# Contrived and Cell Line Sample Considerations

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.

### Demo Data for Commercial Controls

Illumina has provided demo data of some common commercial controls on BaseSpace Sequence Hub on various flow cells:

* TSO 500 ctDNA v2: [1.5B](https://ilmn-tsotest.basespace.illumina.com/s/RU9IFRUTOckR) (NovaSeq X)
* TSO 500 ctDNA v2: [10B](https://ilmn-tsotest.basespace.illumina.com/s/RU9IFRUTOckR) (NovaSeq X)
* TSO 500 ctDNA v2: [S2](https://basespace.illumina.com/s/v0x6nsUx9H3l) (NovaSeq 6000)
* TSO 500 v1: NextSeq [550](https://basespace.illumina.com/s/tVsV7XGADNUg)

### **Known Limitations with Commercial Controls**

#### **TSO 500 ctDNA v2**

<table><thead><tr><th width="180.40625">Product Name</th><th width="146.375">Variant Class</th><th width="292.859375">Description</th><th>SW Version Impacted</th></tr></thead><tbody><tr><td>Seraseq ctDNA Complete Mutation Mix AF0.5%; Material Number 0710-0531</td><td>Small Variants</td><td>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.</td><td>All</td></tr><tr><td>Seraseq ctDNA Complete Mutation Mix AF0.5%; Material Number 0710-0531</td><td>TMB</td><td>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).</td><td>All</td></tr><tr><td>Twist cfDNA Pan-Cancer Reference Standard v2</td><td>Fusions</td><td>False negatives due to a known issue on high efficiently libraries.</td><td>Resolved 2.6.1+</td></tr><tr><td>Seraseq ctDNA Reference Material v2 (multiple VAF)</td><td>Fusions</td><td>False negatives due to a known issue on high efficiently libraries.</td><td>Resolved 2.6.1+</td></tr></tbody></table>

#### **TSO 500 v1 and v2**

<table><thead><tr><th width="181.109375">Product Name</th><th width="150.11328125">Variant Class</th><th width="292.796875">Description</th><th>SW Version Impacted</th></tr></thead><tbody><tr><td>Seraseq® FFPE BRCA1/2 LGR Reference Material Material Number 0730-0564</td><td>BRCA LR</td><td>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.</td><td>All</td></tr></tbody></table>