Freeze thaw stability of TruSight Oncology 500 (TSO500) ctDNA input and reagents

Will repeated freeze-thaw cycles will impact the cfDNA/ctDNA quantity and impact assay performance? Illumina has not tested repeated freeze thaws of cfDNA samples. In general, it is important to limit the number of freeze thaws for cfDNA/ctDNA and commercial control samples, and this must be validated by each lab according to the samples used and sample handling workflows. Repeated freeze-thaw cycles may impact input quality and, therefore, quantity that can be converted to library.

Are the enrichment reagents stable for longer than the 8 freeze-thaw cycles stated in the protocol (assuming two rounds of hybridization for each batch, with a freeze-thaw in between)? The 8 freeze-thaw cycles for hybridization (hyb) reagents includes the typical in-use scenario, where reagents would go through an additional freeze/thaw between first and second hyb. In testing, Illumina included in-use freeze-thaw cycles (exposed the reagents to an overnight freeze between the day one hyb setup and day two hyb setup) and counted this overnight storage as part of the in-use testing for a single freeze-thaw event. Additionally, the kit provides 2 tubes for the majority of the reagents, with each tube being sufficient for 24 libraries, so labs can limit freeze thaws by thawing only one tube if 24 libraries or fewer are being prepared. Reagents that are provided as a single tube are lower risk to experience impacts from freeze-thaw cycles.

For any feedback or questions regarding this article (Illumina Knowledge Article #2919), contact Illumina Technical Support [e-mail].