Performance Testing

The following sections describe performance testing methods.

Analytical Performance Testing

Illumina tests the analytical performance of variant calling for TruSight Oncology 500 and TruSight Oncology 500 ctDNA assays using an approach that covers the entire workflow including library preparation, sequencing, and secondary analysis. This approach is used to test a diverse selection of variants. When the variant calling pipeline is expanded to call a new variant class, this approach is always used.

Some versions of the DRAGEN TruSight™ Oncology 500 Analysis Software and DRAGEN TruSight™ Oncology 500 ctDNA Analysis Software include results generated by features tested in silico and by beta features. Beta features have not been fully evaluated for performance, see Beta Features for more details.

In Silico Testing Methods

Illumina uses in silico testing to the test the ability of the software to call an expanded scope of clinically relevant variants, including rare variants. In silico testing is used as a complementary method to analytical performance testing with wet lab step to expand the scope of testing. For example, while Illumina has analytically verified the performance of the software for calling complex variants in EGFR, the in silico testing approach characterizes the ability of the software to call complex variants in other genes.

For in silico testing of the DRAGEN TruSight Oncology 500 Analysis Software, variants of interest are compiled from public databases like COSMIC and ClinVar. Each variant is simulated at different VAF levels by, depending on the variant class, spiking in mutant reads into a normal FFPE background (for sequence variants) or by increasing or decreasing the coverage of exons in the normal FFPE sample (for CNVs, for example, exon-level CNVs). The simulated reads match the expected quality of typical FFPE samples, such as fragment length, error rate, and family size. After the simulation, the software processes samples with spiked-in variants and determines the results. This approach does not include library prep and sequencing of tumor FFPE samples that include the rare variants of interest. The software reports these variants, but analytical verification was not performed.

In silico tested features for DRAGEN TruSight Oncology 500 Analysis Software and DRAGEN TruSight Oncology 500 ctDNA Analysis Software are summarized in the table below.

Name	in Silico Tested Features
DRAGEN TruSight Oncology 500 Analysis Software for TruSight Oncology 500 and TruSight Oncology 500 HT (v2.1+)	Complex variants in genes beyond EGFR Insertions and deletions > 25 bp CNV amplifications CNV deletions Variants in intron-exon junctions (2 bp – 10 bp into introns)
DRAGEN TruSight Oncology 500 Analysis Software for TruSight Oncology 500 HT (v2.1+)	Exon-level CNVs in BRCA1 and BRCA2
DRAGEN TruSight Oncology 500 ctDNA Analysis Software (v2.1+)	Complex variants in genes beyond EGFR Insertions and deletions > 25 bp

Beta Features

DRAGEN TruSight Oncology 500 Analysis Software also includes beta features. The features have not been verified by Illumina due to limited access to samples or lack of an appropriate orthogonal method to perform testing, and, the use of in silico testing alone was not sufficient for verification purposes.

Customers are responsible for evaluating and demonstrating performance of any beta features they choose to implement. Beta features are indicated as such in the CombinedVariantOutput.tsv file produced by the DRAGEN TruSight Oncology 500 Analysis Software. Illumina will continue to evaluate beta features with intent to fully release upon completion of verification for each feature.

Beta features are summarized in the table below.

Beta feature results are included in the Combined Variant Output file and other files. However, disclaimers that the results are generated by beta features are only provided in the Combined Variant Output file.

Name	Beta Features
DRAGEN TruSight Oncology 500 Analysis Software (v2.6.2+) for : TruSight Oncology 500 + HRD Add-on TruSight Oncology 500 v2	Absolute copy numbers (ACN) Gene-level loss of heterozygosity (LOH) events
DRAGEN TruSight Oncology 500 Analysis Software (v2.5.2 - 2.6.1) for: TruSight Oncology 500 + HRD Add-on	Tumor fraction Ploidy Absolute copy numbers (ACN) Gene-level loss of heterozygosity (LOH) events