What is Ion AmpliSeq Chemistry? - Seq It Out #6

what is ion ampliseq chemistry and how does it work let's find out what this newer 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 sing with sin genome in days rather than months or even years however for many applications this large amount of data becomes overkill creating processing and informatics headaches and adds additional costs 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 a specific genomic region of interest can result in significant savings in terms of time effort and money and perhaps most importantly it can speed up getting to that answer you're looking for so what is targeted sequencing and how does ion ampliseq chemistry fit into that 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 enable specific regions to be pulled out of the genome however these methods were limited on their specificity resulting in representation of regions of the no interest additionally relatively large amounts of starting to nomic DNA were required for these traditional hype methods leveraging over 10 years in PCR assay design and pre amplification methods developed by our applied Biosystems colleagues ion ampliseq targeted enrichment offers a vast improvement over these early approaches delivering a unique highly multiplex PCR based workflow with clear specificity in uniformity benefit additionally extremely low DNA input amounts can be amplified through the PCR process as low as 10 nanograms so what is the ion ampliseq approach and how does it work and what makes it so remarkable well let's take a look at our lab book at its most basic level an ion ampliseq panel consists of a pool of oolagah nucleotide primer pairs each pair designed to amplify a specific genomic regions 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 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 the 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 multiplex reactions per sample to achieve full coverage in contrast of mutation hotspot is dying 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 175 base pairs works best with degraded sample sources such as FFPE but come with a trade-off of coverage within a design now you may be thinking alright this is all very interesting but I still don't know how to create a design we'll never fear because I on tauron offers a variety of pre-designed panels designed with input from the expert in the field and if none of these panels meet your needs custom design is made super-easy using our online ample seek designer tool now that you know a little bit about what AI enables say 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'm sure you have more questions so submit your questions at thermo Fisher com forward slash ask and subscribe to our channel to see more videos like this and remember when in doubt just seek it out

Channel: Thermo Fisher Scientific

Views: 26,411

Rating: 4.7468352 out of 5

Keywords: genomics, NGS, Ion AmpliSeq, Thermo Fisher Scientific, Ion Torrent, Next gen Sequencing, Next Generation Sequencing, Targeted Sequencing, Generation, Semiconductor sequencing, primer design, Life technologies, Thermo Fisher, Seq It Out, science tutorial, primer designer, AmplISeq Panel, AmplISeq Designer, science education, Next, Sequencing, genetics, primer designer tool

Id: D3Dv7QHpLcs

Length: 4min 48sec (288 seconds)

Published: Wed Aug 26 2015