Performance Testing

The following sections describe performance testing methods.

Analytical Performance Testing

Illumina tests the analytical performance of variant calling 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.

This version of the software includes results generated by features tested in silico and by beta features. Beta features have not been fully evaluated for performance, see Beta Features.

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, variants of interest are extracted 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.

DRAGEN TruSight Oncology 500 Analysis Software includes the following features that were tested in silico for both TruSight Oncology 500 and TruSight Oncology 500 HT:

• 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)

In addition, the following features were tested in silico for TruSight Oncology 500 HT:

• Exon-level CNVs in BRCA1 and BRCA2

Beta Features

This version of DRAGEN TruSight Oncology 500 Analysis Software includes beta features which 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 is 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. Illumina will continue to evaluate beta features with intent to fully release upon completion of verification for each feature.

This version includes the following beta features that may be used with the TruSight Oncology 500 HRD Assay:

• Tumor fraction (beta)

• Ploidy (beta)

• Absolute copy numbers (ACN) (beta)

• Gene-level loss of heterozygosity (LOH) events (beta)

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.