{"id":7462,"date":"2016-02-01T14:26:30","date_gmt":"2016-02-01T19:26:30","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/?p=7462"},"modified":"2016-05-12T22:49:28","modified_gmt":"2016-05-12T22:49:28","slug":"developing-the-minor-variant-finder","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/developing-the-minor-variant-finder\/","title":{"rendered":"Developing the Minor Variant Finder"},"content":{"rendered":"<p><img loading=\"lazy\" decoding=\"async\" class=\"alignright wp-image-7464 size-medium\" src=\"http:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/02\/IMG_07091-300x217.jpg\" alt=\"IMG_0709\" width=\"300\" height=\"217\" srcset=\"https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/02\/IMG_07091-300x217.jpg 300w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/02\/IMG_07091-768x555.jpg 768w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/02\/IMG_07091-1024x740.jpg 1024w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/02\/IMG_07091-1250x903.jpg 1250w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/02\/IMG_07091-400x289.jpg 400w\" sizes=\"auto, (max-width: 300px) 100vw, 300px\" \/>The hope of our <a href=\"http:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/sequencing\/sanger-sequencing\/sanger-dna-sequencing\/sanger-sequencing-data-analysis\/minor-variant-finder-software.html?Socid=social_btb\">project<\/a> team was that we could find a way to pick off 10% minor variants using Sanger sequencing.\u00a0 \u00a0\u00a0These hopes were based on an insight of Edgar Schreiber\u2019s that the difference in minor\/major peak amplitude ratios between a test and control sample should stand out at base positions where variants are located.\u00a0 He had some preliminary results that looked promising.\u00a0\u00a0 With the general perception that the limit of detection for <a href=\"http:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/sequencing\/dna-sequencing\/targeted-sequencing\/targeted-sequencing-sanger-sequencing.html?Socid=Social_btb\">Sanger sequencing<\/a> is around 20%, this seemed to be a worthy goal especially considering that people are using NGS to find minor variants associated with cancer and have few alternatives to validate these results.\u00a0 And what if we could do better than 10%?\u00a0 Maybe we could provide a cheaper way to detect minor variants on a tried and true gold-standard sequencing platform.<\/p>\n<p>So as I investigated further I noticed that the noise underlying Sanger sequencing data appeared to be awfully similar between control and test samples.\u00a0 It appeared that the noise was largely determined by the primary base sequence that was common between the control and test.\u00a0 This was an exciting find!\u00a0 It suggested that I could remove that noise from the test by using the control to model\u00a0the noise.\u00a0\u00a0 After devising a way to do this in a somewhat optimized fashion I discovered that we could detect 2.5% variants, even 1.25% variants!\u00a0 \u00a0\u00a0This was enticing!\u00a0 All this required was the use of existing standard\u00a0chemistry\u00a0to process the samples, a methodical application of the protocols to be sure that control and test samples are treated identically, both forward and reverse sequencing orientations, and running the data through software that implemented the noise removal algorithm and applied a machine learning algorithm to find the variants.<\/p>\n<p>It turned out that we could not reliably detect variants at 2.5% and lower.\u00a0 We were able to reach &gt;95% sensitivity and &gt;99% specificity for 5% variants.\u00a0\u00a0 Is this limitation caused by limitations of the software algorithm or the reagent technologies or the limitations of the instruments involved?\u00a0 We have not isolated the critical variables underlying the limitations yet.\u00a0 Though algorithms can offer cheap solutions to issues caused by instruments or reagents (software manufacturing costs are nearly nil), there are hard physical limitations.\u00a0 But, as this story illustrates, even complex systems that have been around for more than a decade might still be operating at levels that have not reached those physical limits.\u00a0\u00a0 We just have to be lucky enough to discover those nuggets amenable to algorithmic solutions and collaborate with our peers in chemistry and instrumentation to engineer performance breakthroughs for the system as a whole.<\/p>\n<p>&#8212;<\/p>\n<p>Harrison Leong, Ph.D.<br \/>\nSr. Staff Engineer, Software<\/p>\n","protected":false},"excerpt":{"rendered":"<p>The hope of our project team was that we could find a way to pick off 10% minor variants using Sanger sequencing.\u00a0 \u00a0\u00a0These hopes were based on an insight of Edgar Schreiber\u2019s that the difference in minor\/major peak amplitude ratios between a test and control sample should stand out at base positions where variants are<\/p>\n","protected":false},"author":122,"featured_media":7465,"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":[101],"tags":[17,189,25],"division":[],"class_list":{"0":"post-7462","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-product-news","8":"tag-applied-biosystems","9":"tag-bioinformatics-software","10":"tag-sanger-sequencing","11":"entry"},"_selected_authors":"","_selected_reviewers":"","acf":[],"yoast_head":"<!-- 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While immunohistochemistry remains central\u2026","rel":"","context":"In &quot;Cancer Research&quot;","block_context":{"text":"Cancer Research","link":"https:\/\/admin.acceleratingscience.com\/behindthebench\/cancer-research\/"},"img":{"alt_text":"Clinical Pathologist in a Molecular Diagnostics Laboratory","src":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/01\/Bartsch-1.jpg?resize=350%2C200&ssl=1","width":350,"height":200,"srcset":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/01\/Bartsch-1.jpg?resize=350%2C200&ssl=1 1x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/01\/Bartsch-1.jpg?resize=525%2C300&ssl=1 1.5x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/01\/Bartsch-1.jpg?resize=700%2C400&ssl=1 2x"},"classes":[]},{"id":19223,"url":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/innovations-in-qpcr-technology\/","url_meta":{"origin":7462,"position":2},"title":"Innovations in qPCR Technology: What Researchers Need to Know","author":"Ashleigh Barlow","date":"October 29, 2025","format":false,"excerpt":"https:\/\/youtu.be\/z9Kb1xAnNcg?si=TVUIc8rUxZUOhCqA Real-time PCR, also known as quantitative polymerase chain reaction (qPCR) technology, has been a cornerstone of molecular biology research for decades. With continuous advancements, qPCR remains a critical tool for researchers worldwide. Damien Luk, Senior Director of Product Management at Thermo Fisher Scientific, shared his insights on the future\u2026","rel":"","context":"In &quot;Developmental Biology&quot;","block_context":{"text":"Developmental Biology","link":"https:\/\/admin.acceleratingscience.com\/behindthebench\/developmental-biology\/"},"img":{"alt_text":"A lineup of Thermo Fisher Scientific QuantStudio real-time PCR systems, including QuantStudio 3, 5, 6 Pro, 7 Pro, and 12K Flex instruments, displayed against a blue gradient background.","src":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/10\/qPCR-Blog-Cover-Image.jpg?resize=350%2C200&ssl=1","width":350,"height":200,"srcset":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/10\/qPCR-Blog-Cover-Image.jpg?resize=350%2C200&ssl=1 1x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/10\/qPCR-Blog-Cover-Image.jpg?resize=525%2C300&ssl=1 1.5x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/10\/qPCR-Blog-Cover-Image.jpg?resize=700%2C400&ssl=1 2x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/10\/qPCR-Blog-Cover-Image.jpg?resize=1050%2C600&ssl=1 3x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/10\/qPCR-Blog-Cover-Image.jpg?resize=1400%2C800&ssl=1 4x"},"classes":[]},{"id":19883,"url":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/building-a-better-sample-qc-workflow-from-extraction-to-sequencing\/","url_meta":{"origin":7462,"position":3},"title":"Building a Better Sample QC Workflow: From Extraction to Sequencing","author":"Marlene Gasdia-Cochrane","date":"August 25, 2026","format":false,"excerpt":"Article Summary Successful genomics experiments don't begin with sequencing\u2014they begin with sample quality. Every downstream application, from PCR to next-generation sequencing (NGS), depends on starting with nucleic acids that are accurately quantified and free from contaminants that could compromise results. Establishing a consistent quality control workflow helps laboratories improve reproducibility\u2026","rel":"","context":"In &quot;General&quot;","block_context":{"text":"General","link":"https:\/\/admin.acceleratingscience.com\/behindthebench\/general\/"},"img":{"alt_text":"A pipette drops a liquid into a petri dish with a dna sequence background, science concept. It suggests medical or genetic research.","src":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/iStock-2216731026_petridish-scaled.jpg?resize=350%2C200&ssl=1","width":350,"height":200,"srcset":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/iStock-2216731026_petridish-scaled.jpg?resize=350%2C200&ssl=1 1x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/iStock-2216731026_petridish-scaled.jpg?resize=525%2C300&ssl=1 1.5x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/iStock-2216731026_petridish-scaled.jpg?resize=700%2C400&ssl=1 2x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/iStock-2216731026_petridish-scaled.jpg?resize=1050%2C600&ssl=1 3x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/08\/iStock-2216731026_petridish-scaled.jpg?resize=1400%2C800&ssl=1 4x"},"classes":[]},{"id":20004,"url":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/a-guide-to-nucleic-acid-quality-control-dna-and-rna-purity\/","url_meta":{"origin":7462,"position":4},"title":"A Guide to Nucleic Acid Quality Control: DNA and RNA Purity","author":"Marlene Gasdia-Cochrane","date":"September 22, 2026","format":false,"excerpt":"Article Summary Nucleic acid quality control helps researchers evaluate DNA and RNA concentration and purity before downstream applications such as PCR, qPCR, and next-generation sequencing (NGS). A standardized workflow combining reliable extraction, concentration and purity assessment, application-appropriate quantification, and consistent laboratory practices can help identify sample issues early, improve reproducibility,\u2026","rel":"","context":"In &quot;General&quot;","block_context":{"text":"General","link":"https:\/\/admin.acceleratingscience.com\/behindthebench\/general\/"},"img":{"alt_text":"Close-up shot of a dna sequence on a screen, a digital representation of genetic code, used for analysis.","src":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/iStock-2212923489_dnasequence-scaled.jpg?resize=350%2C200&ssl=1","width":350,"height":200,"srcset":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/iStock-2212923489_dnasequence-scaled.jpg?resize=350%2C200&ssl=1 1x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/iStock-2212923489_dnasequence-scaled.jpg?resize=525%2C300&ssl=1 1.5x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/iStock-2212923489_dnasequence-scaled.jpg?resize=700%2C400&ssl=1 2x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/iStock-2212923489_dnasequence-scaled.jpg?resize=1050%2C600&ssl=1 3x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/iStock-2212923489_dnasequence-scaled.jpg?resize=1400%2C800&ssl=1 4x"},"classes":[]},{"id":20019,"url":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/qpcr-dpcr-ngs-cancer-research\/","url_meta":{"origin":7462,"position":5},"title":"From tumor heterogeneity to low-frequency variants: How qPCR, dPCR, and NGS work together in cancer research","author":"Ashleigh Barlow","date":"September 26, 2026","format":false,"excerpt":"Dr. Javier Hernandez Losa explains how combining broad genomic profiling with targeted, sensitive confirmation can help researchers address some of cancer biology\u2019s most difficult analytical challenges. A Q&A with Dr. Javier Hernandez Losa, Director of the Molecular Biology Laboratory, Department of Pathology, Hospital Universitario Vall d\u2019Hebron, Barcelona Cancer research increasingly\u2026","rel":"","context":"In &quot;Cancer Research&quot;","block_context":{"text":"Cancer Research","link":"https:\/\/admin.acceleratingscience.com\/behindthebench\/cancer-research\/"},"img":{"alt_text":"Dr. Javier Hernandez Losa, Director of the Molecular Biology Laboratory at Hospital Universitario Vall d'Hebron in Barcelona.","src":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/Picture133.jpg?resize=350%2C200&ssl=1","width":350,"height":200,"srcset":"https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/Picture133.jpg?resize=350%2C200&ssl=1 1x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/Picture133.jpg?resize=525%2C300&ssl=1 1.5x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/Picture133.jpg?resize=700%2C400&ssl=1 2x, https:\/\/i0.wp.com\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2026\/09\/Picture133.jpg?resize=1050%2C600&ssl=1 3x"},"classes":[]}],"jetpack_sharing_enabled":true,"jetpack_featured_media_url":"https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/02\/IMG_0722.jpg","_links":{"self":[{"href":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/wp-json\/wp\/v2\/posts\/7462","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/wp-json\/wp\/v2\/users\/122"}],"replies":[{"embeddable":true,"href":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/wp-json\/wp\/v2\/comments?post=7462"}],"version-history":[{"count":0,"href":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/wp-json\/wp\/v2\/posts\/7462\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/wp-json\/wp\/v2\/media\/7465"}],"wp:attachment":[{"href":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/wp-json\/wp\/v2\/media?parent=7462"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/wp-json\/wp\/v2\/categories?post=7462"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/wp-json\/wp\/v2\/tags?post=7462"},{"taxonomy":"division","embeddable":true,"href":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/wp-json\/wp\/v2\/division?post=7462"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}