Abstract
Challenges with distinguishing circulating tumor DNA (ctDNA) from next-generation sequencing (NGS) artifacts limits variant searches to established solid tumor mutations. Here we show early and random PCR errors are a principal source of NGS noise that persist despite duplex molecular barcoding, removal of artifacts due to clonal hematopoiesis of indeterminate potential, and suppression of patterned errors. We also demonstrate sample duplicates are necessary to eliminate the stochastic noise associated with NGS. Integration of sample duplicates into NGS analytics may broaden ctDNA applications by removing NGS-related errors that confound identification of true very low frequency variants during searches for ctDNA without a priori knowledge of specific mutations to target.
Highlights
Cell-free DNA is an emerging molecular tool for non-invasive diagnosis and disease monitoring in a variety of human cancers [1]
We found that the overall ligation efficiency of the duplex adapter was higher compared to the singleton adapter (~74% vs. ~58%, respectively; S3 Fig)
This observation may be due, at least in part, to the predominantly single-stranded character of the singleton adapter structure (~56 nt unpaired compared to ~19 nt unpaired in the duplex adapter; S2a and S1a Figs, respectively) which may interfere with ligation on account of secondary structure formation, reduced affinity to ligase, and/or increased adapter dimer formation [16]
Summary
Cell-free DNA is an emerging molecular tool for non-invasive diagnosis and disease monitoring in a variety of human cancers [1]. DNA released into the blood without a protective membrane is known as circulating cell-free DNA (ccfDNA). Mutations specific to a cancer are represented in the portion of ccfDNA derived from tumor cells and has been termed circulating tumor DNA (ctDNA). The ccfDNA pool is overwhelmingly composed of normal DNA originating from healthy cells [2]. The proportion of ctDNA variants within this pool varies widely based on disease severity [3, 4]. CtDNA applications have been largely constrained to detection of known tumor variants [8]
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