The chip is then ready for sequencing.Įach well of the chip can be thought as of the smallest pH meter in the world. The ISP solution is loaded onto the chip, then flowed over it by centrifuging it in different directions, in order to attempt to get as many ISPs into wells as possible.
Each well is just large enough to fit a single ISP. Each of the chips contains a top layer covered in tiny wells. There are three different types of chips for the Personal Genome Machine (PGM) – 314, 316, 318 – and five different types for their GeneStudio S5 system (510, 520, 530, 540, 550), offering enough coverage for a single sample of a hotspot panel, all the way up to enough coverage for a specimen of exome sequencing. The size of the chip is determined by the number of bases needing to be sequenced.
This mixture of control ISPs and specimen ISPs is then loaded onto the chip. The primer is annealed to each of the amplicons on every ISP. Next, control Ion Sphere Particles are added to the mix, along with sequencing primer, which is complimentary to one of the adapter sequences added back in library preparation. When we left off, the final product of the clonal amplification had been prepared – Ion Sphere Particles (ISPs) covered in single stranded amplicons (hopefully all of the same amplicon). Next time, we will review the Illumina sequencing process.
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This post we will discuss the technology and process behind the Ion Torrent sequencing step. We’ve finally made it to the sequencing step of the NGS workflow.
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