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

Content Provider	PubMed Central
Author	De, Leeneer Kim Schrijver, Joachim De Clement, Lieven Baetens, Machteld Lefever, Steve Keulenaer, Sarah De Van, Criekinge Wim Dieter, Deforce Van, Nieuwerburgh Filip Bekaert, Sofie Pattyn, Filip Wilde, Bram De Coucke, Paul Jo, Vandesompele Claes, Kathleen Hellemans, Jan
Editor	Shioda, Toshi
Copyright Year	2011
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.
Related Links	http://dx.doi.org/10.1371/journal.pone.0025531
Starting Page	25531
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	9
Volume Number	6
Language	English
Publisher	Public Library of Science
Publisher Date	2011-09-30
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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