{"id":10179,"date":"2017-02-15T20:41:57","date_gmt":"2017-02-15T20:41:57","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/behindthebench\/?p=10179"},"modified":"2017-02-15T20:41:57","modified_gmt":"2017-02-15T20:41:57","slug":"str-analysis-using-thresholds-forensic-focus-5","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/str-analysis-using-thresholds-forensic-focus-5\/","title":{"rendered":"STR Analysis Using Thresholds &#8211; Forensic Focus #5"},"content":{"rendered":"<p>Thresholds.\u00a0 Why do we spend so much time worrying about them?\u00a0 What are they, and how do I set them?<\/p>\n<p>Thresholds allow you to assess data quality.\u00a0 For example, on an Applied Biosystems 3500 series capillary electrophoresis instrument, peaks heights correlate to signal intensity or relative fluorescent units (Or RFU\u2019s). You can have confidence in peaks that reach or exceed your threshold.<\/p>\n<p><iframe loading=\"lazy\" width=\"760\" height=\"428\" src=\"https:\/\/www.youtube.com\/embed\/lbCQmMbfIoM?feature=oembed\" frameborder=\"0\" allow=\"accelerometer; autoplay; encrypted-media; gyroscope; picture-in-picture\" allowfullscreen><\/iframe><\/p>\n<p>There are two basic types of thresholds used in forensic DNA analysis; analytical and stochastic.\u00a0 An analytical threshold is the lowest RFU value at which DNA can be distinguished from noise.\u00a0 Signal above may be a DNA peak or an artifact; signal below is generally disregarded. To establish analytical thresholds, we measure the background noise of the chemistry and instrument.\u00a0\u00a0 Historically, different analytical thresholds were set for each dye because the noise varied, especially when DNA was present.\u00a0 Nowadays, kits like the Applied Biosystems GlobalFiler\u2122 amplification kit produce minimal noise, whether you are amplifying a negative control or a 1 ng DNA sample. Also, the GlobalFiler kit has very little dye-specific variation, so a single analytical threshold may be sufficient.<\/p>\n<p>We\u2019ve seen that the\u00a0 GlobalFiler kit on the 3500 instrument\u00a0 has a clean baseline;\u00a0 now let\u2019s establish a reasonable analytical threshold.\u00a0 This requires an understanding of the 3500 series instruments.\u00a0 Because the 3500 scale is increased compared to the legacy instruments, noise-based analytical thresholds may complicate data analysis and interpretation. For example, samples with 500 pg or more of input DNA may generate pull-up peaks that exceed a low, noise-based analytical threshold. In complex mixtures, these pull-up peaks may be difficult to differentiate from minor component DNA peaks.<\/p>\n<p>Can\u2019t choose\u00a0 where to set the threshold?<\/p>\n<p>Well, analytical thresholds should establish a balance between allele preservation and artifact editing, resulting in confident, accurate allele calls.\u00a0 Now let\u2019s talk about stochastic thresholds.\u00a0\u00a0\u00a0 The stochastic threshold applies to low DNA input data where stochastic effects may be in play.\u00a0 You can reasonably assume that a peak above the stochastic threshold is not impacted by stochastic effects, therefore dropout of a sister allele is unlikely. Conversely, you may not have much confidence that a single peak below the stochastic threshold is a true homozygote. Examples of stochastic effects are allele drop-out, allele drop-in, sister allele imbalance, and elevated stutter.\u00a0 These effects occur more frequently in low-level DNA samples.<\/p>\n<p>Set your stochastic threshold carefully! Thresholds set too low may lead to incorrect genotype calls due to stochastic effects, while thresholds set too high may lead to the loss of reliable information. The area between the analytical and stochastic thresholds is what we call \u201cthe gray zone\u201d.\u00a0 DNA in the gray zone is in the stochastic region, therefore use caution when analyzing and interpreting Gray Zone data.<\/p>\n<p>In discussing thresholds, we\u2019ve been evaluating samples with complex mixtures and low-level DNA.\u00a0 But not all samples require such intense analysis.\u00a0 For example, reference samples from known individuals generally contain plenty of high-quality, single-source DNA. For these samples, database labs may use a direct amplification.\u00a0 To further streamline the direct workflow, database labs may choose to implement a single threshold for analysis.<\/p>\n<p>Well, that about covers it for setting thresholds.\u00a0 If you have any questions or would like to see more such videos, click on the link below,<\/p>\n<p>And don\u2019t forget- when in doubt, you can always refer Back to Bases!<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Thresholds.\u00a0 Why do we spend so much time worrying about them?\u00a0 What are they, and how do I set them? Thresholds allow you to assess data quality.\u00a0 For example, on an Applied Biosystems 3500 series capillary electrophoresis instrument, peaks heights correlate to signal intensity or relative fluorescent units (Or RFU\u2019s). You can have confidence in<\/p>\n","protected":false},"author":107,"featured_media":10180,"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":[71,108],"tags":[17,204],"division":[],"class_list":{"0":"post-10179","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-forensics","8":"category-lab-tips-and-tricks","9":"tag-applied-biosystems","10":"tag-forensic-focus","11":"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>STR Analysis Using Thresholds - Forensic Focus #5 - 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\/str-analysis-using-thresholds-forensic-focus-5\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"STR Analysis Using Thresholds - Forensic Focus #5\" \/>\n<meta property=\"og:description\" content=\"Thresholds.\u00a0 Why do we spend so much time worrying about them?\u00a0 What are they, and how do I set them? 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