNA sequence conjugated to the same bead and this happens on every bead PCR is important because if we only had one strand of DNA releasing one hydrogen ion at a time when a nucleotide is incorporated our chip won't be able to detect it it just isn't sensitive enough it will however be able to detect thousands of hydrogen ions being released at the same time from all of these copies then we break the emulsion and load the beads onto a chip each ion chip contains millions of micro wells which will each hold a single bead later will flood the micro Wells with nucleotides one type at a time and this is what a single well looks like this chip has a semiconductor membrane that will detect when a nucleotide is incorporated because it will detect the hydrogen ion beneath the micro L is an eye on sensitive layer and below it is an is Fe T sensor plate is FD T stands for ion sensitive field effect transistor which is a transistor used for measuring ion concentrations in solution so in our case when the hydrogen ion concentration changes in the well it will result in a change in the current through the transistor again a single micro L contains the template DNA strand we want to sequence along with polymer E's and primers we flip the micro Wells with one of four nucleotides at a time appear is a zoomed in diagram of a polymerase elongating a single DNA template and it's currently stalled till we add the right nucleotide that complements the template we're trying to figure out if the nucleotide we added does not complement the next based on the template there will be no release of hydrogen and therefore no polymerization reaction for example if we flip the well with guanine no voltage is detected which means that the base wasn't incorporated so we'll need to wash out the unattached nucleotides before the next cycle of nucleotides are added to avoid confusing which base is actually being added in this example adding adenine and cytosine also didn't change the voltage so we have to wash it away if the nucleotide does complement the next base on the template it'll be incorporated to the growing complementary strand and a hydrogen ion will be released this will change the pH of the solution which triggers the is Fe T ion sensor to send a series of electrical pulses to the computer that will be used later on to translate into a DNA sequence but if the nucleotide complements several bases in a row more hydrogen will be released and thus a higher voltage will be recorded in the case of two of the same base in a row the voltage will be double the normal for example if there are two guanine bases in a row two cytosines will be incorporated which means twice the amount of hydrogen's will be released and the higher voltage the signal processing and assembling the DNA sequence is carried out by a software because we're using electronics to determine whether a reaction occurs we don't actually need to use any specific kind of nucleotide like the fluorescently labelled nucleotides used in smart sequencing or any fancy optics making Ion Torrent sequencing fast efficient and adaptable compared to other sequencers