How to Detect and Analyze Alternative Splicing with RNA-seq

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Ensure adequate sequencing depth.\n\nAlign the RNA-seq reads to a reference genome using spliced aligners like STAR, HISAT2, or TopHat2, which can identify splice junctions.","upVoteCount":0,"author":{"@type":"Person","url":"https://uk.linkedin.com/in/omerfarukagca","name":"Omer Agca","image":"https://media.licdn.com/dms/image/v2/D4E03AQFqBCFo7beZsg/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1699220859698?e=2147483647&v=beta&t=et53nxom2cYgWAuM3bLVpa-eVcoStvE9G_UQYYEMrdk","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}}},{"@type":"Comment","text":"Our ability to accurately define an quantify similar transcripts is inherently limited when using the short reads traditionally favored for RNA-Seq. At Edinburgh Genomics we are excited about the possibilities of PacBio's new Kinnex kit, which allows the sequencing of multiple complete transcripts in a single HiFi read. Combined with the higher throughput of the new Revio sequencer, this makes the long read RNA-Seq analysis far more practical and affordable.","upVoteCount":0,"author":{"@type":"Person","url":"https://uk.linkedin.com/in/frances-turner-7797bb233","name":"Frances Turner","image":"https://media.licdn.com/dms/image/v2/D4E03AQFq2Pq4AVakoQ/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1721740335702?e=2147483647&v=beta&t=HP42P8LJDv120cm55tJQKjTJE6oRq1yKVKpN7R69a50","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}}},{"@type":"Comment","text":"After alignment, assemble the reads into transcript models using tools such as Cufflinks, StringTie, or Trinity to provide potential isoforms produced by alternative splicing.\n\nUse PCR (especially RT-PCR) with primers flanking potential splice junctions to validate predicted splicing events. Sequencing of PCR products, like Sanger sequencing, can further validate the alternative splice variants.\n\nUse tools like IGV (Integrative Genomics Viewer), Sashimi plots (available in MISO), or JBrowse for visualization of splicing patterns and events.","upVoteCount":0,"author":{"@type":"Person","url":"https://uk.linkedin.com/in/omerfarukagca","name":"Omer Agca","image":"https://media.licdn.com/dms/image/v2/D4E03AQFqBCFo7beZsg/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1699220859698?e=2147483647&v=beta&t=et53nxom2cYgWAuM3bLVpa-eVcoStvE9G_UQYYEMrdk","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}}},{"@type":"Comment","text":"Use tools like MISO (Mixture of Isoforms), rMATS, and SpliceTrap to detect and quantify specific alternative splicing events, comparing splice junction reads and exon-exon junctions.","upVoteCount":0,"author":{"@type":"Person","url":"https://uk.linkedin.com/in/omerfarukagca","name":"Omer Agca","image":"https://media.licdn.com/dms/image/v2/D4E03AQFqBCFo7beZsg/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1699220859698?e=2147483647&v=beta&t=et53nxom2cYgWAuM3bLVpa-eVcoStvE9G_UQYYEMrdk","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}}},{"@type":"Comment","text":"For studies comparing splicing between conditions or groups, use tools like DEXSeq, rMATS, and SUPPA to identify differentially spliced events between conditions.\n\nDetermine the potential functional implications of splicing variants using tools like ASpedia, SpliceAid, or Human Splicing Finder.\n\nEnsure quality control at each step and, if possible, replicate findings with independent datasets or other experimental methods.","upVoteCount":3,"author":{"@type":"Person","url":"https://uk.linkedin.com/in/omerfarukagca","name":"Omer Agca","image":"https://media.licdn.com/dms/image/v2/D4E03AQFqBCFo7beZsg/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1699220859698?e=2147483647&v=beta&t=et53nxom2cYgWAuM3bLVpa-eVcoStvE9G_UQYYEMrdk","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}}}]} </script>  </head> <body dir="ltr">  <div class="guest-upsells"> <form class="google-auth base-google-auth" action="https://www.linkedin.com/uas/login-submit" method="post"> <input name="loginCsrfParam" value="14cec1c0-54d2-4f33-8063-48dd9efec332" type="hidden"> <input name="session_redirect" value="https://www.linkedin.com/advice/1/how-can-you-identify-alternative-splicing-events-zwqpc" type="hidden"> <input name="trk" value="pulse-article_google-one-tap-submit" type="hidden"> <code 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class="bg-color-surface"></div> </div> <div class="max-w-[1128px] py-2 md:py-4 mx-auto">  <code id="isGuest" style="display: none"></code> <code id="memberUrn" style="display: none"></code> <code id="isNbaRecommendedArticlesDeeplinkEnabled" style="display: none"></code> <code id="isDynamicTextareaEnabled" style="display: none"></code> <article class="article-main relative flex-grow">  <section class="core-section-container my-3 relative">    <div class="core-section-container__content break-words">  <div class="md:row pb-6 md:flex article-content-row-reduced"> <div class="column-article-segment-content md:column md:mb-8 md:mr-4 md:w-1/2 md:flex-1 md:pr-[88px]"> <div> <div class="article-segment-content-container ">  <section class="mb-2"> <ol itemscope itemtype="http://schema.org/BreadcrumbList" class="breadcrumbs "> <li itemprop="itemListElement" itemscope itemtype="http://schema.org/ListItem" class="breadcrumb inline-block mr-0.5 font-sans text-sm babybear:text-xs text-color-text after:content-['/'] after:text-color-text-low-emphasis after:ml-0.5 after:no-underline last:mr-0 last:after:hidden"> <a itemprop="item" class="breadcrumb__link inline-block link-no-visited-state !text-color-text-low-emphasis" href="https://www.linkedin.com/pulse/topics/home/" data-tracking-control-name="breadcrumb" data-tracking-will-navigate> <span class="font-normal" itemprop="name">All</span> </a> <meta itemprop="position" content="1"> </li> <li itemprop="itemListElement" itemscope itemtype="http://schema.org/ListItem" class="breadcrumb inline-block mr-0.5 font-sans text-sm babybear:text-xs text-color-text after:content-['/'] after:text-color-text-low-emphasis after:ml-0.5 after:no-underline last:mr-0 last:after:hidden"> <a aria-current="page" itemprop="item" class="breadcrumb__link inline-block link-no-visited-state !text-color-text" href="https://www.linkedin.com/pulse/topics/biotechnology-s784/" data-tracking-control-name="breadcrumb" data-tracking-will-navigate> <span class="font-semibold" itemprop="name">Biotechnology</span> </a> <meta itemprop="position" content="2"> </li> </ol> </section> <h1 class="pulse-title text-display-md text-color-text lg:text-display-lg font-bold w-11/12 ">How can you identify alternative splicing events with RNA-seq data?</h1>  <div class="powered-by-ai py-3 text-sm text-color-text-secondary">Powered by AI and the LinkedIn community</div> <section class="table-of-content pb-6"> <div class="group menu-item relative flex cursor-pointer flex-row justify-start border-0 pl-0" data-chunk-header-id="preprocessing-and-alignment"> <h3 class="text-color-text group-hover:text-color-text-brand">1</h3> <div class="ml-2 mr-6 mt-0.5 min-w-[75%]"> <a class="fragmented-links font-normal !text-color-text underline decoration-1 underline-offset-4 group-hover:!text-color-text-brand" href="#preprocessing-and-alignment" data-tracking-will-navigate data-tracking-control-name="article-ssr-frontend-x-article__toc-article-segment-navigation">Preprocessing and alignment </a>  </div> <icon class="invisible absolute right-2 top-1/2 flex -translate-y-2/4 items-center justify-between rounded-full border-1 border-solid border-color-container-brand p-1 group-hover:visible" data-delayed-url="https://static.licdn.com/aero-v1/sc/h/6q8fxh9snmcwlh1wxg6fktwgf" data-svg-class-name="w-2 h-2 text-color-text-brand"></icon> </div> <div class="group menu-item relative flex cursor-pointer flex-row justify-start border-0 pl-0" data-chunk-header-id="splicing-event-annotation"> <h3 class="text-color-text group-hover:text-color-text-brand">2</h3> <div class="ml-2 mr-6 mt-0.5 min-w-[75%]"> <a class="fragmented-links font-normal !text-color-text underline decoration-1 underline-offset-4 group-hover:!text-color-text-brand" href="#splicing-event-annotation" data-tracking-will-navigate data-tracking-control-name="article-ssr-frontend-x-article__toc-article-segment-navigation">Splicing event annotation </a>  </div> <icon class="invisible absolute right-2 top-1/2 flex -translate-y-2/4 items-center justify-between rounded-full border-1 border-solid border-color-container-brand p-1 group-hover:visible" data-delayed-url="https://static.licdn.com/aero-v1/sc/h/6q8fxh9snmcwlh1wxg6fktwgf" data-svg-class-name="w-2 h-2 text-color-text-brand"></icon> </div> <div class="group menu-item relative flex cursor-pointer flex-row justify-start border-0 pl-0" data-chunk-header-id="splicing-event-quantification"> <h3 class="text-color-text group-hover:text-color-text-brand">3</h3> <div class="ml-2 mr-6 mt-0.5 min-w-[75%]"> <a class="fragmented-links font-normal !text-color-text underline decoration-1 underline-offset-4 group-hover:!text-color-text-brand" href="#splicing-event-quantification" data-tracking-will-navigate data-tracking-control-name="article-ssr-frontend-x-article__toc-article-segment-navigation">Splicing event quantification </a>  </div> <icon class="invisible absolute right-2 top-1/2 flex -translate-y-2/4 items-center justify-between rounded-full border-1 border-solid border-color-container-brand p-1 group-hover:visible" data-delayed-url="https://static.licdn.com/aero-v1/sc/h/6q8fxh9snmcwlh1wxg6fktwgf" data-svg-class-name="w-2 h-2 text-color-text-brand"></icon> </div> <div class="group menu-item relative flex cursor-pointer flex-row justify-start border-0 pl-0" data-chunk-header-id="splicing-event-differential-analysis"> <h3 class="text-color-text group-hover:text-color-text-brand">4</h3> <div class="ml-2 mr-6 mt-0.5 min-w-[75%]"> <a class="fragmented-links font-normal !text-color-text underline decoration-1 underline-offset-4 group-hover:!text-color-text-brand" href="#splicing-event-differential-analysis" data-tracking-will-navigate data-tracking-control-name="article-ssr-frontend-x-article__toc-article-segment-navigation">Splicing event differential analysis </a>  </div> <icon class="invisible absolute right-2 top-1/2 flex -translate-y-2/4 items-center justify-between rounded-full border-1 border-solid border-color-container-brand p-1 group-hover:visible" data-delayed-url="https://static.licdn.com/aero-v1/sc/h/6q8fxh9snmcwlh1wxg6fktwgf" data-svg-class-name="w-2 h-2 text-color-text-brand"></icon> </div> <div class="group menu-item relative flex cursor-pointer flex-row justify-start border-0 pl-0" data-chunk-header-id="here’s-what-else-to-consider"> <h3 class="text-color-text group-hover:text-color-text-brand">5</h3> <div class="ml-2 mr-6 mt-0.5 min-w-[75%]"> <a class="fragmented-links font-normal !text-color-text underline decoration-1 underline-offset-4 group-hover:!text-color-text-brand" href="#here’s-what-else-to-consider" data-tracking-will-navigate data-tracking-control-name="article-ssr-frontend-x-article__toc-article-segment-navigation">Here’s what else to consider </a> <p class="inline-promotion-text mt-0.5 text-xs font-normal text-color-text-low-emphasis">Be the first to add your personal experience</p> </div> <icon class="invisible absolute right-2 top-1/2 flex -translate-y-2/4 items-center justify-between rounded-full border-1 border-solid border-color-container-brand p-1 group-hover:visible" data-delayed-url="https://static.licdn.com/aero-v1/sc/h/6q8fxh9snmcwlh1wxg6fktwgf" data-svg-class-name="w-2 h-2 text-color-text-brand"></icon> </div> </section> <div class="x-article-introduction"> <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class>Alternative splicing is a process that allows a single gene to produce multiple variants of messenger RNA (mRNA), which can encode different proteins or regulate gene expression. It is a key mechanism for increasing the diversity and complexity of the proteome and the transcriptome, and it is involved in many biological functions and diseases. However, identifying and quantifying alternative splicing events from RNA-seq data, which is a high-throughput technique that sequences and measures the abundance of RNA molecules in a sample, can be challenging and requires specialized tools and methods. 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data-delayed-url="https://media.licdn.com/dms/image/v2/D4E03AQFq2Pq4AVakoQ/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1721740335702?e=2147483647&v=beta&t=HP42P8LJDv120cm55tJQKjTJE6oRq1yKVKpN7R69a50" data-ghost-classes="bg-color-entity-ghost-background" data-ghost-url="https://static.licdn.com/aero-v1/sc/h/9c8pery4andzj6ohjkjp54ma2" alt="Contributor profile photo"> </figure> <figure class="top-[4px] hidden lg:inline-flex"> <img class="inline-block relative rounded-[50%] w-6 h-6 " data-delayed-url="https://media.licdn.com/dms/image/v2/D4E03AQFq2Pq4AVakoQ/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1721740335702?e=2147483647&v=beta&t=HP42P8LJDv120cm55tJQKjTJE6oRq1yKVKpN7R69a50" data-ghost-classes="bg-color-entity-ghost-background" data-ghost-url="https://static.licdn.com/aero-v1/sc/h/9c8pery4andzj6ohjkjp54ma2" alt="Contributor profile photo"> </figure> <div class="min-w-0 ml-2"> <div class="flex items-center"> <span class="mr-0.5 truncate text-sm font-bold text-color-text lg:text-md"> Frances Turner </span>  </div> <p class="m-0 truncate text-sm text-color-text-low-emphasis md:text-md" data-test-id="contribution-lock-up-headline"> Bioinformatician at The University of Edinburgh </p> </div> </a> <div class="flex items-center">  <div class="ellipsis-menu absolute right-0 top-0 contribution-control-menu relative" data-feed-action="show-comment-dropdown" data-id="comment__ellipsis-menu" data-feed-control="control_menu" data-tracking-control-name="comment_control_menu" data-feed-action-type="expandControl" data-feed-action-category="expand"> <div class="collapsible-dropdown flex items-center relative hyphens-auto"> <button class="ellipsis-menu__trigger collapsible-dropdown__button btn-md btn-tertiary cursor-pointer !py-[6px] !px-1 flex items-center rounded-[50%] ellipsis-menu__trigger--small w-[32px] h-[32px] flex justify-center w-[40px] min-w-[40px]" aria-expanded="false" aria-label="Open menu" 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You'll no longer see this contribution</p> </template> </li>  </ul>  </div> </div> <code id="contributionCopyLink" style="display: none"></code> <code id="contributionCopyFail" style="display: none"></code> <code id="contributionCopyUnsupported" style="display: none"></code> </div> </div> <div class="contribution__container left__border -mb-2 -mt-1 animate-show ml-0 pl-0 md:ml-[-41px] md:pl-[32px] pr-0 lg:ml-[-33px] lg:pl-[40px] lg:pr-1 pt-2 pb-4"> <div class="flex flex-col justify-between lg:flex-col"> <div class="flex items-center justify-between gap-1"> </div> </div> <div class="contribution__text-wrapper relative"> <p class="contribution__text mb-1 whitespace-pre-wrap pt-1 text-md leading-open text-color-text md:mb-0 md:pt-0 line-clamp-5 lg:text-lg" data-tracking-control-name="comment_text">Our ability to accurately define an quantify similar transcripts is inherently limited when using the short reads traditionally favored for RNA-Seq. At Edinburgh Genomics we are excited about the possibilities of PacBio's new Kinnex kit, which allows the sequencing of multiple complete transcripts in a single HiFi read. Combined with the higher throughput of the new Revio sequencer, this makes the long read RNA-Seq analysis far more practical and affordable.</p> <button class="contribution__see-more-button absolute bottom-0 right-0 hidden cursor-pointer border-0 bg-color-surface p-0 text-md leading-open text-color-text-secondary hover:text-color-action hover:underline lg:text-lg" data-tracking-control-name="comment_expand" data-feed-control="comment_expand" data-feed-action-type="expandContributionText" data-feed-action-category="EXPAND" role="button" type="button">…see more</button> </div>  <div class="contribution__social-action-bar relative right-1 flex flex-row items-center justify-start gap-1"> <div class="contribution-social-activity inline-flex items-center justify-start"> <code id="i18n_reaction_selection_state" style="display: none"></code> <div class="reactions-menu flex flex-row relative items-center justify-center select-none [-webkit-user-select:none] pointer-events-auto 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md:block">Funny</span> </div> </div> </template>  </div>  </div>  </div>  </li> </ul>  </div> </div> </div> <code id="loginUrl" style="display: none"></code> <div class="article-segment" data-is-logged-in="false" data-thread-urn="urn:li:articleSegment:(urn:li:linkedInArticle:7120190234788593664,7120190236550184960)" data-test-id="article-segment" data-total-count="1" data-tracking-prefix="article-ssr-frontend-x-article" role="presentation"> <div class="md:row pb-6 md:flex"> <div class="column-article-segment-content md:column md:mb-8 md:mr-4 md:w-1/2 md:flex-1 md:pr-[88px]"> <div class="md:pt-6 "> <div class="article-segment-content-container "> <div id="content-urn_li_articleSegment__urn_li_linkedInArticle_7120190234788593664_7120190236550184960_"> <div class="article-main__content" data-test-id="publishing-text-block"> <h2 class="flex"> <a id="splicing-event-annotation" href="#splicing-event-annotation" class="dummy-anchor invisible absolute mt-[-100px]"></a> <span class="header-index pr-1.5">2</span> <span class>Splicing event annotation</span> </h2> </div> <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class>The next step is to annotate the splicing events that are present in your RNA-seq data. A splicing event is a specific way of joining exons or skipping introns in a gene. There are different types of splicing events, such as exon skipping, alternative 5' or 3' splice sites, intron retention, or mutually exclusive exons. To annotate the splicing events, you need to use a tool that can compare your alignment file to a reference annotation file, such as GTF or GFF3, that contains the gene and transcript structures. Some of the tools that can do this are rMATS, SUPPA2, or SpliceR.</span> </p> </div> </div> </div> </div> </div> <div class="column-article-segment-contributions md:column md:pb-[200px] md:w-1/2"> <div class="article-segment-contributions-header-container bg-color-canvas-tint md:ml-[-12px] md:pt-6 lg:ml-0 mt-1 ">  <div class="add-your-perspective-section relative flex flex-row items-center " data-segment-urn="urn:li:articleSegment:(urn:li:linkedInArticle:7120190234788593664,7120190236550184960)" data-test-add-contribution> <code id="add_your_perspective_tracking_prefix" style="display: none"></code> <code id="isGuest" style="display: none"></code> <code id="showFuseErrorMessage" style="display: none"></code> <div class="mb-2"> <a class="add-your-perspective-button btn-sm flex flex-col items-start justify-center lg:btn-md bg-color-surface-accent-tint-1 hover:bg-color-container-tertiary-hover " 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You'll no longer see this contribution</p> </template> </li>  </ul>  </div> </div> <code id="contributionCopyLink" style="display: none"></code> <code id="contributionCopyFail" style="display: none"></code> <code id="contributionCopyUnsupported" style="display: none"></code> </div> </div> <div class="contribution__container left__border -mb-2 -mt-1 animate-show ml-0 pl-0 md:ml-[-41px] md:pl-[32px] pr-0 lg:ml-[-33px] lg:pl-[40px] lg:pr-1 pt-2 pb-4"> <div class="flex flex-col justify-between lg:flex-col"> <div class="flex items-center justify-between gap-1"> </div> </div> <div class="contribution__text-wrapper relative"> <p class="contribution__text mb-1 whitespace-pre-wrap pt-1 text-md leading-open text-color-text md:mb-0 md:pt-0 line-clamp-5 lg:text-lg" data-tracking-control-name="comment_text">After alignment, assemble the reads into transcript models using tools such as Cufflinks, StringTie, or Trinity to provide potential isoforms produced by alternative splicing. Use PCR (especially RT-PCR) with primers flanking potential splice junctions to validate predicted splicing events. Sequencing of PCR products, like Sanger sequencing, can further validate the alternative splice variants. Use tools like IGV (Integrative Genomics Viewer), Sashimi plots (available in MISO), or JBrowse for visualization of splicing patterns and events.</p> <button class="contribution__see-more-button absolute bottom-0 right-0 hidden cursor-pointer border-0 bg-color-surface p-0 text-md leading-open text-color-text-secondary hover:text-color-action hover:underline lg:text-lg" data-tracking-control-name="comment_expand" data-feed-control="comment_expand" data-feed-action-type="expandContributionText" data-feed-action-category="EXPAND" role="button" type="button">…see more</button> </div>  <div class="contribution__social-action-bar relative right-1 flex flex-row items-center justify-start gap-1"> <div class="contribution-social-activity inline-flex items-center justify-start"> <code id="i18n_reaction_selection_state" style="display: none"></code> <div class="reactions-menu flex flex-row relative items-center justify-center select-none [-webkit-user-select:none] pointer-events-auto 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md:block">Funny</span> </div> </div> </template>  </div>  </div>  </div>  </li> </ul>  </div> </div> </div> <code id="loginUrl" style="display: none"></code> <div class="article-segment" data-is-logged-in="false" data-thread-urn="urn:li:articleSegment:(urn:li:linkedInArticle:7120190234788593664,7120190236592148480)" data-test-id="article-segment" data-total-count="1" data-tracking-prefix="article-ssr-frontend-x-article" role="presentation"> <div class="md:row pb-6 md:flex"> <div class="column-article-segment-content md:column md:mb-8 md:mr-4 md:w-1/2 md:flex-1 md:pr-[88px]"> <div class="md:pt-6 "> <div class="article-segment-content-container "> <div id="content-urn_li_articleSegment__urn_li_linkedInArticle_7120190234788593664_7120190236592148480_"> <div class="article-main__content" data-test-id="publishing-text-block"> <h2 class="flex"> <a id="splicing-event-quantification" href="#splicing-event-quantification" class="dummy-anchor invisible absolute mt-[-100px]"></a> <span class="header-index pr-1.5">3</span> <span class>Splicing event quantification</span> </h2> </div> <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class>The next step is to quantify the splicing events that are annotated in your RNA-seq data. This means estimating the relative abundance or frequency of each splicing event in each sample or condition. To do this, you need to use a tool that can calculate the number of reads that support each splicing event or each splice junction, and normalize them by the total number of reads or the gene expression level. 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md:block">Funny</span> </div> </div> </template>  </div>  </div>  </div>  </li> </ul>  </div> </div> </div> <code id="loginUrl" style="display: none"></code> <div class="article-segment" data-is-logged-in="false" data-thread-urn="urn:li:articleSegment:(urn:li:linkedInArticle:7120190234788593664,7120190236604735488)" data-test-id="article-segment" data-total-count="1" data-tracking-prefix="article-ssr-frontend-x-article" role="presentation"> <div class="md:row pb-6 md:flex"> <div class="column-article-segment-content md:column md:mb-8 md:mr-4 md:w-1/2 md:flex-1 md:pr-[88px]"> <div class="md:pt-6 "> <div class="article-segment-content-container "> <div id="content-urn_li_articleSegment__urn_li_linkedInArticle_7120190234788593664_7120190236604735488_"> <div class="article-main__content" data-test-id="publishing-text-block"> <h2 class="flex"> <a id="splicing-event-differential-analysis" href="#splicing-event-differential-analysis" class="dummy-anchor invisible absolute mt-[-100px]"></a> <span class="header-index pr-1.5">4</span> <span class>Splicing event differential analysis</span> </h2> </div> <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class>The final step is to perform differential analysis of the splicing events that are quantified in your RNA-seq data. This means testing whether the abundance or frequency of each splicing event is significantly different between two or more samples or conditions, such as different tissues, developmental stages, or treatments. To do this, you need to use a tool that can apply statistical methods and models to account for the variability and noise in the RNA-seq data, and adjust for multiple testing and confounding factors. 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Ensure quality control at each step and, if possible, replicate findings with independent datasets or other experimental methods.</p> <button class="contribution__see-more-button absolute bottom-0 right-0 hidden cursor-pointer border-0 bg-color-surface p-0 text-md leading-open text-color-text-secondary hover:text-color-action hover:underline lg:text-lg" data-tracking-control-name="comment_expand" data-feed-control="comment_expand" data-feed-action-type="expandContributionText" data-feed-action-category="EXPAND" role="button" type="button">…see more</button> </div>  <div class="contribution__social-action-bar relative right-1 flex flex-row items-center justify-start gap-1"> <div class="contribution-social-activity inline-flex items-center justify-start"> <code id="i18n_reaction_selection_state" style="display: none"></code> <div class="reactions-menu flex flex-row relative items-center justify-center select-none [-webkit-user-select:none] pointer-events-auto 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How to Detect and Analyze Alternative Splicing with RNA-seq