{"id":6249,"date":"2015-10-22T20:18:54","date_gmt":"2015-10-23T00:18:54","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/?p=6249"},"modified":"2020-05-28T19:06:42","modified_gmt":"2020-05-28T19:06:42","slug":"next-generation-sequencing-library-preparation-seq-it-out-10","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/next-generation-sequencing-library-preparation-seq-it-out-10\/","title":{"rendered":"Next Generation Sequencing Library Preparation &#8211; Seq It Out #10"},"content":{"rendered":"<p>What do you call a collection of millions of DNA fragments sharing the same short sequences on the 5\u2019 and 3\u2019 ends? The answer is a next generation sequencing, or NGS, library.\u00a0 Today, we are going to focus on the four basic steps of NGS library preparation that can be broadly applied across different preparation methods.<\/p>\n<p><iframe loading=\"lazy\" width=\"760\" height=\"428\" src=\"https:\/\/www.youtube.com\/embed\/_yC0Bzw3WbQ?feature=oembed\" frameborder=\"0\" allow=\"accelerometer; autoplay; encrypted-media; gyroscope; picture-in-picture\" allowfullscreen><\/iframe><\/p>\n<p>A key step in the NGS workflow is preparing the input for sequencing, known as creating a library.\u00a0 An NGS library is a collection of similarly sized DNA fragments with known adapter sequences added to the 5\u2019 and 3\u2019 ends.\u00a0 A library corresponds to a single sample and multiple libraries, each with their own unique adapter sequences, can be pooled and sequenced in the same sequencing run.<\/p>\n<p>NGS library preparation has four general steps: 1. DNA Fragmentation or Target Selection, 2. Addition of adapter sequences, 3. Size selection, and 4. Final library quantification and QC.<\/p>\n<p>The first step, DNA fragmentation or <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.html?socid=social_btb\">Target Selection<\/a>.\u00a0 In order to get the starting DNA into smaller pieces, isolated DNA may be fragmented using physical or enzymatic methods.\u00a0 These libraries are referred to as fragment libraries.\u00a0 Alternatively, if the sequence of specific DNA targets is known, PCR amplification of those targets may be used to produce DNA amplicons within the desired size range. These libraries are referred to as amplicon libraries.<\/p>\n<p>Next, specific DNA adapter sequences are annealed to the 5\u2019 and 3\u2019 ends of the fragmented or amplicon DNA.\u00a0 The double-stranded DNA adapters are approximately 20 to 40bp fragments that contain known sequences.\u00a0 Generally, there are two different adapter sequences that can anneal to the DNA fragments in either the 5\u2019 or 3\u2019 orientation.\u00a0 One adapter sequence contains the primer annealing site for the sequencing primer, while the second adapter sequence is generally used to anchor the DNA fragment to a surface for sequencing; for example beads or a solid surface containing a complimentary DNA sequence.<\/p>\n<p>Now we have our DNA fragments with known adapter sequences on either end.\u00a0 The next step is to select the library fragment sizes we need for our sequencing run.\u00a0 There are two common size selection methods, the first is a gel electrophoresis based-method, while the second is bead-based size selection method.\u00a0 For the gel-based method, the adapted library fragments are run on a gel to separate the fragments by size and the band corresponding to the size of interest is collected.\u00a0 Using the bead-based method, magnetic beads are used with varying concentrations of buffers to isolate the DNA fragment sizes of interest.\u00a0 Final library fragment size is important for efficient, high quality DNA sequencing downstream.<\/p>\n<p>Bonus, when preparing <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-construct-library.html?socid=Social_btb\">amplicon libraries<\/a>, size selection is usually not necessary, as long as the PCR products were already designed to be within the desired size range.\u00a0 We are almost done!\u00a0 The last and very important step is library quantification and QC.\u00a0 Accurate library quantification is important for successful template preparation and sequencing.\u00a0 There are a few library quantification methods commonly used. The first is analysis by the <a href=\"http:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/sequencing\/sanger-sequencing.html?ICID=hero3-sanger?socid=social_btb\">Bioanalyzer<\/a> system.\u00a0 This method gives you both library concentration and fragment size information.\u00a0 The second is <a href=\"http:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/pcr\/real-time-pcr.html?socid=socia_btb\">qPCR<\/a>.\u00a0 This method provides the most accurate library quantification information, as it only measures amplifiable library fragments, but lacks library size information. \u00a0Which method you prefer is entirely up to you (and probably what\u2019s available in your lab).<\/p>\n<p>And that\u2019s NGS library preparation in a nutshell. Of course there are variations on this theme depending on your application, for example, gene expression or DNA methylation analysis, but the fundamentals stay the same. \u00a0Since your library quality dictates the success of all downstream processes from template preparation to sequencing, understanding the library preparation is important to help ensure you get the highest quality sequencing data.<\/p>\n<p>I hope this video was helpful on NGS library, and I am sure you\u2019ll have more questions.\u00a0Submit your question at <a href=\"http:\/\/www.thermofisher.com\/ask\">thermofisher.com\/ask<\/a> and subscribe to our channel to see more videos like this.<\/p>\n<p>And remember, when in doubt, just Seq It Out<\/p>\n","protected":false},"excerpt":{"rendered":"<p>What do you call a collection of millions of DNA fragments sharing the same short sequences on the 5\u2019 and 3\u2019 ends? The answer is a next generation sequencing, or NGS, library.\u00a0 Today, we are going to focus on the four basic steps of NGS library preparation that can be broadly applied across different preparation<\/p>\n","protected":false},"author":114,"featured_media":7181,"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],"tags":[11,6,43,176,74],"division":[],"class_list":{"0":"post-6249","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-lab-tips-and-tricks","8":"tag-ion-torrent","9":"tag-next-generation-sequencing","10":"tag-pcr","11":"tag-seqitout","12":"tag-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>Next Generation Sequencing Library Preparation - Seq It Out #10<\/title>\n<meta name=\"description\" content=\"Let&#039;s focus on the four basic steps of Next Generation Sequencing library preparation that can be broadly applied across different preparation methods.\" \/>\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\/next-generation-sequencing-library-preparation-seq-it-out-10\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Next Generation Sequencing Library Preparation - Seq It Out #10\" \/>\n<meta property=\"og:description\" content=\"Let&#039;s focus on the four basic steps of Next Generation Sequencing library preparation that can be broadly applied across different preparation methods.\" \/>\n<meta property=\"og:url\" content=\"https:\/\/www.thermofisher.com\/blog\/behindthebench\/next-generation-sequencing-library-preparation-seq-it-out-10\/\" \/>\n<meta property=\"og:site_name\" content=\"Behind the Bench\" \/>\n<meta property=\"article:publisher\" content=\"https:\/\/www.facebook.com\/thermofisher\" \/>\n<meta property=\"article:published_time\" content=\"2015-10-23T00:18:54+00:00\" \/>\n<meta property=\"article:modified_time\" content=\"2020-05-28T19:06:42+00:00\" \/>\n<meta property=\"og:image\" content=\"http:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2015\/10\/ngs-library-prep.png\" \/>\n\t<meta property=\"og:image:width\" content=\"499\" \/>\n\t<meta property=\"og:image:height\" content=\"279\" \/>\n\t<meta property=\"og:image:type\" content=\"image\/png\" \/>\n<meta name=\"author\" content=\"Natalie Gurson\" \/>\n<meta name=\"twitter:card\" content=\"summary_large_image\" \/>\n<meta name=\"twitter:creator\" content=\"@thermofisher\" \/>\n<meta name=\"twitter:site\" content=\"@thermofisher\" \/>\n<meta name=\"twitter:label1\" content=\"Written by\" \/>\n\t<meta name=\"twitter:data1\" content=\"Natalie Gurson\" \/>\n\t<meta name=\"twitter:label2\" content=\"Est. reading time\" \/>\n\t<meta name=\"twitter:data2\" content=\"3 minutes\" \/>\n<script type=\"application\/ld+json\" class=\"yoast-schema-graph\">{\"@context\":\"https:\\\/\\\/schema.org\",\"@graph\":[{\"@type\":\"Article\",\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/behindthebench\\\/next-generation-sequencing-library-preparation-seq-it-out-10\\\/#article\",\"isPartOf\":{\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/behindthebench\\\/next-generation-sequencing-library-preparation-seq-it-out-10\\\/\"},\"author\":{\"name\":\"Natalie Gurson\",\"@id\":\"https:\\\/\\\/admin.acceleratingscience.com\\\/behindthebench\\\/#\\\/schema\\\/person\\\/a8d8159d870d7ca4bde182af286a7ed8\"},\"headline\":\"Next Generation Sequencing Library Preparation &#8211; 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