Brief communicationValidation of next-generation sequencing technologies in genetic diagnosis of dementia
Introduction
Genetic diagnosis of the inherited dementias currently relies on sequential Sanger sequencing of genes selected on a clinical basis. This process is costly, time-consuming, and gene tests are variably available, contributing to the limited ascertainment of inherited dementia in the population (Stevens et al., 2011). Here we show that next-generation sequencing (NGS) technology offers a 1-step, accurate, and cost-effective method of screening many causal genes simultaneously, and is likely to be a transformational technology in early-onset dementia diagnosis (Rehm, 2013).
Section snippets
Methods
We used Life Technology's Ion Torrent PGM sequencer with a polymerase chain reaction (PCR) amplicon-based library preparation (AmpliSeq) and Illumina's MiSeq with a PCR amplicon-based (TrueSeq custom amplicon) target enrichment to screen for variants across 16 dementia disease genes (PRNP, PSEN1, PSEN2, APP, GRN, MAPT, TREM2, CHMP2B, CSF1R, FUS, ITM2B, NOTCH3, SERPINI1, TARDBP, TYROBP, and VCP) (Bettens et al., 2013, Rademakers et al., 2012). These sequencing-based technologies were coupled
Results
In the blinded study, the Ion Torrent correctly identified all control samples. Of the 85 case samples, 82 had the expected mutations or polymorphisms identified during primary analysis (sensitivity 96.5%, specificity 100%), including deletion of the entire GRN gene in 1 case. The 3 variants not initially detected were 2 samples with an identical APP p.V717G mutation and an APP duplication. The APP p.V717G mutations were present at the overlap between 2 amplicons and because of a software
Discussion
Using our current testing strategy and allowing for the fact that the APP p.V717G mutations were not identified because of a straightforward and remediable software problem, we demonstrated extremely high sensitivity and specificity. The present recurrent consumable costs are estimated at approximately £60 ($90) per sample using the Ion Torrent and £120 ($180) using the Illumina MiSeq. NGS has many benefits including vastly reduced costs compared with Sanger sequencing of individual genes, a
Disclosure statement
The authors declare no conflicts of interest. All subjects included in this study gave informed consent for genetic research. The work was approved by the London - Queen Square Research Ethics Committee.
Acknowledgements
This work was funded by the Medical Research Council Prion Unit and the NIHR Queen Square Dementia Biomedical Research Unit.
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