{"id":9275,"date":"2015-08-26T20:13:51","date_gmt":"2015-08-26T20:13:51","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/behindthebench\/?p=9275"},"modified":"2018-10-18T23:33:29","modified_gmt":"2018-10-18T23:33:29","slug":"what-is-ion-ampliseq-chemistry-how-does-it-work-seq-it-out-6","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/what-is-ion-ampliseq-chemistry-how-does-it-work-seq-it-out-6\/","title":{"rendered":"What is Ion AmpliSeq\u2122 Chemistry &amp; How Does it Work? &#8211; Seq It Out #6"},"content":{"rendered":"<p><span lang=\"EN-GB\">\u201cWhat is <a href=\"http:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/sequencing\/next-generation-sequencing\/ion-torrent-next-generation-sequencing-workflow\/ion-torrent-next-generation-sequencing-select-targets\/ampliseq-target-selection.html?cid=social_btb_targetseq\" target=\"_blank\" rel=\"nofollow\">Ion AmpliSeq\u2122 Chemistry<\/a> &amp; how does it work?\u201d Let\u2019s find out what this new technology can do for you.<\/span><\/p>\n<p><span lang=\"EN-GB\"><a href=\"http:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/sequencing\/next-generation-sequencing.html?cid=social_btb_targetseq\" target=\"_blank\" rel=\"nofollow\">Next Generation Sequencing<\/a> 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.<\/span><\/p>\n<p><iframe loading=\"lazy\" width=\"760\" height=\"428\" src=\"https:\/\/www.youtube.com\/embed\/D3Dv7QHpLcs?feature=oembed\" frameborder=\"0\" allow=\"accelerometer; autoplay; encrypted-media; gyroscope; picture-in-picture\" allowfullscreen><\/iframe><\/p>\n<p><span lang=\"EN-GB\">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.\u00a0 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.\u00a0 And, perhaps most importantly, can speed up getting to that answer you\u2019re looking for. So what is targeted sequencing and how does Ion AmpliSeq\u2122 chemistry fit into the picture?\u00a0 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 &#8211;\u00a0 A good example might be the targeting of oncogenes and tumor suppressors in a cancer research study.<\/span><\/p>\n<p><span lang=\"EN-GB\">Early approaches, using hybridization based techniques, enabled specific regions to be pulled out of the genome.\u00a0 However, these methods were limited in their specificity resulting in representation of regions of no interest.\u00a0 Additionally, relatively large amounts of starting genomic DNA were required for these traditional hyb methods.\u00a0 Leveraging over ten years in <a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/pcr\/real-time-pcr\/real-time-pcr-assays.html\/?cid=social_btb_targetseq\" target=\"_blank\" rel=\"nofollow\">PCR assay design<\/a> and preamplification methods developed by our <a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/brands\/applied-biosystems.html?cid=social_btb_abseq\" target=\"_blank\" rel=\"nofollow\">Applied Biosystems<\/a> colleagues, Ion AmpliSeq\u2122 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.<\/span><\/p>\n<p><span lang=\"EN-GB\">So what is the Ion AmpliSeq\u2122 approach, how does it work, and what makes it so remarkable?<\/span><\/p>\n<p><strong><span lang=\"EN-GB\">Let\u2019s take a look at our lab book<\/span><\/strong><\/p>\n<p><span lang=\"EN-GB\">At its most basic level, an Ion AmpliSeq\u2122 panel consists of a pool of oligonucleotide primer pairs, each pair designed to amplify a specified genomic region.\u00a0 Unique to this approach is the ability to multiplex up to 24,000 primer pairs in a single PCR reaction!<\/span><\/p>\n<p><span lang=\"EN-GB\">Following simple PCR amplification of the selected genomic regions, remaining primers are digested and a library containing the remaining amplicons is prepared for sequencing.<\/span><\/p>\n<p><span lang=\"EN-GB\">It is worth mentioning two key implications to how the design of an Ion AmpliSeq\u2122 Panel is approached.<\/span><\/p>\n<p><span lang=\"EN-GB\">First, a panel can be designed to interrogate all bases across a gene or can be focused on specific mutation hotspots.<\/span><\/p>\n<p><span lang=\"EN-GB\">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.\u00a0 As a result, this approach will require two separate PCR multiplexed reactions per sample to achieve full coverage.<\/span><\/p>\n<p><span lang=\"EN-GB\">In contrast, a <a href=\"http:\/\/www.thermofisher.com\/order\/catalog\/product\/4475346?cid=social_btb_targetseq\" target=\"_blank\" rel=\"nofollow\">mutation hotspot design<\/a> typically results in non-overlapping amplicons whose primers can be accommodated in a single multiplexed reaction.<\/span><\/p>\n<p><span lang=\"EN-GB\">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.<\/span><\/p>\n<p><span lang=\"EN-GB\">Now you may be thinking \u201cthis all very interesting but I still don\u2019t know how to create a design\u201d? Never fear, <a href=\"http:\/\/www.thermofisher.com\/us\/en\/home\/brands\/ion-torrent.html?cid=social_btb_targetseq\" target=\"_blank\" rel=\"nofollow\">Ion Torrent<\/a> 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\u2122 targeted sequencing has to offer and how it works, feel free to play with our design tool \u2013 after all it is completely free to try.<\/span><\/p>\n<p><span lang=\"EN-GB\">I hope this video was helpful on explaining the Ion AmpliSeq\u2122 Chemistry, and <\/span>I am sure you\u2019ll have more questions.<\/p>\n<p>Submit your question at <a href=\"http:\/\/www.thermofisher.com\/us\/en\/home\/products-and-services\/promotions\/ask.html?cid=social_btb\" target=\"_blank\" rel=\"nofollow\"><span lang=\"EN-US\">thermofisher.com\/ask<\/span><\/a> and subscribe to our channel to see more videos like this.<\/p>\n<p>And remember, when in doubt, just <a href=\"https:\/\/www.youtube.com\/playlist?list=PLGlvFEwL2wDFVLBA1egJFwHyetpVDPExK\" target=\"_blank\" rel=\"nofollow\">Seq It Out<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"<p>\u201cWhat is Ion AmpliSeq\u2122 Chemistry &amp; how does it work?\u201d Let\u2019s 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,<\/p>\n","protected":false},"author":114,"featured_media":0,"comment_status":"open","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_monsterinsights_skip_tracking":false,"_genesis_hide_title":false,"_genesis_hide_breadcrumbs":false,"_genesis_hide_singular_image":false,"_genesis_hide_footer_widgets":false,"_genesis_custom_body_class":"","_genesis_custom_post_class":"","_genesis_layout":"","_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[108,101],"tags":[11,6,176,74,156],"division":[],"class_list":{"0":"post-9275","1":"post","2":"type-post","3":"status-publish","4":"format-standard","6":"category-lab-tips-and-tricks","7":"category-product-news","8":"tag-ion-torrent","9":"tag-next-generation-sequencing","10":"tag-seqitout","11":"tag-sequencing","12":"tag-targeted-dna-sequencing","13":"entry"},"_selected_authors":"","_selected_reviewers":"","acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO Premium plugin v27.8 (Yoast SEO v27.8) - https:\/\/yoast.com\/product\/yoast-seo-premium-wordpress\/ -->\n<title>What is Ion AmpliSeq\u2122 Chemistry &amp; How Does it Work? - Seq It Out #6 - Behind the Bench<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.thermofisher.com\/blog\/behindthebench\/what-is-ion-ampliseq-chemistry-how-does-it-work-seq-it-out-6\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"What is Ion AmpliSeq\u2122 Chemistry &amp; How Does it Work? - Seq It Out #6\" \/>\n<meta property=\"og:description\" content=\"\u201cWhat is Ion AmpliSeq\u2122 Chemistry &amp; how does it work?\u201d Let\u2019s find out what this new technology can do for you. 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