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Practical tools to implement massive parallel pyrosequencing of PCR products in next generation molecular diagnostics

Kim De Leeneer (UGent) , Joachim De Schrijver (UGent) , Lieven Clement (UGent) , Machteld Baetens (UGent) , Steve Lefever (UGent) , Sarah De Keulenaer (UGent) , Wim Van Criekinge (UGent) , Dieter Deforce (UGent) , Filip Van Nieuwerburgh (UGent) , Sofie Bekaert (UGent) , et al.
(2011) PLOS ONE. 6(9).
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Bioinformatics: from nucleotids to networks (N2N)
Abstract
Despite improvements in terms of sequence quality and price per basepair, Sanger sequencing remains restricted to screening of individual disease genes. The development of massively parallel sequencing (MPS) technologies heralded an era in which molecular diagnostics for multigenic disorders becomes reality. Here, we outline different PCR amplification based strategies for the screening of a multitude of genes in a patient cohort. We performed a thorough evaluation in terms of set-up, coverage and sequencing variants on the data of 10 GS-FLX experiments (over 200 patients). Crucially, we determined the actual coverage that is required for reliable diagnostic results using MPS, and provide a tool to calculate the number of patients that can be screened in a single run. Finally, we provide an overview of factors contributing to false negative or false positive mutation calls and suggest ways to maximize sensitivity and specificity, both important in a routine setting. By describing practical strategies for screening of multigenic disorders in a multitude of samples and providing answers to questions about minimum required coverage, the number of patients that can be screened in a single run and the factors that may affect sensitivity and specificity we hope to facilitate the implementation of MPS technology in molecular diagnostics. A
Keywords
IDENTIFICATION, MELTING ANALYSIS, DNA

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Chicago
De Leeneer, Kim, Joachim De Schrijver, Lieven Clement, Machteld Baetens, Steve Lefever, Sarah De Keulenaer, Wim Van Criekinge, et al. 2011. “Practical Tools to Implement Massive Parallel Pyrosequencing of PCR Products in Next Generation Molecular Diagnostics.” Plos One 6 (9).
APA
De Leeneer, K., De Schrijver, J., Clement, L., Baetens, M., Lefever, S., De Keulenaer, S., Van Criekinge, W., et al. (2011). Practical tools to implement massive parallel pyrosequencing of PCR products in next generation molecular diagnostics. PLOS ONE, 6(9).
Vancouver
1.
De Leeneer K, De Schrijver J, Clement L, Baetens M, Lefever S, De Keulenaer S, et al. Practical tools to implement massive parallel pyrosequencing of PCR products in next generation molecular diagnostics. PLOS ONE. 2011;6(9).
MLA
De Leeneer, Kim, Joachim De Schrijver, Lieven Clement, et al. “Practical Tools to Implement Massive Parallel Pyrosequencing of PCR Products in Next Generation Molecular Diagnostics.” PLOS ONE 6.9 (2011): n. pag. Print.
@article{2043761,
  abstract     = {Despite improvements in terms of sequence quality and price per basepair, Sanger sequencing remains restricted to screening of individual disease genes. The development of massively parallel sequencing (MPS) technologies heralded an era in which molecular diagnostics for multigenic disorders becomes reality. Here, we outline different PCR amplification based strategies for the screening of a multitude of genes in a patient cohort. We performed a thorough evaluation in terms of set-up, coverage and sequencing variants on the data of 10 GS-FLX experiments (over 200 patients). Crucially, we determined the actual coverage that is required for reliable diagnostic results using MPS, and provide a tool to calculate the number of patients that can be screened in a single run. Finally, we provide an overview of factors contributing to false negative or false positive mutation calls and suggest ways to maximize sensitivity and specificity, both important in a routine setting. By describing practical strategies for screening of multigenic disorders in a multitude of samples and providing answers to questions about minimum required coverage, the number of patients that can be screened in a single run and the factors that may affect sensitivity and specificity we hope to facilitate the implementation of MPS technology in molecular diagnostics. A},
  articleno    = {e25531},
  author       = {De Leeneer, Kim and De Schrijver, Joachim and Clement, Lieven and Baetens, Machteld and Lefever, Steve and De Keulenaer, Sarah and Van Criekinge, Wim and Deforce, Dieter and Van Nieuwerburgh, Filip and Bekaert, Sofie and Pattyn, Filip and De Wilde, Bram and Coucke, Paul and Vandesompele, Jo and Claes, Kathleen and HELLEMANS, JAN},
  issn         = {1932-6203},
  journal      = {PLOS ONE},
  keyword      = {IDENTIFICATION,MELTING ANALYSIS,DNA},
  language     = {eng},
  number       = {9},
  pages        = {7},
  title        = {Practical tools to implement massive parallel pyrosequencing of PCR products in next generation molecular diagnostics},
  url          = {http://dx.doi.org/10.1371/journal.pone.0025531},
  volume       = {6},
  year         = {2011},
}

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