Commercial Control Use with TSO 500 ctDNA
The page aggregates materials to help plan analytical studies with commercial controls for TruSight Oncology ctDNA v2 assay.
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.
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.
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 for Seraseq ctDNA Complete Mutation Mix AF0.5%, Certificate of Analysis 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.
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 section for known limitations and deviations in performance, including due to the lot-to-lot variations.
Use assay performance materials provided by Illumina:
Application note on Using lower input amounts with TruSight™ Oncology ctDNA v2
Application notes demonstrating performance for select instruments: NextSeq 2000 System, NovaSeq X System (for NovaSeq 6000, see TSO 500 ctDNA v2 data sheet).
Demo Data 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; 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, 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 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 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 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.
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.
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