The molecular analysis of BRCA1 and BRCA2: Next-generation sequencing supersedes conventional approaches

D’Argenio, Valeria; Esposito, Maria Valeria; Telese, Antonella; Precone, Vincenza; Starnone, Flavio; Nunziato, Marcella; Cantiello, Piergiuseppe; Iorio, Mariangela; Evangelista, Eloisa; D’Aiuto, Massimiliano; Calabrese, Alessandra; Frisso, Giulia; D’Aiuto, Giuseppe; Salvatore, Francesco

doi:10.1016/j.cca.2015.03.045

Cited by 56 publications

(50 citation statements)

References 17 publications

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“…NGS-based analysis of the BRCA1 and BRCA2 genes were previously reported, including PCR-based library preparation on the Roche sequencer, 10 PCR-based on Ion-Torrent sequencer, 4,12 PCR-based on SOLiD and IoneTorrent sequencers, 13 and RainDance microdroplet …”

Section: Discussionmentioning

confidence: 99%

“…Recent improvements to NGS technologies that facilitate massively parallel analysis of multiple genes have enabled the implementation of more cost-effective, rapid, and high-throughput methods to allow precise identification of BRCA1 and BRCA2 mutations in these high-risk patients, as well as testing of more comprehensive gene panels for cancer predisposition syndromes. 4,9,10 NGS is also capable of sensitive detection of sequence variants and may also be used for detection of copy number variants (CNVs), such as exon deletions and duplications that currently require use of MLPA or other copy number technologies.…”

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confidence: 99%

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Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis

Schenkel

Kerkhof

Stuart

et al. 2016

The Journal of Molecular Diagnostics

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Advances in next-generation sequencing (NGS) have facilitated parallel analysis of multiple genes enabling the implementation of cost-effective, rapid, and high-throughput methods for the molecular diagnosis of multiple genetic conditions, including the identification of BRCA1 and BRCA2 mutations in high-risk patients for hereditary breast and ovarian cancer. We clinically validated a NGS pipeline designed to replace Sanger sequencing and multiplex ligation-dependent probe amplification analysis and to facilitate detection of sequence and copy number alterations in a single test focusing on a BRCA1/BRCA2 gene analysis panel. Our custom capture library covers 46 exons, including BRCA1 exons 2, 3, and 5 to 24 and BRCA2 exons 2 to 27, with 20 nucleotides of intronic regions both 5' and 3' of each exon. We analyzed 402 retrospective patients, with previous Sanger sequencing and multiplex ligation-dependent probe amplification results, and 240 clinical prospective patients. One-hundred eighty-three unique variants, including sequence and copy number variants, were detected in the retrospective (n = 95) and prospective (n = 88) cohorts. This standardized NGS pipeline demonstrated 100% sensitivity and 100% specificity, uniformity, and high-depth nucleotide coverage per sample (approximately 7000 reads per nucleotide). Subsequently, the NGS pipeline was applied to the analysis of larger gene panels, which have shown similar uniformity, sample-to-sample reproducibility in coverage distribution, and sensitivity and specificity for detection of sequence and copy number variants.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis

Schenkel

Kerkhof

Stuart

et al. 2016

The Journal of Molecular Diagnostics

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show abstract

“…Focused cancer gene-panels have been most frequently used to date due to cost-efficiency, because the raw data handling and flow of data through bioinformatics pipelines is relatively simple, the demand for server data storage is limited and because they decrease the chance of finding variants of underdetermined/unknown significance (VUS), which are difficult to interpret in clinical management (see also Section 7). In this context, the most straightforward way to overcome these challenges is achieved by restricting the NGS sequencing to the high risk-genes only (targeted re-sequencing), which still presents some advantages and has higher sensitivity compared with traditional methods [61,62]. Although the use of WES and WGS has been less frequent in the past, the decreasing costs and the improvements in pipeline analyses are making these strategies increasingly more suitable and it is envisaged that they will be the preferred approach in the near future.…”

Section: Inherited Cancer Syndromesmentioning

confidence: 99%

Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification

Kamps

Brandão

Bosch

et al. 2017

IJMS

387

294

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Next-generation sequencing (NGS) technology has expanded in the last decades with significant improvements in the reliability, sequencing chemistry, pipeline analyses, data interpretation and costs. Such advances make the use of NGS feasible in clinical practice today. This review describes the recent technological developments in NGS applied to the field of oncology. A number of clinical applications are reviewed, i.e., mutation detection in inherited cancer syndromes based on DNA-sequencing, detection of spliceogenic variants based on RNA-sequencing, DNA-sequencing to identify risk modifiers and application for pre-implantation genetic diagnosis, cancer somatic mutation analysis, pharmacogenetics and liquid biopsy. Conclusive remarks, clinical limitations, implications and ethical considerations that relate to the different applications are provided.

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“…Our lab experience and several recent papers have demonstrated how NGS methods are adequate to detect point mutations and indels in BRCA1 / BRCA2 genes, revolutionizing this genetic analysis and reducing time and costs 4–6. This approach in fact is suitable in routine diagnostic workflow, since it is faster and more sensitive than denaturing high-performance liquid chromatography/Sanger sequencing methods.…”

Section: Ngs Analysis For Solid Cancer Diagnosismentioning

confidence: 90%

Next-generation sequencing: advances and applications in cancer diagnosis

Serratì¹,

Summa²,

Pilato³

et al. 2016

OTT

158

113

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Technological advances have led to the introduction of next-generation sequencing (NGS) platforms in cancer investigation. NGS allows massive parallel sequencing that affords maximal tumor genomic assessment. NGS approaches are different, and concern DNA and RNA analysis. DNA sequencing includes whole-genome, whole-exome, and targeted sequencing, which focuses on a selection of genes of interest for a specific disease. RNA sequencing facilitates the detection of alternative gene-spliced transcripts, posttranscriptional modifications, gene fusion, mutations/single-nucleotide polymorphisms, small and long noncoding RNAs, and changes in gene expression. Most applications are in the cancer research field, but lately NGS technology has been revolutionizing cancer molecular diagnostics, due to the many advantages it offers compared to traditional methods. There is greater knowledge on solid cancer diagnostics, and recent interest has been shown also in the field of hematologic cancer. In this review, we report the latest data on NGS diagnostic/predictive clinical applications in solid and hematologic cancers. Moreover, since the amount of NGS data produced is very large and their interpretation is very complex, we briefly discuss two bioinformatic aspects, variant-calling accuracy and copy-number variation detection, which are gaining a lot of importance in cancer-diagnostic assessment.

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The molecular analysis of BRCA1 and BRCA2: Next-generation sequencing supersedes conventional approaches

Abstract: Next-generation sequencing is more sensitive, faster, easier to use and less expensive than the conventional Sanger method. Consequently, it is a reliable procedure for the routine molecular screening of the BRCA1/2 genes.

Cited by 56 publications

References 17 publications

Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis

Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis

Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification

Next-generation sequencing: advances and applications in cancer diagnosis

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