![]() After replication, the strands are denatured creating two single strands. The fragment then folds over and anneals to the second universal primer. The combined libraries are flowed over the slide at the beginning of the run and they anneal to the universal primer. This universal primers are, again, complimentary to the adapters added during the library preparation. A flow cell is essentially a glass slide that has universal primer anchored all over it. The multiple copies are created in close proximity to each other, just as with clonal amplification, but instead of using a separate ISP for each specimen, a separate location on the flow cell is used. Illumina’s method of template preparation is termed cluster generation by bridge amplification and actually takes place on the MiSeq a step before the sequencing step. Illumina MiSeq “Cluster Generation by Bridge Amplification” Optimizing the concentration takes a few runs and the concentration can be different for each instrument in the lab. This ISP is called “polyclonal” and the data from it will get thrown out. In the end, more than one fragment gets amplified on the ISP. Conversely, if the concentration is too high, there is a possibility of more than one sample amplicon ending up in the droplet of oil. If the concentration is too low, obviously not enough amplicons will be amplified on the ISPs, and the end result will be not enough data. One thing to consider is the concentration of the combined libraries that are added at the beginning of the template preparation. The strands that are not anchored to the ISP by the universal primer will be lost, leaving each ISP single stranded and ready for priming in the sequencing step. These amplicons will replicate all over the ISP, and as a final step, NaOH will be added to separate the strands. This, along with millions of other ISPs in droplets of oil, will undergo cycles of PCR, with the primers on the ISP priming the specimen’s amplicon. Through a series of steps, ideally, what is produced is a droplet of oil containing one ISP, one sample’s amplicon, and the components of the master mix. The primers on the ISP are complementary to one of the adapters added during library preparation so that only the universal primer is necessary on the ISPs, instead of each individual gene-specific primer. In order for this ISP to create enough of a signal to be detected by the instrument, it must have many copies of the fragment all over the surface of the ISP.Īt the beginning of the clonal amplification step, a specific concentration of combined libraries is added to the instrument, along with all the components of a standard PCR (buffer, dNTPs, polymerase) with the addition of the Ion Sphere Particles, which provide the primer, and oil. Eventually this ISP will be deposited in a well on a chip and be sequenced. This looks like a bead with primers all over the surface of it. In the Ion Torrent method of template preparation, the multiple copies are created on an Ion Sphere Particle or ISP. Ion Torrent “Template Preparation by Emulsion PCR” or “Clonal Amplification” Again, this technique is platform specific, so each has a different way to achieve this goal. This occurs for each amplicon in the specimen’s library. ![]() The main goal of this step is to create multiple copies of the same amplicon in close proximity so that when it is sequenced, it creates a strong enough signal to be detected. The next step in NGS preparation is template preparation. These are what compose a specimen’s “library.” The final product of this work is a collection of amplicons that have been amplified, barcoded, tagged with the appropriate platform adapters and purified. Specifically, we reviewed how Ion Torrent and MiSeq libraries may be prepared for DNA amplicon sequencing. Over the last two blogs we have seen why NGS is being used in a Molecular Diagnostics Lab and how library preparation is executed. Hello again – let’s continue our discussion of Next Generation, or Massively Parallel, Sequencing and how it is performed. ![]()
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