Practical Tools to Implement Massive Parallel Pyrosequencing of PCR Products in Next Generation Molecular Diagnostics

Leeneer, Kim De; Schrijver, Joachim De; Clement, Lieven; Baetens, Machteld; Lefever, Steve; Keulenaer, Sarah De; Criekinge, Wim Van; Deforce, Dieter; Nieuwerburgh, Filip Van; Bekaert, Sofie; Pattyn, Filip; Wilde, Bram De; Coucke, Paul; Vandesompele, Jo; Claes, Kathleen; Hellemans, Jan

doi:10.1371/journal.pone.0025531

Cited by 37 publications

(35 citation statements)

References 21 publications

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“…The main disadvantage of pyrosequencing relative to other NGS technologies is the accuracy of length determination in homopolymers. 12,17,21 In pyrosequencing, the light-intensity signal observed in each cycle is proportional to the actual number of incorporated nucleotides, which is the base for homopolymer length calling. The accuracy of this method decreases with homopolymer length, which may eventually generate artefactual insertions and deletions in long homopolymers.…”

Section: Discussionmentioning

confidence: 99%

Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes

et al. 2012

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Next-generation sequencing (NGS) is changing genetic diagnosis due to its huge sequencing capacity and cost-effectiveness. The aim of this study was to develop an NGS-based workflow for routine diagnostics for hereditary breast and ovarian cancer syndrome (HBOCS), to improve genetic testing for BRCA1 and BRCA2. A NGS-based workflow was designed using BRCA MASTR kit amplicon libraries followed by GS Junior pyrosequencing. Data analysis combined Variant Identification Pipeline freely available software and ad hoc R scripts, including a cascade of filters to generate coverage and variant calling reports. A BRCA homopolymer assay was performed in parallel. A research scheme was designed in two parts. A Training Set of 28 DNA samples containing 23 unique pathogenic mutations and 213 other variants (33 unique) was used. The workflow was validated in a set of 14 samples from HBOCS families in parallel with the current diagnostic workflow (Validation Set). The NGS-based workflow developed permitted the identification of all pathogenic mutations and genetic variants, including those located in or close to homopolymers. The use of NGS for detecting copy-number alterations was also investigated. The workflow meets the sensitivity and specificity requirements for the genetic diagnosis of HBOCS and improves on the cost-effectiveness of current approaches.

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Section: Discussionmentioning

confidence: 99%

Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes

et al. 2012

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“…Variants were selected to be called when Ͼ15% in Ͼ15 total reads for the base being interrogated. A 15% mutation filter was chosen because a heterozygous variant had been statistically determined to be identified 99.995% of the time when there was 30-fold coverage (23). Regions with less than 30-fold coverage were analyzed by Sanger sequencing.…”

Section: Ngs Data Analysismentioning

confidence: 99%

A Comprehensive Next Generation Sequencing–Based Genetic Testing Strategy To Improve Diagnosis of Inherited Pheochromocytoma and Paraganglioma

Rattenberry

Vialard

Yeung

et al. 2013

The Journal of Clinical Endocrinology &Amp; Metabolism

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Context: Pheochromocytomas and paragangliomas are notable for a high frequency of inherited cases, many of which present as apparently sporadic tumors.Objective: The objective of this study was to establish a comprehensive next generation sequencing (NGS)-based strategy for the diagnosis of patients with pheochromocytoma and paraganglioma by testing simultaneously for mutations in MAX, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL. Design: After the methodology for the assay was designed and established, it was validated on DNA samples with known genotype and then patients were studied prospectively. Setting:The study was performed in a diagnostic genetics laboratory.Patients: DNA samples from 205 individuals affected with adrenal or extraadrenal pheochromocytoma/head and neck paraganglioma (PPGL/HNPGL) were analyzed. A proof-of-principle study was performed using 85 samples known to contain a variant in 1 or more of the genes to be tested, followed by prospective analysis of an additional 120 samples. Main Outcome Measures:We assessed the ability to use an NGS-based method to perform comprehensive analysis of genes implicated in inherited PPGL/HNPGL. Results:The proof-of-principle study showed that the NGS assay and analysis gave a sensitivity of 98.7%. A pathogenic mutation was identified in 16.6% of the prospective analysis cohort of 120 patients. Conclusions:A comprehensive NGS-based strategy for the analysis of genes associated with predisposition to PPGL and HNPGL was established, validated, and introduced into diagnostic service. The new assay provides simultaneous analysis of 9 genes and allows more rapid and cost-effective mutation detection than the previously used conventional Sanger sequencing-based methodology. (J Clin Endocrinol Metab 98: E1248 -E1256, 2013) P heochromocytomas and paragangliomas are rare, mostly benign, tumors that are notable for a high frequency of inherited cases (1-5). Both pheochromocytomas (which arise from the adrenal medulla) and paragangliomas (also known as extraadrenal pheochromocytomas) (together referred to as PPGLs) usually present with symptoms caused by the cardiovascular effects of excess catecholamine secretion. Head and neck paragangliomas (HNPGLs) are generally nonfunctional and are derived from parasympathetic ganglia. Before 2000, about 10% of PPGLs were thought to be inherited (6) and mostly associated with defined clinical syndromes: von

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“…On the other hand, we can confidently state that variants with an allelic ratio of o15% are false positives (somatic analyses excluded), a finding in accordance with those of other platforms. 19,20 Depth of coverage is another prominent point to consider, because the lower the depth, the higher the risk of missing a variant, by sequencing failure and/or bioinformatics filtering. Conversely, the Streamlined ion torrent PGM diagnostics J Tarabeux et al higher the depth, the higher the probability of discriminating false positives (ie, random errors) from true positives.…”

Section: Bioinformaticsmentioning

confidence: 99%

Streamlined ion torrent PGM-based diagnostics: BRCA1 and BRCA2 genes as a model

et al. 2013

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To meet challenges in terms of throughput and turnaround time, many diagnostic laboratories are shifting from Sanger sequencing to higher throughput next-generation sequencing (NGS) platforms. Bearing in mind that the performance and quality criteria expected from NGS in diagnostic or research settings are strikingly different, we have developed an Ion Torrent's PGM-based routine diagnostic procedure for BRCA1/2 sequencing. The procedure was first tested on a training set of 62 control samples, and then blindly validated on 77 samples in parallel with our routine technique. The training set was composed of difficult cases, for example, insertions and/or deletions of various sizes, large-scale rearrangements and, obviously, mutations occurring in homopolymer regions. We also compared two bioinformatic solutions in this diagnostic context, an in-house academic pipeline and the commercially available NextGene software (Softgenetics). NextGene analysis provided higher sensitivity, as four previously undetected single-nucleotide variations were found. Regarding specificity, an average of 1.5 confirmatory Sanger sequencings per patient was needed for complete BRCA1/2 screening. Large-scale rearrangements were identified by two distinct analyses, that is, bioinformatics and fragment analysis with electrophoresis profile comparison. Turnaround time was enhanced, as a series of 30 patients were sequenced by one technician, making the results available for the clinician in 10 working days following blood sampling. BRCA1/2 genes are a good model, representative of the difficulties commonly encountered in diagnostic settings, which is why we believe our findings are of interest for the whole community, and the pipeline described can be adapted by any user of PGM for diagnostic purposes.

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Practical Tools to Implement Massive Parallel Pyrosequencing of PCR Products in Next Generation Molecular Diagnostics

Cited by 37 publications

References 21 publications

Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes

Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes

A Comprehensive Next Generation Sequencing–Based Genetic Testing Strategy To Improve Diagnosis of Inherited Pheochromocytoma and Paraganglioma

Streamlined ion torrent PGM-based diagnostics: BRCA1 and BRCA2 genes as a model

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