Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing

Ståhlberg, Anders; Krzyzanowski, Paul M.; Jackson, Jennifer; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E.

doi:10.1093/nar/gkw224

Cited by 114 publications

(116 citation statements)

References 26 publications

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“…Raw FastQ files were subsequently processed as described[28] using Debarcer Version 0.3.0 (https://github.com/oicr-gsi/debarcer). Sequence reads with the same barcode were grouped into families for each amplicon.…”

Section: Methodsmentioning

confidence: 99%

Recurrent promoter mutations in melanoma are defined by an extended context-specific mutational signature

et al. 2017

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Sequencing of whole tumor genomes holds the promise of revealing functional somatic regulatory mutations, such as those described in the TERT promoter. Recurrent promoter mutations have been identified in many additional genes and appear to be particularly common in melanoma, but convincing functional data such as influence on gene expression has been more elusive. Here, we show that frequently recurring promoter mutations in melanoma occur almost exclusively at cytosines flanked by a distinct sequence signature, TTCCG, with TERT as a notable exception. In active, but not inactive, promoters, mutation frequencies for cytosines at the 5' end of this ETS-like motif were considerably higher than expected based on a UV trinucleotide mutational signature. Additional analyses solidify this pattern as an extended context-specific mutational signature that mediates an exceptional position-specific vulnerability to UV mutagenesis, arguing against positive selection. We further use ultra-sensitive amplicon sequencing to demonstrate that cell cultures exposed to UV light quickly develop subclonal mutations specifically in affected positions. Our findings have implications for the interpretation of somatic mutations in regulatory regions, and underscore the importance of genomic context and extended sequence patterns to accurately describe mutational signatures in cancer. Author summaryCancer is caused by somatic mutations that alter cell behavior. While such mutations typically occur in protein-coding genes, recent studies describe individual positions in gene regulatory regions (promoters) that are recurrently mutated in many independent tumors. This suggests that positive selection could be acting on these non-coding mutations, and that they may contribute to carcinogenesis. However, proper interpretation of recurrent mutations requires a detailed understanding of how such mutations arise in the absence of selection pressures, referred to as mutational heterogeneity. In this paper, we describe a distinct sequence signature that characterizes nearly all highly recurrent promoter mutations in melanoma. Additional analyses support that this sequence mediates an exceptional local vulnerability to UV-induced mutagenesis, explaining why mutations are frequently observed in these positions. Importantly, cultured cells exposed to UV light quickly developed mutations specifically in the expected sites. Our results have important implications for the interpretation of recurrent somatic mutation patterns in non-coding DNA.

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Section: Methodsmentioning

confidence: 99%

Recurrent promoter mutations in melanoma are defined by an extended context-specific mutational signature

et al. 2017

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“…DNA from primary tumor samples and matched normal DNA from buffy coats were sequenced using whole‐exome or targeted next‐generation sequencing panels and mutations were identified using previously published methods . The Mutect2 and Strelka algorithms were used to generate variant call files consisting of several somatic mutations .…”

Section: Methodsmentioning

confidence: 99%

“…Reads that result from polymerase and sequencing error can be identified and discounted, as they occur only in a subset of the raw reads from an individual barcode family. Consequently, barcoding greatly reduces background sequencing noise and enables rare variant allele detection …”

Section: Methodsmentioning

confidence: 99%

“…Despite this interest, circulating tumor DNA remains challenging to detect and to quantify as it represents a small fraction of total plasma circulating cell‐free DNA. Novel PCR‐based sequencing approaches have improved our ability to detect rare tumor mutations present in circulating tumor DNA against the total circulating cell‐free DNA background …”

Section: Introductionmentioning

confidence: 99%

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Plasma circulating tumor DNA as a potential tool for disease monitoring in head and neck cancer

et al. 2018

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Background Recommendations for perioperative therapy in head and neck cancer are not explicit and recurrence occurs frequently. Circulating tumor DNA is an emerging cancer biomarker, but has not been extensively explored for detection of recurrence in head and neck cancer. Methods Patients diagnosed with head and neck squamous cell carcinoma were recruited into the study protocol. Tumors were sequenced to identify patient‐specific mutations. Mutations were then identified in plasma circulating tumor DNA from pre‐treatment blood samples and longitudinally during standard follow‐up. Circulating tumor DNA status during follow‐up was correlated to disease recurrence. Results Samples were taken from eight patients. Tumor mutations were verified in seven patients. Baseline circulating tumor DNA was positive in six patients. Recurrence occurred in four patients, two of whom had detectable circulating tumor DNA prior to recurrence. Conclusion Circulating tumor DNA is a potential tool for disease and recurrence monitoring following curative therapy in head and neck cancer, allowing for better prognostication, and/or modification of treatment strategies.

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“…Data were analyzed using Debarcer as described [12,13]. At least three reads with the same barcode (consensus 3) were required to form a valid barcode family.…”

Section: Methodsmentioning

confidence: 99%

Considerations and quality controls when analyzing cell-free tumor DNA

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et al. 2019

Biomolecular Detection and Quantification

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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.

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Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing

Cited by 114 publications

References 26 publications

Recurrent promoter mutations in melanoma are defined by an extended context-specific mutational signature

Recurrent promoter mutations in melanoma are defined by an extended context-specific mutational signature

Plasma circulating tumor DNA as a potential tool for disease monitoring in head and neck cancer

Considerations and quality controls when analyzing cell-free tumor DNA

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