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
Introduction
Cardiomyopathies (CM) are primitive disorders in which the heart muscle is structurally and functionally abnormal in the absence of coronary artery disease, hypertension, valvular disease, and congenital heart disease sufficient to explain the observed myocardial abnormality [1]. Among the different known phenotypic groups, hypertrophic (HCM) and dilated cardiomyopathies (DCM) are the most frequent with an estimated prevalence of 1/500 and 1/2500, respectively [2]. These heterogeneous diseases are characterized mostly with autosomal-dominant transmission, adult onset, and reduced penetrance. Although identification of underlying etiology is important for management, therapy, and screening of at risk family members, molecular diagnosis remains difficult due to the large number of causative genes and the high rate of private mutations. Genetic causation in these disorders is attributed to over 40 genes (many of which overlap between phenotypic groups). Although genetic cause has been identified in familial HCM in up to 65% of cases, identifiable genetic cause in other familial cardiomyopathies has been lower (e.g. DCM 30%), and even less in sporadic cases.
Mutational screening of HCM and DCM cohorts is mostly performed by direct capillary sequencing. Despite development of scanning methods such as DHPLC or HRM [3], [4], [5], [6], targeted Sanger sequencing panels using traditional individual exon-by-exon sequencing remain expensive and time consuming, and massively parallel NextGen approaches are beginning to supplant Sanger sequencing [7]. This evolution is crucial as the prevalence of some cardiomyopathies is high. Previous studies have already reported the efficiency of NGS approaches to sequence HCM or DCM gene panels on GS-FLX Titanium, SOLID, or Illumina NGS sequencers [7], [8], [9], [10], [11]. Most of them were performed using capture libraries based on an enrichment of regions of interest. However, without automation, generation of these libraries remains more expensive and time-consuming than libraries based on multiplex PCR.
To date, the two main bench-top NGS platforms able of Gb-scale DNA sequencing with relatively short run times are MiSeq (Illumina) and Ion PGM™ (Life Technologies) sequencers [12]. Thus, the aim of our study was to evaluate the efficiency of a fast protocol based on custom AmpliSeq panel and Ion PGM™ Sequencer for sequencing most prevalent CM-causing genes (MYH7, MYBPC3, MYL2, LMNA, SCN5A, TNNT2, TNNI3 and TPM1). A cohort of previously studied 75 CM patients was screened in order to evaluate this strategy in terms of sensibility, specificity, practicability and cost. In silico analysis, based on previously reported suggestions, was performed using the NextGENe® software [7], [13].
Section snippets
Patients
Genomic DNA samples from HCM or DCM patients with previously characterized genetic variants were used to determine sensitivity and specificity of this NGS mutation detection approach. The study was conducted in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained for all cases.
Library preparation
Genomic DNAs were tested by NGS sequencing using a custom design for the cardiomyopathy panel, based on an AmpliSeq strategy (LifeTechnologies, Carlsbad, CA, USA). The panel,
Results
A cohort of 75 patients previously studied by our HRM/sequencing strategy was used to evaluate a NGS workflow designed according to recent publications [7], [13]. Five separate runs, containing 15 patient samples each, were performed. The coverage statistics were comparable between both runs. Each 316-chip generated an average of 2.5 million reads resulting in an average of 19.4 million Q20 bases per sample sequenced (Q20 = 99% chance correct base called). The mean depth was near from 400 reads and
Discussion
Next-Generation sequencing allows analysis of substantially larger genomic regions at a lower cost than conventional capillary Sanger sequencing. Consequently, this methodology could be considered as a high throughput mutation detection method for genes for which large cohorts of patients have to be investigated such as cardiomyopathies [7], [8], [9], [10], [11]. For the first time in the molecular diagnosis of cardiomyopathies, a NGS workflow based on a custom AmpliSeq library and Ion Torrent
Acknowledgements
This work was supported by the French Ministry of Research (Diagnosis Network on Neuromuscular Diseases).
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