> For the complete documentation index, see [llms.txt](https://help.tso500software.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://help.tso500software.illumina.com/performance-testing/commercial-control-use-with-tso-500-ctdna.md). # Commercial Control Use with TSO 500 ctDNA ### Best Practices * Use 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, or fusions, we would not recommend using this sample for validation or as a positive control for SNV or fusions. * Plan the studies taking into account that the actual VAFs in the control do not match exactly the targeted VAFs and can vary from lot to lot based on the manufacture’s process for creating the controls. The recall could be lower for variants with VAF below the assay’s LOD. Include dilutions above LOD, at LOD, and below LOD. {% hint style="warning" %} Sensitivity 95% for variants at 0.5% VAF means that a variant with 0.5% VAF (Limit of detection (LOD)) can be detected at least 95% of the time, for example, in 95 out of 100 replicates. If VAF is lower, the sensitivity is lower and the variant is detected less consistently, less than 95% of the time. {% endhint %} * Consult the manufacturer’s CoA for particular lots to determine their stated VAF for the targets in question as well as also understand the limitations and variability in the manufacturer’s method of quantitation of the targets (such as ddPCR or other method). Examples: [technical report](https://www.seracare.com/globalassets/seracare-resources/tpr-0710-0531-seraseq-ctdna-complete-mm-af-0.5-10636957.pdf) for Seraseq ctDNA Complete Mutation Mix AF0.5%, [Certificate of Analysis](https://www.seracare.com/globalassets/seracare-resources/coa-0710-3099-seraseq-ctdna-mutation-mix-v4-af-0.5-10687645.pdf) for Seraseq® ctDNA Mutation Mix v4 AF0.5%. * Confirm assay’s LOD. LOD values for each variant class are provided in the [TSO 500 ctDNA v2 data sheet](https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/trusight-oncology-500-ctdna-v2-m-gl-02196/tso500-ctdna-v2-data-sheet-m-gl-02196.pdf). * Use a pilot study to identify observed VAFs with TSO 500 ctDNA v2 assays before planning a larger study. VAFs determined by TSO 500 ctDNA v2 assay can be found in the “Allele Frequency” column in the Combined Variant Output file. * Understand the variability inherent in the control sample and that some variability will also exist due to the library preparation. * Run multiple replicates of the control, especially when near LoD, to account for sample to sample and run to run variability. * Refer to the [Known Limitations with Commercial Controls](#known-limitations-with-commercial-controls) section for known limitations and deviations in performance, including due to the lot-to-lot variations. * Use assay performance materials provided by Illumina: * [TSO 500 ctDNA v2 data sheet](https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/trusight-oncology-500-ctdna-v2-m-gl-02196/tso500-ctdna-v2-data-sheet-m-gl-02196.pdf) * Application note on [Using lower input amounts with TruSight™ Oncology ctDNA v2](https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/trusight-oncology-500-ctdna-v2-input-titration-tech-note-m-gl-02229/tso-500-ctdna-v2-input-titration-tech-note-m-gl-02229.pdf) * Application notes demonstrating performance for select instruments: [NextSeq 2000 System](https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/tso-500-ctdna-v2-nextseq-2000-tech-note-m-gl-03798/tso-500-ctdna-v2-nextseq-2000-tech-note-m-gl-03798.pdf), [NovaSeq X System](https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/tso500-ctdna-v2-novaseq-6000-x-concordance-tech-note-m-gl-03016/tso500-ctdna-v2-novaseq-6000-x-concordance-tech-note-m-gl-03016.pdf) (for [NovaSeq 6000](https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/trusight-oncology-500-ctdna-v2-m-gl-02196/tso500-ctdna-v2-data-sheet-m-gl-02196.pdf), see [TSO 500 ctDNA v2 data sheet](https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/trusight-oncology-500-ctdna-v2-m-gl-02196/tso500-ctdna-v2-data-sheet-m-gl-02196.pdf)). * Section [TSO 500 ctDNA v2 Performance for Seraseq ctDNA Complete Mutation Mix 0.5%AF](#tso-500-ctdna-v2-performance-for-seraseq-ctdna-complete-mutation-mix-0.5-af) * [Known Limitations with Commercial Controls](/performance-testing/known-limitations-with-commercial-controls.md) section * [Demo Data](/performance-testing/demo-data.md) section ### VAF in Seraseq ctDNA Complete Mutation Mix Factors that impact variant VAF in Seraseq ctDNA Complete Mutation Mix products: * The mutation mix products are formulated so that the target VAFs fall within a specified range [as measured by dPCR](https://digital.seracare.com/cg-faq?_gl=1*phm459*_gcl_au*MTE0MjgxNzQyMC4xNzY5MDM1NzIw); for example, the Seraseq ctDNA Complete Mutation Mix AF0.5% includes variants with dPCR‑measured VAFs between approximately 0.400% and 0.625%. For certain variant classes - such as indels and fusions - a VAF near 0.4% is below the LOD for TSO 500 ctDNA v2, which may result in less consistent detection. * VAFs are measured by dPCR before DNA fragmentation. Subsequent fragmentation can alter the observed VAF. * Technical reports, [see example](https://www.seracare.com/globalassets/seracare-resources/tpr-0710-0531-seraseq-ctdna-complete-mm-af-0.5-10636957.pdf), provide VAFs determined by the Archer® Reveal ctDNA™ 28 kit. VAFs measured by dPCR can differ from VAFs determined by NGS, as well as change after fragmentation In addition, VAFs are assay‑dependent, and VAFs observed using the TSO 500 ctDNA v2 assay may differ from those determined using the Archer® Reveal ctDNA™ 28 assay. * Seraseq ctDNA Complete Mutation Mix products may show some batch‑to‑batch variation in individual variant VAFs while still meeting quality control specifications established by dPCR. Illumina aggregates customer reports regarding variants that are not detected due to lower‑than‑expected VAFs in certain batches and can provide additional information upon request. The newly released next‑generation [Seraseq® ctDNA v4 Reference Materials](https://www.seracare.com/globalassets/seracare-resources/ps-0710-3097.3099.3100.3101-seraseq-ctdna-mutation-mix-v4.pdf) offer several advantages for TSO 500 ctDNA studies: * The VAF is determined using TSO 500 ctDNA instead of the Archer® Reveal ctDNA™ 28 assay. See [example of Certificate of Analysis](https://www.seracare.com/globalassets/seracare-resources/coa-0710-3099-seraseq-ctdna-mutation-mix-v4-af-0.5-10687645.pdf) for Seraseq® ctDNA Mutation Mix v4 AF0.5%. * Improved fragmentation process to lower background noise >10x compared to Seraseq ctDNA Complete products * Expanded number of variants including SNVs, deletions, insertions, INDELs, CNVs, and translocations for the broader coverage. ### MSI testing When designing analytical performance studies for MSI (also referred to as bMSI), Illumina recommends using as a truth set MSI status for real cancer samples or cell lines, established using an orthogonal method, for example, PCR or tissue NGS. Using contrived reference samples with limited number of microsatellite sites is not recommended due to the design of the MSI algorithm, specifically, the use of > 2,300 microsatellite sites with 6-7 bp size. See [MSI algorithm page](/dragen-tso-500-ctdna-guides/dragen-tso-500-ctdna-v2.6/analysis-methods/msi.md) for more details. ### Performance for Seraseq ctDNA Complete Mutation Mix 0.5%AF The table below summarizes the variant calling results by the TSO 500 ctDNA v2 assay for Seraseq ctDNA Complete Mutation Mix AF0.5% (Material Number 0710-0531, Lot 10624818) across 9 customer sites that participated in the early access program. The call rate, and the average observed VAF, depth and read counts are generated across 9 sites and 19 replicates. | Gene | AA Change | Variant type | Call Rate | Expected VAF, %\* | Observed VAF, % | Observed Depth | # Variant Allele Reads | | --------- | ------------------------- | ------------- | --------- | ---------------------- | ---------------- | -------------- | ---------------------- | | AKT1 | p.E17K | SNV | 100% | 0.51 | 0.32 ± 0.09 | 5424 ± 1745 | 17 ± 6 | | ALK | p.F1174L | SNV | 100% | 0.54 | 0.57 ± 0.12 | 3115 ± 832 | 18 ± 6 | | ALK | p.G1202R | SNV | 100% | 0.48 | 0.45 ± 0.12 | 2774 ± 792 | 12 ± 4 | | BRAF | p.V600E | SNV | 100% | 0.46 | 0.5 ± 0.14 | 4405 ± 1294 | 22 ± 7 | | BRCA1 | p.K654fs\*47 | Del | 58% | 0.46 | 0.5 ± 0.09 | 4018 ± 1254 | 20 ± 7 | | BRCA2 | p.R2645fs\*3 | Del | 100% | 0.59 | 0.57 ± 0.11 | 4400 ± 1383 | 25 ± 7 | | EGFR | p.E746\_A750 del ELREA | Del | 100% | 0.64 | 0.51 ± 0.14 | 2663 ± 628 | 14 ± 5 | | EGFR | p.L747\_P753>S | Del | 100% | 0.50 | 0.51 ± 0.12 | 2649 ± 563 | 14 ± 5 | | EGFR | p.L858R | SNV | 100% | 0.53 | 0.49 ± 0.16 | 3605 ± 983 | 18 ± 8 | | EGFR | p.S752\_I759 del SPKANKEI | Del | 100% | 0.50 | 0.54 ± 0.1 | 3932 ± 1257 | 22 ± 9 | | EGFR | p.T790M | SNV | 100% | 0.42 | 0.48 ± 0.12 | 4558 ± 1347 | 22 ± 7 | | ERBB2 | p.A775\_G776 ins YVMA | Ins | 100% | 0.56 | 0.53 ± 0.15 | 4043 ± 1284 | 22 ± 9 | | KIT | p.D816V | SNV | 100% | 0.56 | 0.46 ± 0.12 | 3717 ± 1205 | 17 ± 7 | | KRAS | p.G12C | SNV | 100% | 0.54 | 0.5 ± 0.13 | 2920 ± 604 | 15 ± 5 | | KRAS | p.G12D | SNV | 100% | 0.55 | 0.68 ± 0.18 | 2201 ± 513 | 15 ± 5 | | KRAS | p.Q61H | SNV | 100% | 0.55 | 0.4 ± 0.12 | 3401 ± 975 | 14 ± 6 | | NRAS | p.Q61R | SNV | 100% | 0.56 | 0.37 ± 0.11 | 4953 ± 1514 | 18 ± 7 | | PIK3CA | p.H1047R | SNV | 100% | 0.56 | 0.49 ± 0.12 | 4448 ± 1399 | 21 ± 6 | | PIK3CA | p.N1068fs\*4 | Ins | 97% | 0.52 | 0.49 ± 0.14 | 3108 ± 766 | 15 ± 5 | | ERBB2 | | Amplification | 100% | 2.56 copies or 1.28 FC | 1.31 ± 0.01 (FC) | | | | MET | | Amplification | 100% | 2.41 copies or 1.21 FC | 1.41 ± 0.02 (FC) | | | | MYC | | Amplification | 92% | 2.37 copies or 1.19 FC | 1.2 ± 0.01 (FC) | | | | CD74-ROS1 | | Translocation | 89% | 0.53 | 0.41 ± 0.16 | 4291 ± 1153 | 18 ± 8 | | EML4-ALK | | Translocation | 100% | 0.49 | 0.34 ± 0.12 | 4261 ± 1070 | 14 ± 5 | | NCOA4-RET | | Translocation | 83% | 0.56 | 0.22 ± 0.07 | 3740 ± 1034 | 8 ± 3 | \*Expected VAF (%) provided as detected by dPCR for Seraseq ctDNA Complete Mutation Mix AF0.5% (MN 0710-0531, lot 10624818), [Technical Report.](https://www.seracare.com/globalassets/seracare-resources/tpr-0710-0531-seraseq-ctdna-complete-mm-af-0.5-10624818.pdf) --- # Agent Instructions This documentation is published with GitBook. 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