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Figure 1: A – First DNA is fragmented and capped with synthetic oligonucleotide adaptors; B – An individual fragment is captured by a tiny bead densely coated with a complimentary synthetic primer; C – PCR amplification results in a bead coated with clonal copies of the fragment. This is performed in a nebulised oil/water droplet to keep each bead isolated; D – Once the beads are deposited on the chip surface, the sequencing cocktail is passed across the surface. The Illumina approach uses 4 fluorescent dye tagged nucleotides. An image is taken and deconvoluted to give the growing nucleotide sequence for each spot. The fluorescent dyes must be removed after each cycle. taken of the array at the end of each cycle to record the colour of each spot before the dye is cleaved off, freeing up the 3’ hydroxyl of the growing sequence ready for the next cycle. Because all four bases are added, every bead sequence is extended although an additional cleavage cycle is required. If the cleavage is not efficient, the signal from following cycles progressively diminishes. A third process developed by the company Applied Biosystems uses ligation based sequencing. The cocktail that passes the chip surface contains DNA ligase and a large pool of 8bp fluorescent primers that anneal and ligate to the DNA template with the first 2 bases at the 5’ end determining specificity. The colour of each bead reveals first 2 bases. The last 3 bases of the primer containing the fluorescent dye are then cleaved off to allow the next cycle to proceed. The sequence is therefore built up 2 bases at a time with a 3bp gap in between. Once the first cycle set is complete, the entire strand is removed by melting and the process repeated but using a starting primer that is one base shorter than the previous. This means the sequence starts at n-1. This primer reset is repeated 4 times so that every base is interrogated twice leading to much greater accuracy. The ABI SOLID currently boasts the highest capacity at 30 Giga bases/run and the highest base calling accuracy at 99.99% (1 base in every 10,000 is possibly wrong).
than one base is added – such as in homopolymer stretches – the voltage is proportional up to 6X that of a single base addition. The voltage change is converted immediately to a remarkably simple single digital value. This requires no more than a laptop to record and store. The Ion Torrent also eliminates the need for expensive reagents such as fluorescently labelled terminating nucleotides. Instead, as Rothberg proudly announced: Ion Torrent decided not to fight one billion years of evolution and went with unmodified bases and DNA polymerase! These are dirt cheap so the reduction on cost is just enormous. While currently the throughput of the Ion Torrent (0.5Gb) does not match that of an Illumina HiSeq (10Gb) or the ABI SOLID (30Gb), time will inevitably see the density of the semiconductor wells on the Ion Torrent chip increase (perhaps to the level of today’s microprocessors) and the read lengths extend to the point where this technology rivals or surpasses the current NextGen sequencers – at a fraction of the cost!
The next step in NextGen sequencing In a recent development, the company Ion Torrent headed again by Jonathan Rothberg of 454 Life Sciences fame, has developed a microfabricated semiconductor chip (currently 1.4 million wells are on a single chip) that detect a pH change from the release of a proton during nucleotide addition. The Ion Torrent does away with expensive light, optics’ and lasers’ systems and so is a much cheaper and faster method. Notably it does not require the high resolution images that generate multi-megabyte files after each cycle. Rather, it simply samples each of the 1.4 million wells 50 times a second for any change in voltage that signals that a base has been added and a proton released – a reaction that is completed in 5 seconds. When more 20
New Zealand Association of Science Educators
Figure 2: A typical image of an Illumina chip surface.
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