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.
Vendor | Workflow | Kit example | Catalog example |
IDT | PCR-free | xGen™ UDI-UMI Adapters, 16 rxn | 10006914 |
xGen™ UDI-UMI Adapters, 96 rxn | 10005903 | ||
IDT | Enzyme based normalization | xGen™ Normalase™ Module 96rxn | 10009793 |
Illumina | Bead-based normalization | Nextera XT DNA Library Preparation Kit (24 samples) | FC-131-1024 |
Nextera XT DNA Library Preparation Kit (96 samples) | FC-131-1096 | ||
TruSight Tumor 170 NextSeq Kit (24 Sample Library Prep Kit) | 20028821 | ||
TruSight Oncology 500 DNA Kit (48 Sample Library Prep Kit Only) | 20028213 | ||
TruSight Oncology 500 DNA/RNA Bundle (24 Sample Library Prep Kit Only) | 20028215 |