Reducing Sequence Artifacts in Amplicon-Based Massively Parallel Sequencing of Formalin-Fixed Paraffin-Embedded DNA by Enzymatic Depletion of Uracil-Containing Templates

Do, Hongdo; Wong, Stephen Q.; Li, Jason; Dobrovic, Alexander

doi:10.1373/clinchem.2012.202390

Cited by 96 publications

(116 citation statements)

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“…An alternative approach to reduce false-positive results is treatment of FFPE tissue DNA with uracil glycosylase. 13,25 This can significantly reduce false-positive changes resulting from deamination of cytosine; however, uracil glycosylase is active only at uracil lesions but not thymine lesions resulting from deamination of 5-methyl cytosine. In addition, the C:G>T:A artifact at CpG dinucleotides is resistant to uracil glycosylase treatment.…”

Section: Strategies To Eliminate False-positive Resultsmentioning

confidence: 99%

“…In addition, the C:G>T:A artifact at CpG dinucleotides is resistant to uracil glycosylase treatment. 25 Thus, duplicate experiments offer broader efficacy in elimination of false-positive results, which is very much highlighted by a recent review. 26 Choosing Appropriate Cutoff Value of AAF for Reliable Detection of Somatic Mutations On the basis of the concordance of reproducible variants between two sets of independent experiments, together with known somatic mutations by Sanger sequencing, we suggest using 10% and 7% as the optimal cutoff value of AAF for mutation detection in FFPE tissue and HMW DNA, respectively.…”

Section: Strategies To Eliminate False-positive Resultsmentioning

confidence: 99%

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Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing

Wang

Escudero‐Ibarz

Moody

et al. 2015

The Journal of Molecular Diagnostics

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High-throughput somatic mutation screening using FFPE tissues is a major challenge because of a lack of established methods and validated variant calling algorithms. We aimed to develop a targeted sequencing protocol by Fluidigm multiplex PCR and Illumina sequencing and to establish a companion variant calling algorithm. The experimental protocol and variant calling algorithm were first developed and optimized against a series of somatic mutations (147 substitutions, 12 indels ranging from 1 to 33 bp) in seven genes, previously detected by Sanger sequencing of DNA from 163 FFPE lymphoma biopsy specimens. The optimized experimental protocol and variant calling algorithm were further ascertained in two separate experiments by including the seven genes as a part of larger gene panels (22 or 13 genes) using FFPE and high-molecular-weight lymphoma DNAs, respectively. We found that most false-positive variants were due to DNA degradation, deamination, and Taq polymerase errors, but they were nonreproducible and could be efficiently eliminated by duplicate experiments. A small fraction of false-positive variants appeared in duplicate, but they were at low alternative allele frequencies and could be separated from mutations when appropriate threshold value was used. In conclusion, we established a robust practical approach for high-throughput mutation screening using archival FFPE tissues. (J Mol Diagn 2015, 17: 521e532; http://dx

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Section: Strategies To Eliminate False-positive Resultsmentioning

confidence: 99%

Section: Strategies To Eliminate False-positive Resultsmentioning

confidence: 99%

Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing

Wang

Escudero‐Ibarz

Moody

et al. 2015

The Journal of Molecular Diagnostics

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“…Deamination of 5-mC to thymine causes artifactual C:GϾT:A SNVs because DNA polymerase incorporates an adenine opposite to the thymine lesions. It is not surprising that high levels of artifactual C:GϾT:A SNVs are found at CpG dinucleotide sites in FFPE DNA, strongly indicative of deamination of 5-mC bases (41 ).…”

Section: Deamination Of Cytosine Basesmentioning

confidence: 99%

“…In vitro removal of uracil bases from FFPE DNA using UDG before PCR amplification markedly reduces the artifactual C:GϾT:A SNVs (40 ). This can be as high as 60%-80% in some FFPE DNAs (41 ).…”

Section: Dna-glycosylasesmentioning

confidence: 99%

Sequence Artifacts in DNA from Formalin-Fixed Tissues: Causes and Strategies for Minimization

2015

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BACKGROUND: Precision medicine is dependent on identifying actionable mutations in tumors. Accurate detection of mutations is often problematic in formalinfixed paraffin-embedded (FFPE) tissues. DNA extracted from formalin-fixed tissues is fragmented and also contains DNA lesions that are the sources of sequence artifacts. Sequence artifacts can be difficult to distinguish from true mutations, especially in the context of tumor heterogeneity, and are an increasing interpretive problem in this era of massively parallel sequencing. Understanding of the sources of sequence artifacts in FFPE tissues and implementation of preventative strategies are critical to improve the accurate detection of actionable mutations.CONTENT: This mini-review focuses on DNA template lesions in FFPE tissues as the source of sequence artifacts in molecular analysis. In particular, fragmentation, base modification (including uracil and thymine deriving from cytosine deamination), and abasic sites are discussed as indirect or direct sources of sequence artifacts. We discuss strategies that can be implemented to minimize sequence artifacts and to distinguish true mutations from sequence artifacts. These strategies are applicable for the detection of actionable mutations in both single amplicon and massively parallel amplicon sequencing approaches.

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“…BRCA testing of formalin-fixed paraffin-embedded tumour samples also presents a number of additional challenges to the diagnostics laboratory, including limited quantity and quality of extracted DNA, and artefactual sequence alterations. [20][21][22][23] Genetic counselling is integral to the BRCA testing process. In the final review, Professor Nicoline Hoogerbrugge and Dr Marjolijn Jongmans examine best practices for genetic counselling and BRCA testing, exploring the challenges facing current practice and looking at adapted models of genetic counselling.…”

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confidence: 99%

BRCA to the future: towards best testing practice in the era of personalised healthcare

Capoluongo

2016

Eur J Hum Genet

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The year 2014 marked the twentieth anniversary of the discovery of the breast cancer susceptibility gene, BRCA1. 1,2 Since this discovery, our understanding of pathogenic BRCA variants and the associated increase in lifetime cancer risks has advanced significantly. 3,4 National guidelines have been developed to help clinicians identify patients with an increased risk of pathogenic BRCA mutations, and genetic counselling for risk assessment is now a routine practice. 5,6 The evolution of genetic counselling models has placed increasing importance on the use of multidisciplinary health-care teams, including medical geneticists, genetic counsellors, gynaecologists, surgeons, radiotherapists, medical oncologists and all other professionals involved in a patient's management. There has also been a surge of technological advances and new strategies allowing for the rapid turnaround of BRCA1 and BRCA2 mutation testing. In addition, evaluation of BRCA and other BRCA-like deleterious mutations as potentially actionable tumour targets are underway, such as the development of poly(ADP)-ribose polymerase (PARP) inhibitors in classes four or five BRCA mutation-positive cancers. [7][8][9] The present supplement contains review articles based on presentations from a scientific symposium focused on the evolving BRCA testing landscape (BRCA to the future: towards best testing practice in the era of personalised healthcare) held at the European Human Genetics Conference in Milan, June 2014.In the first review, Professor Dominique Stoppa-Lyonnet discusses the biological effects and clinical implications of pathogenic BRCA mutations. BRCA1 and BRCA2 have key roles in the repair of DNA double-strand breaks via the mechanism of homologous recombination (HR) and ensure genome stability. 10-13 Germline deleterious mutations in BRCA1 or BRCA2 are associated with elevated risk of developing breast and/or ovarian cancer, sometimes with a genotype-phenotype correlation. 3,4,14 BRCA pathogenic mutationpositive ovarian or breast cancers are susceptible to inhibitors of additional DNA damage repair pathways, such as PARP inhibitors. [7][8][9] As the presence of actionable BRCA mutations in patients with breast and/or ovarian cancer becomes increasingly important in clinical management decisions, there is increasing support to expand the screening criteria for BRCA mutation testing, including testing for pathogenic mutations in other HR-deficiency genes.In his review on BRCA testing, Dr Andrew Wallace covers the challenges of BRCA testing from the perspective of a diagnostic laboratory. Demand for BRCA testing is steadily increasing, placing a strain on diagnostics laboratories, particularly in those offering rapid genetic testing at the point of diagnosis. Increasingly, diagnostic laboratories are adopting next-generation sequencing (NGS) technology for BRCA testing, which offers the potential of fast, scalable, costefficient and comprehensive sequencing. [15][16][17][18][19] However, the choice and complexity surrounding NGS means that adopt...

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Reducing Sequence Artifacts in Amplicon-Based Massively Parallel Sequencing of Formalin-Fixed Paraffin-Embedded DNA by Enzymatic Depletion of Uracil-Containing Templates

Cited by 96 publications

References 20 publications

Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing

Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing

Sequence Artifacts in DNA from Formalin-Fixed Tissues: Causes and Strategies for Minimization

BRCA to the future: towards best testing practice in the era of personalised healthcare

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