Evaluation of a new NGS method based on a custom AmpliSeq library and Ion Torrent PGM sequencing for the fast detection of genetic variations in cardiomyopathies.
BACKGROUND
Hypertrophic and dilated cardiomyopathies are common genetic cardiac diseases. Due to large cohorts to investigate, large number of causative genes and high rate of private mutations, mutational screening must be performed using an extremely sensitive and specific detection method.


METHODS
NGS workflow based on a custom AmpliSeq panel was designed for sequencing most prevalent cardiomyopathy-causing genes on the Ion PGM™ Sequencer. A cohort of 75 previously studied patients was screened to evaluate this strategy in terms of sensibility, specificity, practicability and cost. In silico analysis was performed using the NextGENe® software.


RESULTS
Our AmpliSeq custom panel allowed us to efficiently explore 96% of targeted sequences. Using adjusted alignment settings, all genetic variants (57 substitutions, 34 indels) present in covered regions and previously detected by HRM/sequencing were readily identified except a 73-bp MYBPC3 deletion (analytical sensitivity: 98.9%). Uncovered targeted regions were further analysed by a HRM/sequencing strategy. Complete molecular investigation was performed faster and cheaper than with previously used mutation detection methods.


CONCLUSIONS
Finally, these results suggested that our new NGS approach based on Ampliseq libraries and Ion PGM sequencing is a highly efficient, fast and cheap high-throughput mutation detection method that is ready to be deployed in clinical laboratories.
Laboratoire de Cardiogénétique Moléculaire, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, Lyon, France; NGS sequencing platform for molecular diagnosis, Hospices Civils de Lyon, Lyon, France; Université Lyon 1, Lyon, France. Electronic address: gilles.millat@chu-lyon.fr.
Gilles
G
Millat
Laboratoire de Cardiogénétique Moléculaire, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, Lyon, France; NGS sequencing platform for molecular diagnosis, Hospices Civils de Lyon, Lyon, France.
Valérie
V
Chanavat
Laboratoire de Cardiogénétique Moléculaire, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, Lyon, France; NGS sequencing platform for molecular diagnosis, Hospices Civils de Lyon, Lyon, France; Université Lyon 1, Lyon, France.
Robert
R
Rousson
D009202:Cardiomyopathies / Q000235:genetics*; D015331:Cohort Studies; D019295:Computational Biology; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software; D013997:Time Factors
Dilated cardiomyopathy; Hypertrophic cardiomyopathy; Molecular diagnosis; Mutations; NGS sequencing