Cloudbreak Freestyle Compatibility with Third-Party Libraries

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.

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.

A few library types requiring additional processes using the Adept PCR-Free Workflow achieved optimal density when loaded directly in a linear format and sequenced with Cloudbreak Freestyle chemistry (Figure 2).

• Standard – Library with intact ends
• Type 1 – Additional base at 3’ end
• Type 2 – Truncated 1 base
• Type 3 – Blocked 5’ end; PF Polonies (M) – total pass filter polonies in million

Assays Requiring Additional Process

There are a few library types that still require additional processes before sequencing directly with Cloudbreak Freestyle. When preparing libraries using the kits and specified workflow listed in the following table, the prepared libraries are not readily compatible with Cloudbreak Freestyle.

Element recommends amplifying the library with 4-5 cycles of PCR using KAPA HiFi HotStart Library Amp Kit with Primer Mix (Roche, 07958978001), and perform clean-up and quality check. The purified library is then compatible with Cloudbreak Freestyle and can be loaded in linear format for sequencing.