Detecting ultralow-frequency mutations by Duplex Sequencing

Kennedy, Scott R.; Schmitt, Michael; Fox, Edward J.; Kohrn, Brendan F.; Salk, Jesse J.; Ahn, Eun Hyun; Prindle, Marc J.; Kuong, Kawai J.; Shen, Jiang; Risques, Rosa Ana; Loeb, Lawrence A.

doi:10.1038/nprot.2014.170

Cited by 392 publications

(512 citation statements)

References 39 publications

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“…DNA was prepared into libraries for duplex sequencing according to published protocols (7,38). For peritoneal fluid libraries, up to 10 μg of genomic DNA (mean 5.4 μg) was used.…”

Section: Methodsmentioning

confidence: 99%

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Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues

Krimmel

Schmitt

Harrell

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

148

110

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Current sequencing methods are error-prone, which precludes the identification of low frequency mutations for early cancer detection. Duplex sequencing is a sequencing technology that decreases errors by scoring mutations present only in both strands of DNA. Our aim was to determine whether duplex sequencing could detect extremely rare cancer cells present in peritoneal fluid from women with highgrade serous ovarian carcinomas (HGSOCs). These aggressive cancers are typically diagnosed at a late stage and are characterized by TP53 mutations and peritoneal dissemination. We used duplex sequencing to analyze TP53 mutations in 17 peritoneal fluid samples from women with HGSOC and 20 from women without cancer. The tumor TP53 mutation was detected in 94% (16/17) of peritoneal fluid samples from women with HGSOC (frequency as low as 1 mutant per 24,736 normal genomes). Additionally, we detected extremely low frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37). These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cancers from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age-associated, low frequency TP53 mutations were also found in 100% of peripheral blood samples from 15 women with and without ovarian cancer (none with hematologic disorder). Our results demonstrate the ability of duplex sequencing to detect rare cancer cells and provide evidence of widespread, low frequency, age-associated somatic TP53 mutation in noncancerous tissue.TP53 mutations | ultra-deep sequencing | ovarian cancer | clonal hematopoiesis | premalignant mutations T he detection of tumor-specific mutations in clinically accessible samples has enormous potential to transform cancer diagnostics, monitoring, and screening. However, a major limitation is insufficiently accurate sequencing methods. Conventional nextgeneration sequencing (NGS) technologies have a high false positive error rate, which precludes reliable detection of mutations at frequencies <1/100 (1). "Molecular tagging" of single-stranded DNA decreases the rate of false mutations to less than 1 per 10,000 sequenced nucleotides and has been successfully applied to the detection of mutant cancer DNA in a variety of clinical samples (2-6). However, this false positive error rate limits the specificity of this method in challenging situations in which ultra-deep sequencing is needed to detect extremely low frequency mutant molecules (e.g., <1/10,000), as is the case of ovarian cancer DNA in Pap smears (5). Because true mutations are indistinguishable from artifacts, compromised specificity leads to lower sensitivity and overall low diagnostic accuracy. Duplex sequencing is an NGS technology that employs molecular tagging of both strands of DNA independently. True mutations are defined as mutations that are present at the same...

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“…DNA was prepared into libraries for duplex sequencing according to published protocols (7,38). For peritoneal fluid libraries, up to 10 μg of genomic DNA (mean 5.4 μg) was used.…”

Section: Methodsmentioning

confidence: 99%

“…Indexed libraries were pooled and sequenced on an Illumina HiSeq2500. Data processing was performed as previously described (38). Raw reads sharing a common molecular tag were grouped to form SSCS.…”

Section: Methodsmentioning

confidence: 99%

Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues

Krimmel

Schmitt

Harrell

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

148

110

View full text Add to dashboard Cite

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“…Because of its ability to remove sequencing artifacts resulting from DNA damage, as well as amplification and sequencing errors, duplex sequencing enables the detection of mutations as low as 1 in 10 8 nucleotides sequenced (17,18).…”

Section: Significancementioning

confidence: 99%

“…To validate the use of duplex sequencing to detect mutagen-induced mutations, normal fibroblasts (GM05659 and NHF-D) and keratinocytes were exposed to UVB or UVC, cultured for 5-7 d, and harvested. Genomic DNA was then isolated and subjected to a single round of duplex sequencing (18). Target genes were exonic regions of NRAS, UMPS, PIK3CA, EGFR, BRAF, KRAS, F10, TP53, and TYMS (Table S2), several of which were chosen for their importance in skin carcinogenesis.…”

Section: Significancementioning

confidence: 99%

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Why Cockayne syndrome patients do not get cancer despite their DNA repair deficiency

Reid-Bayliss

Arron

Loeb

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are human photosensitive diseases with mutations in the nucleotide excision repair (NER) pathway, which repairs DNA damage from UV exposure. CS is mutated in the transcription-coupled repair (TCR) branch of the NER pathway and exhibits developmental and neurological pathologies. The XP-C group of XP patients have mutations in the global genome repair (GGR) branch of the NER pathway and have a very high incidence of UV-induced skin cancer. Cultured cells from both diseases have similar sensitivity to UV-induced cytotoxicity, but CS patients have never been reported to develop cancer, although they often exhibit photosensitivity. Because cancers are associated with increased mutations, especially when initiated by DNA damage, we examined UV-induced mutagenesis in both XP-C and CS cells, using duplex sequencing for high-sensitivity mutation detection. Duplex sequencing detects rare mutagenic events, independent of selection and in multiple loci, enabling examination of all mutations rather than just those that confer major changes to a specific protein. We found telomerase-positive normal and CS-B cells had increased background mutation frequencies that decreased upon irradiation, purging the population of subclonal variants. Primary XP-C cells had increased UV-induced mutation frequencies compared with normal cells, consistent with their GGR deficiency. CS cells, in contrast, had normal levels of mutagenesis despite their TCR deficiency. The lack of elevated UV-induced mutagenesis in CS cells reveals that their TCR deficiency, although increasing cytotoxicity, is not mutagenic. Therefore the absence of cancer in CS patients results from the absence of UV-induced mutagenesis rather than from enhanced lethality.

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mitoSomatic: a tool for accurate identification of mitochondrial DNA somatic mutations without paired controls

et al. 2022

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Mitochondrial DNA (mtDNA) somatic mutations play important roles in the initiation and progression of cancer. Although next‐generation sequencing (NGS) of paired tumor and control samples has become a common practice to identify tumor‐specific mtDNA mutations, the unique nature of mtDNA and NGS‐associated sequencing bias could cause false‐positive/‐negative somatic mutation calling. Additionally, there are clinical scenarios where matched control tissues are unavailable for comparison. Therefore, a novel approach for accurately identifying somatic mtDNA variants is greatly needed, particularly in the absence of matched controls. In this study, the ground truth mtDNA variants orthogonally validated by triple‐paired tumor, adjacent nontumor, and blood samples were used to develop mitoSomatic, a random forest‐based machine learning tool. We demonstrated that mitoSomatic achieved area under the curve (AUC) values over 0.99 for identifying somatic mtDNA variants without paired control in three tumor types. In addition, mitoSomatic was also applicable in nontumor tissues such as adjacent nontumor and blood samples, suggesting the flexibility of mitoSomatic's classification capability. Furthermore, analysis of triple‐paired samples identified a small group of variants with uncertain somatic/germline origin, whereas application of mitoSomatic significantly facilitated the prediction of their possible source. Finally, a control‐free evaluation of the public pan‐cancer NGS dataset with mitoSomatic revealed a substantial number of variants that were probably misclassified by conventional tumor‐control comparison, further emphasizing the usefulness of mitoSomatic in application. Taken together, our study demonstrates that mitoSomatic is valuable for accurately identifying somatic mtDNA variants in mtDNA NGS data without paired controls, applicable for both tumor and nontumor tissues.

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Detecting ultralow-frequency mutations by Duplex Sequencing

Cited by 392 publications

References 39 publications

Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues

Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues

Why Cockayne syndrome patients do not get cancer despite their DNA repair deficiency

mitoSomatic: a tool for accurate identification of mitochondrial DNA somatic mutations without paired controls

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