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Considerations and quality controls when analyzing cell-free tumor DNA

Journal article
Authors Gustav Johansson
Daniel Andersson
Stefan Filges
Junrui Li
Andreas Muth
T. E. Godfrey
Anders Ståhlberg
Published in Biomolecular Detection and Quantification
Volume 17
ISSN 2214-7535
Publication year 2019
Published at Institute of Clinical Sciences, Department of Surgery
Institute of Biomedicine, Department of Pathology
Sahlgrenska Cancer Center
Language en
Links dx.doi.org/10.1016/j.bdq.2018.12.00...
Keywords Cell-free DNA, Cell-free tumor DNA, DNA barcoding, Liquid biopsy, Mutation detection, Plasma, Quality controls, Sample preprocessing, SiMSen-Seq, cell DNA, circulating tumor DNA, genomic DNA, Article, bioinformatics, cancer diagnosis, controlled study, DNA contamination, DNA extraction, DNA fragmentation, DNA isolation, DNA sequence, gastrointestinal stromal tumor, gene library, gene sequence, genetic analysis, human, human cell, limit of detection, next generation sequencing, polymerase chain reaction, priority journal, quality control, real time polymerase chain reaction, sensitivity and specificity, T-47D cell line
Subject categories Cancer and Oncology

Abstract

Circulating cell-free tumor DNA (ctDNA) is a promising biomarker in cancer. Ultrasensitive technologies enable detection of low (< 0.1%) mutant allele frequencies, a pre-requisite to fully utilize the potential of ctDNA in cancer diagnostics. In addition, the entire liquid biopsy workflow needs to be carefully optimized to enable reliable ctDNA analysis. Here, we discuss important considerations for ctDNA detection in plasma. We show how each experimental step can easily be evaluated using simple quantitative PCR assays, including detection of cellular DNA contamination and PCR inhibition. Furthermore, ctDNA assay performance is also demonstrated to be affected by both DNA fragmentation and target sequence. Finally, we show that quantitative PCR is useful to estimate the required sequencing depth and to monitor DNA losses throughout the workflow. The use of quality control assays enables the development of robust and standardized workflows that facilitate the implementation of ctDNA analysis into clinical routine. © 2019 The Authors

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