Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications

Arbeithuber, Barbara; Makova, Kateryna D.; Tiemann‐Boege, Irene

doi:10.1093/dnares/dsw038

Cited by 52 publications

(63 citation statements)

References 61 publications

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“…Our data show that rare mutation frequencies are significantly lower in DCS analysis in comparison to NGS and SSCS analyses, suggesting that large number of rare mutations detected by NGS and SSCS are mostly artifacts (Figure 3A). Particularly, C>A/G>T transversions, which have been previously reported to be a predominant result of DNA oxidation [11,16], showed the greatest decrease in frequencies with DCS analyses of Duplex Sequencing (Figure 3J,K). These findings validate that DCS can identify and correct artifacts and be applied for accurately detecting rarely occurring mutations.…”

Section: Discussionmentioning

confidence: 72%

“…In addition, we identified the heat-induced artifactual mutations. Although Duplex Sequencing has been used in a previous study to show an increased level of mutation frequency of small selected regions of nuclear genome in DNA incubated at 65°C [11], the same temperature used in this study, exact identities of the heat-induced artifactual mutations have not been presented. Moreover, while types of artifactual mutations have been previously examined, influences of neighboring nucleotide base context on artifactual mutations have not been investigated.…”

Section: Discussionmentioning

confidence: 99%

“…It is also a by-product of normal cellular metabolism [20]. The formation of 8-oxoguanine, particularly 8-oxo-dG has been reported to cause a high level of C>A/G>T mutations [11,16,20–23], whereas deamination of cytosine to uracil is known to produce high levels of C>T/G>A mutations [11,19]. In one study, NGS analysis was done on tumors and matching normal tissues of melanoma and an enzyme-linked immunosorbent assay (ELISA) for 8-Oxo-dG found that the C C G>C A G context have a high potential for being a target of DNA oxidation [22].…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, analyses were done with multiple independent DNA library preparation experiments of an identical biological sample to evaluate the detection consistency, reproducibility, and validity of each sequencing method. Heating samples, a common practice in preparing DNA for molecular biology experiments, can introduce such artifactual mutations [11]. Herein, we present heat-induced artifactual mutations identified using Duplex Sequencing and specific nucleotide contexts that contribute to a high level of heat-induced artifactual mutations.…”

Section: Introductionmentioning

confidence: 99%

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Detection of low-frequency mutations and removal of heat-induced artifactual mutations using Duplex Sequencing

Ahn

Lee

2018

Preprint

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We present a genome-wide comparative and comprehensive analysis of three different 12 sequencing methods (conventional next generation sequencing (NGS), tag-based single strand 13 sequencing (eg. SSCS), and Duplex Sequencing for investigating mitochondrial mutations in 14 human breast epithelial cells. Duplex Sequencing produces a single strand consensus sequence 15 (SSCS) and a duplex consensus sequence (DCS) analysis, respectively. Our study validates that 16 although high-frequency mutations are detectable by all the three sequencing methods with the 17 similar accuracy and reproducibility, rare (low-frequency) mutations are not accurately detectable 18 by NGS and SSCS. Even with conservative bioinformatical modification to overcome the high error 19 rate of NGS, the NGS frequency of rare mutations is 7.0x10 -4 . The frequency is reduced to 1.3x10 -4 20 with SSCS and is further reduced to 1.0x10 -5 using DCS. Rare mutation context spectra obtained 21 from NGS significantly vary across independent experiments, and it is not possible to identify a 22 dominant mutation context. In contrast, rare mutation context spectra are consistently similar in all 23 independent DCS experiments. We have systematically identified heat-induced artifactual 24 mutations and corrected the artifacts using Duplex Sequencing. All of these artifacts are 25 stochastically occurring rare mutations. C>A/G>T, a signature of oxidative damage, is the most 26 increased (170-fold) heat-induced artifactual mutation type. Our results strongly support the claim 27 that Duplex Sequencing accurately detects low-frequency mutations and identifies and corrects 28 artifactual mutations introduced by heating during DNA preparation. 29 Sequence (SSCS), Next-Generation Sequencing (NGS), sequencing error, rare mutations, oxidative 31 DNA damage, heat-induced mutations, mitochondrial DNA, human breast cells 32 33 1. Introduction 34 Next-generation sequencing (NGS) has rapidly transformed entire areas of basic research and 35 therapeutic applications by making large scale genomic studies feasible through reduced cost and 36faster turnaround time [1,2]. NGS has been extensively used to study clonal (high-frequency) 37 mutations, but not subclonal (low-frequency) mutations. A major impediment in investigating 38 subclonal (low-frequency) mutations is that conventional NGS methods have high error rates (10 -2 to 39 10 -3 ), which obscure true mutations that occur less frequently than errors [3,4]. These subclonal 40 mutations may account for the genetic heterogeneity of tumors and tumor recurrence, as well as 41 provide a reservoir for the rapid development of resistance to chemotherapy [5]. 42 Conventional sequencing technologies sequence only a single strand of DNA. In contrast, 43 Duplex Sequencing examines both strands of DNA and scores mutations only if they are present on 44 both strands of the same DNA molecule as complementary substitutions. This significantly reduces 45 sequencing error rates to < 5 x 10 -8 [6-9]. In the first report of Duplex Seq...

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Section: Discussionmentioning

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Detection of low-frequency mutations and removal of heat-induced artifactual mutations using Duplex Sequencing

Ahn

Lee

2018

Preprint

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“…Thus, COLD-ddPCR has been demonstrated to easily and rapidly detect multiple mutations and identify unknown variants in the target sequences. In 2016, Arbeithuber et al [59] demonstrated that DNA repair enzymes could reduce the artificial mutations caused by long heat incubation and the subsequent error in ultrasensitive technologies such as ddPCR. This finding provides direction in optimizing dPCR performance.…”

Section: Perspectivesmentioning

confidence: 99%

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

Yu¹,

Shen²,

Jiang³

et al. 2017

Cell Physiol Biochem

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Gene mutation has been considered a research hotspot, and the rapid development of biomedicine has enabled significant advances in the evaluation of gene mutations. The advent of digital polymerase chain reaction (dPCR) elevates the detection of gene mutations to unprecedented levels of precision, especially in cancer-associated genes. dPCR has been utilized in the detection of tumor markers in cell-free DNA (cfDNA) samples from patients with different types of cancer in samples such as plasma, cerebrospinal fluid, urine and sputum, which confers significant value for dPCR in both clinical applications and basic research. Moreover, dPCR is extensively used in detecting pathogen mutations related to typical features of infectious diseases (e.g., drug resistance) and mutation status of heteroplasmic mitochondrial DNA, which determines the manifestation and progression of mtDNA-related diseases, as well as allows for the prenatal diagnosis of monogenic diseases and the assessment of the genome editing effects. Compared with real-time PCR (qPCR) and sequencing, the higher sensitivity and accuracy of dPCR indicates a great advantage in the detection of rare mutation. As a new technique, dPCR has some limitations, such as the necessity of highly allele-specific probes and a large sample volume. In this review, we summarize the application of dPCR in the detection of human disease-associated gene mutations.

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Bioinformatics Analysis for Cell-Free Tumor DNA Sequencing Data

Chen¹,

Liu²,

Zhou³

2018

Methods in Molecular Biology

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As a major biomarker of liquid biopsy, cell-free tumor DNA (ctDNA), which can be extracted from blood, urine, or other circulating liquids, is able to provide comprehensive genetic information of tumor and better overcome the tumor heterogeneity problem comparing to tissue biopsy. Developed in recent years, next-generation sequencing (NGS) is a widely used technology for analyzing ctDNA. Although the technologies of processing ctDNA samples are mature, the task to detect low mutated allele frequency (MAF) variations from noisy sequencing data remains challenging. In this chapter, the authors will first explain the difficulties of analyzing ctDNA sequencing data, review related technologies, and then present some novel bioinformatics methods for analyzing ctDNA NGS data in better ways.

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Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications

Cited by 52 publications

References 61 publications

Detection of low-frequency mutations and removal of heat-induced artifactual mutations using Duplex Sequencing

Detection of low-frequency mutations and removal of heat-induced artifactual mutations using Duplex Sequencing

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

Bioinformatics Analysis for Cell-Free Tumor DNA Sequencing Data

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