Analytical Validation of qPCR-Based Multivariate Index Assays in a Clinical Laboratory: Practical Challenges and Limitations

Sesler, Cheryl L; Grigorenko, Elena

doi:10.1373/jalm.2017.025924

Cited by 3 publications

(2 citation statements)

References 21 publications

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“…We calculated analytical limit of detection (LOD) as previously reported [ 22 , 39 ]. We made dilution samples by spike the gDNA of FFPE PDAC tissues into NA12878 at the tumor fractions of 0.1%, 0.5%, 1%, 5% and 10%, and the mock spike-in samples were set as 0.…”

Section: Methodsmentioning

confidence: 99%

Cross-platform comparisons for targeted bisulfite sequencing of MGISEQ-2000 and NovaSeq6000

Sun

et al. 2023

Clin Epigenet

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Background An accurate and reproducible next-generation sequencing platform is essential to identify malignancy-related abnormal DNA methylation changes and translate them into clinical applications including cancer detection, prognosis, and surveillance. However, high-quality DNA methylation sequencing has been challenging because poor sequence diversity of the bisulfite-converted libraries severely impairs sequencing quality and yield. In this study, we tested MGISEQ-2000 Sequencer’s capability of DNA methylation sequencing with a published non-invasive pancreatic cancer detection assay, using NovaSeq6000 as the benchmark. Results We sequenced a series of synthetic cell-free DNA (cfDNA) samples with different tumor fractions and found MGISEQ-2000 yielded data with similar quality as NovaSeq6000. The methylation levels measured by MGISEQ-2000 demonstrated high consistency with NovaSeq6000. Moreover, MGISEQ-2000 showed a comparable analytic sensitivity with NovaSeq6000, suggesting its potential for clinical detection. As to evaluate the clinical performance of MGISEQ-2000, we sequenced 24 clinical samples and predicted the pathology of the samples with a clinical diagnosis model, PDACatch classifier. The clinical model performance of MGISEQ-2000’s data was highly consistent with that of NovaSeq6000’s data, with the area under the curve of 1. We also tested the model’s robustness with MGISEQ-2000’s data when reducing the sequencing depth. The results showed that MGISEQ-2000’s data showed matching robustness of the PDACatch classifier with NovaSeq6000’s data. Conclusions Taken together, MGISEQ-2000 demonstrated similar data quality, consistency of the methylation levels, comparable analytic sensitivity, and matching clinical performance, supporting its application in future non-invasive early cancer detection investigations by detecting distinct methylation patterns of cfDNAs.

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Section: Methodsmentioning

confidence: 99%

Cross-platform comparisons for targeted bisulfite sequencing of MGISEQ-2000 and NovaSeq6000

Sun

et al. 2023

Clin Epigenet

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“…Test performance was not affected by four potential interferents in blood, and cross-contamination and sample swaps were correctly detected in more than 1400 sample pairs. Analytical validation of multivariate assays, such as this MCED test, is challenging because the evaluation of test sensitivity using a single analyte-based limit of detection experiment is not directly applicable [23] due to the fact that the MCED test aggregates information from >100,000 genomic regions to assess cancer signal status. As an alternative, test sensitivity with respect to expected VAF of tumor mutations in a sample has been characterized.…”

Section: Plos Onementioning

confidence: 99%

Analytical validation of a multi-cancer early detection test with cancer signal origin using a cell-free DNA–based targeted methylation assay

et al. 2023

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The analytical validation is reported for a targeted methylation-based cell-free DNA multi-cancer early detection test designed to detect cancer and predict the cancer signal origin (tissue of origin). A machine-learning classifier was used to analyze the methylation patterns of >105 genomic targets covering >1 million methylation sites. Analytical sensitivity (limit of detection [95% probability]) was characterized with respect to tumor content by expected variant allele frequency and was determined to be 0.07%-0.17% across five tumor cases and 0.51% for the lymphoid neoplasm case. Test specificity was 99.3% (95% confidence interval, 98.6–99.7%). In the reproducibility and repeatability study, results were consistent in 31/34 (91.2%) pairs with cancer and 17/17 (100%) pairs without cancer; between runs, results were concordant for 129/133 (97.0%) cancer and 37/37 (100%) non-cancer sample pairs. Across 3- to 100-ng input levels of cell-free DNA, cancer was detected in 157/182 (86.3%) cancer samples but not in any of the 62 non-cancer samples. In input titration tests, cancer signal origin was correctly predicted in all tumor samples detected as cancer. No cross-contamination events were observed. No potential interferent (hemoglobin, bilirubin, triglycerides, genomic DNA) affected performance. The results of this analytical validation study support continued clinical development of a targeted methylation cell-free DNA multi-cancer early detection test.

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Evaluation of cell-free DNA approaches for multi-cancer early detection

Jamshidi¹,

Liu²,

Klein³

et al. 2022

Cancer Cell

132

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Analytical Validation of qPCR-Based Multivariate Index Assays in a Clinical Laboratory: Practical Challenges and Limitations

Cited by 3 publications

References 21 publications

Cross-platform comparisons for targeted bisulfite sequencing of MGISEQ-2000 and NovaSeq6000

Cross-platform comparisons for targeted bisulfite sequencing of MGISEQ-2000 and NovaSeq6000

Analytical validation of a multi-cancer early detection test with cancer signal origin using a cell-free DNA–based targeted methylation assay

Evaluation of cell-free DNA approaches for multi-cancer early detection

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