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.
Highlights
A multitude of laboratory technologies for the detection of DNA mutations have been developed over the last decades
Calculation of coverage depth in function of sensitivity With Sanger sequencing a two-fold coverage is considered to be sufficient for molecular diagnostics, provided that sequences are of high quality
Because Massively parallel sequencing (MPS) is based on the sequencing of single, clonally amplified molecules, sampling effects need to be taken into account at low coverage
Summary
A multitude of laboratory technologies for the detection of DNA mutations have been developed over the last decades. Most frequently a combination of a mutation scanning technique, followed by Sanger sequencing of the abnormal DNA fragments is used. Sanger sequencing [5] of DNA fragments remains the preferred method for mutation analysis because of its superior sensitivity and specificity and the detailed sequence information that can be obtained in a single step approach. Expansion of molecular diagnostics to the realm of multigenic disorders requires the implementation of new methods with increased mutation detection efficiency but without a decrease in cost efficiency. Parallel sequencing (MPS) technologies (see [6,7] for an overview) are an interesting alternative because of their higher throughput and lower cost per base as compared to Sanger sequencing. Throughput and cost for MPS technologies per base are rapidly evolving (from 0.1 Gb per run for the Roche Genome Sequencer at the end of 2006 to 150–300 Gb per run for Illumina’s HiSeq2000 and ABI’s 5500XL platform in 2011) at a speed vastly surpassing the evolution rate seen in semiconductor industries (Moore’s law)
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