“What is Ion AmpliSeq™ Chemistry & how does it work?” Let’s find out what this new technology can do for you.
Next Generation Sequencing is now producing more sequence, faster than anyone had ever envisioned just 10 years ago making it now possible to sequence a genome in days rather than months or years.
However, for many applications this large amount of data becomes overkill, creating processing and informatics headaches, and adds additional cost with little to no benefit. Rather, for many of the more standard questions being asked in the lab, a focused approach, or targeted sequencing method, that zeroes in on specific genomic regions of interest, can result in significant savings in terms of time, effort, and money. And, perhaps most importantly, can speed up getting to that answer you’re looking for. So what is targeted sequencing and how does Ion AmpliSeq™ chemistry fit into the picture? Simply put, using current genomic knowledge, a targeted sequencing approach introduces a sequence enrichment step focusing in on genes or even genetic variants of interest – A good example might be the targeting of oncogenes and tumor suppressors in a cancer research study.
Early approaches, using hybridization based techniques, enabled specific regions to be pulled out of the genome. However, these methods were limited in their specificity resulting in representation of regions of no interest. Additionally, relatively large amounts of starting genomic DNA were required for these traditional hyb methods. Leveraging over ten years in PCR assay design and preamplification methods developed by our Applied Biosystems colleagues, Ion AmpliSeq™ targeted enrichment offers a vast improvement over these early approaches delivering a unique, highly multiplexed PCR based workflow with clear specificity and uniformity benefits. Additionally, extremely low DNA input amounts can be amplified through the PCR process, as low as 10 ng.
So what is the Ion AmpliSeq™ approach, how does it work, and what makes it so remarkable?
Let’s take a look at our lab book
At its most basic level, an Ion AmpliSeq™ panel consists of a pool of oligonucleotide primer pairs, each pair designed to amplify a specified genomic region. Unique to this approach is the ability to multiplex up to 24,000 primer pairs in a single PCR reaction!
Following simple PCR amplification of the selected genomic regions, remaining primers are digested and a library containing the remaining amplicons is prepared for sequencing.
It is worth mentioning two key implications to how the design of an Ion AmpliSeq™ Panel is approached.
First, a panel can be designed to interrogate all bases across a gene or can be focused on specific mutation hotspots.
Since a gene design relies on the tiling of overlapping amplicons across the sequence of interest, the consequence is that overlapping primer pairs must be separated into independent PCR reactions. As a result, this approach will require two separate PCR multiplexed reactions per sample to achieve full coverage.
In contrast, a mutation hotspot design typically results in non-overlapping amplicons whose primers can be accommodated in a single multiplexed reaction.
Lastly, consideration of the sample source is important to final design performance. Shorter amplicons with a maximum length of 175bp work best with degraded sample sources such as FFPE but come with a trade-off of coverage within the design.
Now you may be thinking “this all very interesting but I still don’t know how to create a design”? Never fear, Ion Torrent offers a variety of predesigned panels designed with input from the experts in the field, and if none of these panels meet your needs, custom design is made super easy using our online AmpliSeq Designer tool. Now that you know a little bit about what Ion AmpliSeq™ targeted sequencing has to offer and how it works, feel free to play with our design tool – after all it is completely free to try.
I hope this video was helpful on explaining the Ion AmpliSeq™ Chemistry, and I am sure you’ll have more questions.
Submit your question at thermofisher.com/ask and subscribe to our channel to see more videos like this.
And remember, when in doubt, just Seq It Out