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.
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).
Examples of these library types include:
- 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
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).
Assays Requiring Additional Processing
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 contact Element technical support for assistance.
Table 1. Incompatible assay or workflows
Vendor | Workflow | Kit example | Expected result | Note |
IDT | PCR-free | xGen™ UDI-UMI Adapters, 16 rxn, 96rxn | No polonies (PCR-free workflow only) | PCR-plus workflow is compatible |
IDT | Enzyme based normalization | xGen™ Normalase™ Module 96rxn | No polonies | |
Illumina | Bead-based normalization | Nextera XT DNA Library Preparation Kit (24, 96 samples) TruSight Tumor 170 NextSeq Kit (24 Sample Library Prep Kit) TruSight Oncology 500 DNA Kit (48 Sample Library Prep Kit Only) TruSight Oncology 500 DNA/RNA Bundle (24 Sample Library Prep Kit Only) | No polonies | The final library is compatible if it does NOT go through bead-based normalization |
Table 2. Assays that may show variable performance
Vendor | Workflow | Kit example | Expected result |
Illumina | Library prep workflow with amplification module using "PPC" reagent | Illumina DNA Prep with Enrichment Illumina RNA Prep with Enrichment (L) Tagmentation TruSeq ChIP Library Prep TruSeq DNA Nano TruSeq RNA Exome TruSeq RNA Library Prep Kit v2 TruSeq Stranded Total RNA TruSeq Stranded Total RNA with Ribo-Zero rRNA Depletion TruSeq Stranded mRNA TruSight RNA Fusion Panel TruSight RNA Pan-Cancer | Low or variable density |
Custom Sequencing Primers
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 the AVITI user guide for custom sequencing primer purity and concentration requirements to achieve the best sequencing quality.
Table 3 lists third-party libraries with adapters requiring custom sequencing primers. Please contact technical support of these vendors for adapter sequences.
Table 3. Assays requiring custom sequencing primers
Vendor | Assay Kit | Custom Primer Requirements |
10x Genomics | Single Cell Gene Expression Flex | Spike-in corresponding small RNA sequencing primers for Index1 and Read2 primers |
10x Genomics | Visium Spatial Gene Expression for FFPE | Spike-in corresponding small RNA sequencing primers for Index1 and Read2 primers |
10x Genomics | Visium HD | Spike-in corresponding small RNA sequencing primers for Index1 and Read2 primers |
10x Genomics | Libraries with Small RNA adapter for Read 2 – “TS” index set | Spike-in corresponding small RNA sequencing primers for Index1 and Read2 primers |
Illumina | TruSeq Small RNA Library Prep | Spike-in small RNA sequencing primers for Index1, Read1, and Read2. |
Illumina | Illumina PCR-free Library Prep | Custom sequencing primer for Read1 |
NEB | NEBNext Small RNA library | Custom sequencing primer for Read1 |
Revvity | NEXTFLEX Small RNA-Seq Kit v4 with UDIs | Spike-in corresponding small RNA sequencing primers for Index1 and Read2 |
Custom | n/a | Custom sequencing primers accordingly |