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Compatibility with Third-Party Libraries </h1></div></div><div class="divider"></div><div class="page-content"><div class="page-block page-text"><div class="container -narrow"><div class="article"><p>The Element Cloudbreak Freestyle sequencing kit provides a simplified workflow for sequencing third-party libraries on the AVITI™ System. Cloudbreak Freestyle technology enables circularization on flow cell, with direct loading of linear third-party libraries, and eliminates the need for library conversion steps before sequencing. The absence of PCR amplification in library loading and the polony generation process minimizes the potential for mutations or amplification bias and allows for a complete PCR-free library sequencing workflow.</p><figure><img src="https://www.elementbiosciences.com/uploads/pages/_pagesPhoto/Workflows-1-1.png" alt="" /><figcaption>Figure 1. Workflow comparison of three methods to support sequencing of third-party libraries on the AVITI System </figcaption></figure><p>Cloudbreak Freestyle chemistry further enables the compatibility of third-party assays, compared to the Element Adept v1.1 or Rapid PCR-free Workflows. Libraries with missing, additional, or mismatched bases at the ends that block circularization, are now seamlessly compatible with Cloudbreak Freestyle chemistry. </p><figure><img src="https://www.elementbiosciences.com/uploads/pages/_pagesPhoto/PF-Polonies-1-1.jpg" alt="" /><figcaption>Figure 2. Optimal density achieved with Cloudbreak Freestyle chemistry </figcaption></figure><figure></figure><p>Previously, some library types required additional processing when using Cloudbreak for sequencing. Now, when sequenced with Cloudbreak Freestyle chemistry, they produce good density when loaded directly in a linear format (Figure 2; PF Polonies (M) – total pass filter polonies in million).</p><p>Examples of these library types include: </p><ul><li>Standard – Library with intact ends</li><li>Type 1 – Additional base at 5' end</li><li>Type 2 – Truncated 1 base</li><li>Type 3 – Blocked 5’ end</li></ul><p>In general, good practice of lab protocol and experiment design contributes to successful sequencing runs and high sequencing quality. For library preparation, we recommend using HPLC purified adapters for reproducibility and a high percentage of full-length oligo synthesis. When a library contains full-length adapters at both 5’ and 3’ ends, it generates consistent ligation yields and will result in good density with Freestyle chemistry (or high circularization yield with Adept workflow). </p><p></p><h3>Assays Requiring Additional Processing</h3><p>Some library types are not directly compatible or may show variable performance with Cloudbreak Freestyle. If you are preparing libraries using the kits and specified workflows listed in the tables below, please <a href="https://www.elementbiosciences.com/contact">contact Element technical support</a> for assistance.</p><p>Table 1. Incompatible assay or workflows</p><table><tbody><tr><td width="55" valign="top"><p><strong>Vendor</strong></p></td><td width="90" valign="top"><p><strong>Workflow</strong></p></td><td width="293" valign="top"><p><strong>Kit example</strong></p></td><td width="130" valign="top"><p><strong>Expected result</strong></p></td><td width="90" valign="top"><p><strong>Note</strong></p></td></tr><tr><td width="55" valign="top"><p>IDT</p></td><td width="90" valign="top"><p>PCR-free</p></td><td width="293" valign="top"><p>xGen™ UDI-UMI Adapters, 16 rxn, 96rxn</p></td><td width="130" valign="top"><p>No polonies (PCR-free workflow only)</p></td><td width="90" valign="top"><p>PCR-plus workflow is compatible</p></td></tr><tr><td width="55" valign="top"><p>IDT</p></td><td width="90" valign="top"><p>Enzyme based normalization</p></td><td width="293" valign="top"><p>xGen™ Normalase™ Module 96rxn</p></td><td width="130" valign="top"><p>No polonies</p></td><td width="90" valign="top"></td></tr><tr><td width="55" valign="top"><p>Illumina</p></td><td width="90" valign="top"><p>Bead-based normalization</p></td><td width="293" valign="top"><p>Nextera XT DNA Library Preparation Kit (24, 96 samples)</p><p>TruSight Tumor 170 NextSeq Kit (24 Sample Library Prep Kit)</p><p>TruSight Oncology 500 DNA Kit (48 Sample Library Prep Kit Only)</p><p>TruSight Oncology 500 DNA/RNA Bundle (24 Sample Library Prep Kit Only)</p></td><td width="130" valign="top"><p>No polonies</p></td><td width="90" valign="top"><p>The final library is compatible if it does NOT go through bead-based normalization</p></td></tr></tbody></table><p>Table 2. Assays that may show variable performance</p><table><tbody><tr><td width="66" valign="top"><p><strong>Vendor</strong></p></td><td width="92" valign="top"><p><strong>Workflow</strong></p></td><td width="339" valign="top"><p><strong>Kit example</strong></p></td><td width="140" valign="top"><p><strong>Expected result</strong></p></td></tr><tr><td width="66" valign="top"><p>Illumina</p></td><td width="200" valign="top"><p>Library prep workflow with amplification module using "PPC" reagent</p></td><td width="339" valign="top"><p>Illumina DNA Prep with Enrichment</p><p>Illumina RNA Prep with Enrichment (L) Tagmentation</p><p>TruSeq ChIP Library Prep</p><p>TruSeq DNA Nano</p><p>TruSeq RNA Exome</p><p>TruSeq RNA Library Prep Kit v2</p><p>TruSeq Stranded Total RNA</p><p>TruSeq Stranded Total RNA with Ribo-Zero rRNA Depletion</p><p>TruSeq Stranded mRNA </p><p>TruSight RNA Fusion Panel</p><p>TruSight RNA Pan-Cancer</p></td><td width="140" valign="top"><p>Low or variable density </p></td></tr></tbody></table><h3 id="custom-sequencing-primers">Custom Sequencing Primers</h3><p>Cloudbreak Freestyle sequencing cartridges support sequencing of Elevate, TruSeq, and Nextera adapter libraries. Libraries with custom adapter sequences require a spike-in or complete swap of sequencing primers for sequencing. Follow <a href="https://go.elementbiosciences.com/aviti-system-user-guide-ma-00008" target="_blank" rel="noopener">the AVITI user guide</a> for custom sequencing primer purity and concentration requirements to achieve the best sequencing quality. </p><p><strong>Update October 1, 2024:</strong></p><p>Effective from sequencing cartridge lot number<strong> 2409210001 and beyond</strong>, Cloudbreak Freestyle cartridges now come pre-loaded with additional Read1, Read2, and Index1 primers compatible with common small RNA adapters. This upgrade ensures direct compatibility and streamlines workflows for assays such as PerkinElmer NEXTFLEX small RNA v3, 10X Genomics Fixed RNA Profiling (Flex), 10x Genomics Visium for FFPE, and Visium HD. This upgrade allows users to omit the previously required primer spike-in when sequencing compatible libraries. All Cloudbreak Freestyle sequencing cartridges including lot # 2409210001 and higher are compatible. See Table 4 below.</p><p>Table 3 lists third-party libraries with adapters requiring custom sequencing primers. Please contact technical support of these vendors for adapter sequences. </p><p>Table 3. Assays requiring custom sequencing primers </p><table><tbody><tr><td width="200" valign="top"><p><strong>Vendor</strong></p></td><td width="200" valign="top"><p><strong>Assay Kit</strong></p></td><td width="450" valign="top"><p><strong>Custom Primer Requirements</strong></p></td></tr><tr><td width="200" valign="top"><p>10x Genomics </p></td><td width="200" valign="top"><p>Single Cell Gene Expression Flex </p></td><td width="450" valign="top"><p>Spike-in corresponding small RNA sequencing primers for Index1 and Read2 primers; <br />Recent lots of Cloudbreak Freestyle cartridges contain common small RNA adapters and are directly compatible, see below for compatible lot numbers <br /></p></td></tr><tr><td width="200" valign="top"><p>10x Genomics </p></td><td width="200" valign="top"><p>Visium Spatial Gene Expression for FFPE </p></td><td width="450" valign="top"><p>Spike-in corresponding small RNA sequencing primers for Index1 and Read2 primers; </p><p>Recent lots of Cloudbreak Freestyle cartridges contain common small RNA adapters and are directly compatible, see below for compatible lot numbers <br /></p></td></tr><tr><td width="200" valign="top"><p>10x Genomics </p></td><td width="200" valign="top"><p>Visium HD </p></td><td width="450" valign="top"><p>Spike-in corresponding small RNA sequencing primers for Index1 and Read2 primers; <br />Recent lots of Cloudbreak Freestyle cartridges contain common small RNA adapters and are directly compatible, see below for compatible lot numbers <br /></p></td></tr><tr><td width="200" valign="top"><p>10x Genomics </p></td><td width="200" valign="top"><p>Libraries with Small RNA adapter for Read 2 – “TS” index set </p></td><td width="450" valign="top"><p>Spike-in corresponding small RNA sequencing primers for Index1 and Read2 primers; <br />Recent lots of Cloudbreak Freestyle cartridges contain common small RNA adapters and are directly compatible, see below for compatible lot numbers <br /></p></td></tr><tr><td width="200" valign="top"><p>Illumina </p></td><td width="200" valign="top"><p>TruSeq Small RNA Library Prep </p></td><td width="450" valign="top"><p>Spike-in small RNA sequencing primers for Index1, Read1, and Read2; <br />Recent lots of Cloudbreak Freestyle cartridges contain common small RNA adapters and are directly compatible, see below for compatible lot numbers <br /></p></td></tr><tr><td width="200" valign="top"><p>Illumina </p></td><td width="200" valign="top"><p>Illumina PCR-free Library Prep </p></td><td width="450" valign="top"><p>Custom sequencing primer for Read1 </p></td></tr><tr><td width="200" valign="top"><p>NEB </p></td><td width="200" valign="top"><p>NEBNext Small RNA library </p></td><td width="450" valign="top"><p>Custom sequencing primer for Read1 </p></td></tr><tr><td width="200" valign="top"><p>Revvity </p></td><td width="200" valign="top"><p>NEXTFLEX Small RNA-Seq Kit v4 with UDIs </p></td><td width="450" valign="top"><p>Spike-in corresponding small RNA sequencing primers for Index1 and Read2; <br />Recent lots of Cloudbreak Freestyle cartridges contain common small RNA adapters and are directly compatible, see below for compatible lot numbers <br /></p></td></tr><tr><td width="200" valign="top"><p>Custom </p></td><td width="200" valign="top"><p>n/a </p></td><td width="450" valign="top"><p>Custom sequencing primers accordingly </p></td></tr></tbody></table><p>Table 4: Cloudbreak Freestyle kits updated, by part number</p><table><tbody><tr><td width="200" valign="top"><p><strong>Product </strong><br /><strong>Number</strong></p></td><td width="200" valign="top"><p><strong>Product Name</strong></p></td><td width="450" valign="top"><p><strong>Kit updated to include small RNA sequencing primers Read1, Read2, and Index1 </strong></p></td></tr><tr><td width="200" valign="top"><p>860-00014 </p></td><td width="200" valign="top"><p>AVITI 2x75 Sequencing Kit Cloudbreak FS Medium Output </p></td><td width="450" valign="top"><p>Sequencing cartridge Lot 2409210001 and higher include updated primers </p></td></tr><tr><td width="200" valign="top"><p>860-00015 </p></td><td width="200" valign="top"><p>AVITI 2x75 Sequencing Kit Cloudbreak FS High Output </p></td><td width="450" valign="top"><p>Sequencing cartridge Lot 2409210001 and higher include updated primers </p></td></tr><tr><td width="200" valign="top"><p>860-00011 </p></td><td width="200" valign="top"><p>AVITI 2x150 Sequencing Kit Cloudbreak FS Low Output </p></td><td width="450" valign="top"><p>Sequencing cartridge Lot 2409210001 and higher include updated primers </p></td></tr><tr><td width="200" valign="top"><p>860-00012 </p></td><td width="200" valign="top"><p>AVITI 2x150 Sequencing Kit Cloudbreak FS Medium Output </p></td><td width="450" valign="top"><p>Sequencing cartridge Lot 2409210001 and higher include updated primers </p></td></tr><tr><td width="200" valign="top"><p>860-00013 </p></td><td width="200" valign="top"><p>AVITI 2x150 Sequencing Kit Cloudbreak FS High Output </p></td><td width="450" valign="top"><p>Sequencing cartridge Lot 2409210001 and higher include updated primers </p></td></tr><tr><td width="200" valign="top"><p>860-00016 </p></td><td width="200" valign="top"><p>AVITI 2x300 Sequencing Kit Cloudbreak FS Medium Output </p></td><td width="450" valign="top"><p>Sequencing cartridge Lot 2409210001 and higher include updated primers </p></td></tr><tr><td width="200" valign="top"><p>860-00017 </p></td><td width="200" valign="top"><p>AVITI 2x300 Sequencing Kit Cloudbreak FS High Output </p></td><td width="450" valign="top"><p>Sequencing cartridge Lot 2409210001 and higher include updated primers </p></td></tr></tbody></table></div></div></div></div></main><footer class="footer" data-ref="footer" role="contentinfo"><div class="footer-content container"><div class="footer-row"><div class="footer-left"><div class="footer-left__wrapper"><div class="footer-tagline">Your Science Can’t Wait</div><div class="footer-social"><a href="https://www.linkedin.com/company/elembio" class="footer-social__link" target="_blank"><svg><use xlink:href="/assets/img/icons/glyphs.svg?v=#linkedin"></use></svg></a><a href="https://twitter.com/elembio" class="footer-social__link" target="_blank"><svg><use xlink:href="/assets/img/icons/glyphs.svg?v=#twitter"></use></svg></a><a href="https://www.youtube.com/@elembio" class="footer-social__link" target="_blank"><svg><use xlink:href="/assets/img/icons/glyphs.svg?v=#youtube"></use></svg></a></div></div></div><div class="footer-right"><picture class="footer-aviti"><source srcset="https://www.elementbiosciences.com/assets/img/footer-aviti-mobile.png" media="(max-width: 454px)"><img src="https://www.elementbiosciences.com/assets/img/footer-aviti-desktop.png" width="1024" height="680" alt="AVITI" ></picture><div class="footer-cta"><div class="footer-cta__wrapper"><strong class="footer-cta__heading"> Have a question? <svg><use xlink:href="/assets/img/icons/glyphs.svg?v=#mail"></use></svg></strong><div class="footer-cta__links"><a href="https://www.elementbiosciences.com/contact" class="button -white -light">Contact Us</a></div></div></div></div></div></div><div class="footer-legal"><div class="container"><div class="footer-legal__wrapper"><span class="footer-legal__copywrite">© Copyright 2024 Element Biosciences</span><div class="footer-legal__links"><a href="https://www.elementbiosciences.com/legal/terms" class="footer-legal__link"> Terms of Use </a><a href="https://www.elementbiosciences.com/uploads/legal/Element-Biosciences-Privacy-Notice-09-05-2024.pdf" class="footer-legal__link" > Privacy Notice </a><a href="https://www.elementbiosciences.com/uploads/legal/Patents-08.19.2024.pdf" class="footer-legal__link" > Patents </a><a href="#" class="footer-legal__link" data-ref="osano-cookie-preferences" > Do Not Sell or Share My Personal Information </a></div></div></div></div></footer></div></body></html>