1) University of Gothenburg, Sweden;
2) Sahlgrenska University Hospital, Sweden;
Liquid biopsy and detection of tumor associated-mutations in cell-free circulating DNA (ctDNA) often requires the ability to identify single nucleotide variants at allele frequencies below 0.1%. Standard sequencing protocols cannot achieve this level of sensitivity due to background noise from DNA damage, polymerase induced errors. Addition of unique molecular identifiers allows identification and removal of errors responsible for this background noise. 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. We show that quantitative PCR is useful to estimate the required sequencing depth and to monitor DNA losses throughout the workflow. Theoretically, high fidelity enzymes will reduce error rates in barcoded NGS but this has not been thoroughly explored. We evaluated the impact of polymerase fidelity on the magnitude of error reduction at different steps of barcoded NGS library construction. The use of quality control assays enables the development of robust and standardized workflows that facilitate the implementation of ctDNA analysis into clinical routine.
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