TruSight Oncology 500 ctDNA FAQs

For analysis on the TruSight Oncology 500 ctDNA Local App, follow the sample sheet instructions in the TruSight Oncology 500 ctDNA Local App User Guide.

The TruSight Oncology 500 ctDNA Local App Sample Sheet template can be found in the TruSight Oncology 500 ctDNA Product Files or in the resource bundle of the TruSight Oncology 500 ctDNA software.

The resource bundle is located in the /staging/illumina/TSO500_ctDNA/resources/ directory.

Yes, both kits use the same manifest.    

The kit enables the preparation of 48 libraries from cfDNA input.

Single nucleotide variants (SNVs), multi-nucleotide variants (MNVs), copy number alterations including deletions and amplifications in 59 genes, gene rearrangements in 23 genes, tumor mutational burden (TMB) and microsatellite instability (MSI). See the TruSight Oncology 500 ctDNA Gene List for list of 59 genes for copy number alterations.

TruSight Oncology 500 ctDNA includes 523 genes. Of these genes, the panel covers all exons for 522.

See the Denature and Dilute libraries guide for your sequencing system.

For a list of compatible sequencing systems and multiplexing compatibility, see the TruSight Oncology 500 ctDNA compatibility page.

We recommend quantifying cfDNA before starting the assay using a capillary electrophoresis-based method, such as the Fragment Analyzer (Agilent 5300 system with High Sensitivity Large Fragment Analysis Kit) or TapeStation (Agilent 4200 TapeStation System with Agilent Cell-free DNA Screen Tape Assay).

The Bioanalyzer is not recommended due to the peak quantification method used (based on both lower and upper marker). Quantification will be based on size which only includes the cfDNA fraction (75-250 bp on Fragment Analyzer and 125-300 bp on TapeStation) to ensure optimal input into the assay.

Customers can use their own pipeline. However, we do not provide resources to facilitate this.

TruSight Oncology 500 ctDNA enrichment probes were designed to tile across both exonic and selective intronic regions to capture potential breakpoints. Some intronic regions are difficult to adequately cover as they can be very large and would require probe coverage that would dramatically increase panel size and reduce throughput. Therefore, only select gene fusions will be reported from the TruSight Oncology 500 ctDNA assay.

Approximately 18-22 hours for S4 runs with 24 libraries and approximately 9-12 hours for S2 runs with 8 libraries.

No, the algorithm settings cannot be changed.

The baseline used for background subtraction of noise was regenerated with cfDNA samples to account for differences in the intended sample types. Additionally, variant calling sensitivity thresholds were modified to enable detection/calling of lower allele frequency variants.

Only one run can be analyzed at a time on a DRAGEN server.

The TruSight Oncology 500 ctDNA analysis workflow uses an off-instrument analysis software run on the DRAGEN server that was developed to analyze data produced by the TruSight Oncology 500 ctDNA assay. This analysis software generates sample outputs which include high level sample metrics, variants detected, TMB (tumor mutational burden) and MSI (microsatellite instability) scores.

A BaseSpace Sequence Hub evaluation app is also available. This application is intended for assessment use only and Illumina has no obligation to provide technical support for this App. Access will be limited to 30 days.

For a list of compatible analysis software, see the TruSight Oncology 500 ctDNA compatibility page.