Using cell lines or contrived samples for validation or as positive controls may result in inconsistent or unusual results depending on how the samples were constructed. Illumina recommends using commercial controls only for the variant class they are intended for. For example, if the sample is contrived for CNVs but not explicitly for SNV, MSI, TMB, or fusions, we would not recommend using this sample for validation or as a positive control for SNV, MSI, TMB, or fusions.
Illumina has provided demo data of some common commercial controls on BaseSpace Sequence Hub on various flow cells:
TSO 500 ctDNA v2: (NovaSeq X)
TSO 500 ctDNA v2: (NovaSeq X)
TSO 500 ctDNA v2: (NovaSeq 6000)
TSO 500 v1: NextSeq
Seraseq ctDNA Reference Material v2 (multiple VAF)
Fusions
False negatives due to a known issue on high efficiently libraries.
Resolved 2.6.1+
Seraseq ctDNA Complete Mutation Mix AF0.5%; Material Number 0710-0531
Small Variants
Low recall on Deletion chr17 41245586 CT>C due to a high noise region adjacent to a homoploymer. Under review to be addressed in the future SW versions.
All
Seraseq ctDNA Complete Mutation Mix AF0.5%; Material Number 0710-0531
TMB
Not recommended for TMB validation due to many small variants having VAF close to the TMB limit of detection. Small changes in preparation and dilution result in large changes in TMB scores (behavior not observed in clinical samples).
All
Twist cfDNA Pan-Cancer Reference Standard v2
Fusions
False negatives due to a known issue on high efficiently libraries.
Seraseq® FFPE BRCA1/2 LGR Reference Material Material Number 0730-0564
BRCA LR
Not recommended for LR validation due to 1) high level of noise 2) method of contriving resulting in some artifacts. We have found Seraseq BRCA 1/2 Exon Deletions DNA Mix Material Number 0730-0570 to work well.
All
Resolved 2.6.1+
The following sections describe performance testing methods.
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 TruSightâ„¢ Oncology 500 Analysis Software and DRAGEN TruSightâ„¢ 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 for more details.
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.
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
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
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.
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
Variants in intron-exon junctions (2 bp – 10 bp into introns)