How should I prepare my long insert libraries for Cloudbreak Freestyle™ Sequencing?

When running libraries using Cloudbreak™ or Cloudbreak Freestyle sequencing cartridges, typically we recommend library fragments ranging from 175 – 1000 bp. Larger library fragments (1 – 2 kb) can be  sequenced on the AVITI™ platforms but require additional considerations. 

• Consult the latest Cloudbreak Sequencing User Guide for loading recommendations. Use the 2 x 300 loading concentrations for Large (> 700 bp) library sizes as a starting point for optimization.  
  • Note: Loading recommendations do not change between AVITI and AVITI24™ systems, even when utilizing AOS 3.4 or later. We do not expect a boost in output for these runs.
• For best performance with longer insert libraries, target an output of 600–700M reads per run, which corresponds to approximately 60–70% output of a standard run. 
• Due to the difference in sizes between PhiX and larger libraries, we recommend keeping the PhiX spike-in to 0.1 pM or lower to target ~1-5% alignment.
• We recommend configuring these runs as 2 x 75 or 2 x 150. Under Advanced Run Settings, select the Long Insert recipe. This recipe adjusts amplification parameters to improve performance for long-insert libraries. 

For more information regarding sequencing libraries with longer inserts, please see these additional resources: 

• Poster: Impact of Insert Length on Variant Quality 
• Which sequencing recipe should I use for my library insert size? 
• 2 x 300 Long-Insert Human WGS with the Elevate Workflow Data Set