Sequencing small genomic targets with high efficiency and extreme accuracy

Schmitt, Michael; Fox, Edward J.; Prindle, Marc J.; Reid-Bayliss, Kate S; True, Lawrence D.; Radich, Jerald P.; Loeb, Lawrence A.

doi:10.1038/nmeth.3351

Cited by 133 publications

(146 citation statements)

References 24 publications

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“…dNTP pools | colon cancer | DNA replication fidelity R ecent advances in whole-genome sequencing have revealed that human cancers often contain thousands of subclonal mutations (1)(2)(3), lending support to the mutator phenotype hypothesis that postulates that an elevation in spontaneous mutation rate is an early step in cancer evolution (4,5). The three major determinants of DNA replication fidelity that control the spontaneous mutation rate are nucleotide selectivity by DNA polymerases, proofreading by replicative DNA polymerases, and the mismatch repair (MMR) system (6).…”

mentioning

confidence: 99%

Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance

Rentoft

Lindell

Tran

et al. 2016

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

103

155

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Even small variations in dNTP concentrations decrease DNA replication fidelity, and this observation prompted us to analyze genomic cancer data for mutations in enzymes involved in dNTP metabolism. We found that sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), a deoxyribonucleoside triphosphate triphosphohydrolase that decreases dNTP pools, is frequently mutated in colon cancers, that these mutations negatively affect SAMHD1 activity, and that several SAMHD1 mutations are found in tumors with defective mismatch repair. We show that minor changes in dNTP pools in combination with inactivated mismatch repair dramatically increase mutation rates. Determination of dNTP pools in mouse embryos revealed that inactivation of one SAMHD1 allele is sufficient to elevate dNTP pools. These observations suggest that heterozygous cancer-associated SAMHD1 mutations increase mutation rates in cancer cells.

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mentioning

confidence: 99%

Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance

Rentoft

Lindell

Tran

et al. 2016

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

103

155

View full text Add to dashboard Cite

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“…An important consideration when designing a selector is that sequencing cost is proportional to the size of the region being sequenced. It should be noted, that targeting very small regions is associated with its own hurdles because the enrichment efficiency of a single capture are typically on the order of 10 4 -fold, so for the targets smaller, than approximately 100 kb additional steps, such as two rounds of capture may be necessary to ensure sufficiently high on-target mapping rate (82). For patient-specific applications or cancers with a high mutation rate (or recurrence in a small number of genes), this may allow deep sequencing to be achieved at a low cost using "bench-top" NGS devices.…”

Section: Ligation and Hybridization-capture Methodsmentioning

confidence: 99%

“…For example, the authors of one of such approach, Schmitt and colleagues estimated the error rate of 3.8 Â 10 À10 in a model experiment (88). The same group demonstrated the robustness of their approach by detecting a single ABL1 imatinib conferring mutation in a sample from the chronic myeloid leukemia patient (82). The E279K mutation was unambiguously detected at 1% rate, whereas the error rate of raw sequencing data would have either completely or partially obscured this mutation.…”

Section: Error Suppression Methodsmentioning

confidence: 99%

Cell-free DNA (cfDNA): Clinical Significance and Utility in Cancer Shaped By Emerging Technologies

Volik¹,

Alcaide

Morin

et al. 2016

Molecular Cancer Research

295

235

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Precision oncology is predicated upon the ability to detect specific actionable genomic alterations and to monitor their adaptive evolution during treatment to counter resistance. Because of spatial and temporal heterogeneity and comorbidities associated with obtaining tumor tissues, especially in the case of metastatic disease, traditional methods for tumor sampling are impractical for this application. Known to be present in the blood of cancer patients for decades, cell-free DNA (cfDNA) is beginning to inform on tumor genetics, tumor burden, and mechanisms of progression and drug resistance. This substrate is amenable for inexpensive noninvasive testing and thus presents a viable approach to serial sampling for screening and monitoring tumor progression. The fragmentation, low yield, and variable admixture of normal DNA present formidable technical challenges for realization of this potential. This review summarizes the history of cfDNA discovery, its biological properties, and explores emerging technologies for clinically relevant sequence-based analysis of cfDNA in cancer patients. Molecular barcoding (or Unique Molecular Identifier, UMI)-based methods currently appear to offer an optimal balance between sensitivity, flexibility, and cost and constitute a promising approach for clinically relevant assays for near real-time monitoring of treatment-induced mutational adaptations to guide evidence-based precision oncology.

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“…This internal error correction effectively reduces false positive mutations because PCR and sequencing artifacts are very unlikely to occur at both strands of DNA at the same position and with complementary nucleotide changes (theoretical false positive rate is ∼4 × 10 −10 ) (7). Previous studies have demonstrated that duplex sequencing is able to detect a single point mutation among >10 7 sequenced nucleotides (7,8), an unprecedented accuracy that holds significant potential for early cancer detection.High-grade serous ovarian carcinoma (HGSOC) is the most common and most aggressive type of ovarian cancer, with a dismal 5-y survival rate of 10-30% (9). A main cause of poor prognosis is the absence of effective screening tools to detect early-stage disease.…”

mentioning

confidence: 99%

“…Moreover, >95% of TP53 mutations cluster in exons 4-10 (15), which provides a relatively small target to perform cost-efficient ultra-deep sequencing (8). Thus, we used duplex sequencing to sequence TP53 exons 4-10 in peritoneal fluid from patients with HGSOC with known TP53 mutations and control patients without ovarian cancer.…”

mentioning

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

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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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Sequencing small genomic targets with high efficiency and extreme accuracy

Cited by 133 publications

References 24 publications

Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance

Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance

Cell-free DNA (cfDNA): Clinical Significance and Utility in Cancer Shaped By Emerging Technologies

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

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