Elsevier

Clinica Chimica Acta

Volume 433, 10 June 2014, Pages 266-271
Clinica Chimica Acta

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

https://doi.org/10.1016/j.cca.2014.03.032Get rights and content

Highlights

  • Hypertrophic and dilated cardiomyopathies are common genetic cardiac diseases.

  • This NGS workflow is an efficient, fast, cheap and high-throughput detection method.

  • This NGS workflow is based on AmpliSeq libraries.

  • This NGS workflow combines Ion PGM sequencing and NextGENe® analysis.

  • This new NGS approach is ready to be deployed in clinical laboratories.

Abstract

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.

Conclusion

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.

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).

References (17)

There are more references available in the full text version of this article.

Cited by (41)

  • Target-enriched sequencing enables accurate identification of bloodstream infections in whole blood

    2022, Journal of Microbiological Methods
    Citation Excerpt :

    Compared with traditional sequencing to detect pathogens in whole blood, ADS significantly reduces interference from human genomic DNA. Ampliseq detection technology has been reported as a tool for screening tumor genes (Butler et al., 2016; Zhang et al., 2014) and genetic diseases (Millat et al., 2014). In recent years, researchers have applied it to bacterial drug resistance testing from surfaces in the international space station (Urbaniak et al., 2018).

  • Evaluation of GeneMarker<sup>®</sup> HTS for improved alignment of mtDNA MPS data, haplotype determination, and heteroplasmy assessment

    2017, Forensic Science International: Genetics
    Citation Excerpt :

    One software package, mitoSAVE, uses a simple Excel-spreadsheet-based solution [15] that converts the data into haplotypes using phylogenetically-derived nomenclature, and allows for consistent assignment of homopolymeric stretches and sequence motifs [16,17], but does not allow for analysis of the individual sequencing reads in the pileup. The commercially available software package, NextGENe® (SoftGenetics, Inc., State College, PA), has been used by a number of researchers and clinicians to analyze MPS data for the diagnosis of cancer, detection of cardiomyopathies, and evaluation of hereditary hearing loss [18–20]. The software includes numerous user-defined parameters, and allows for detailed analysis of the pileup.

  • A fast and cost-effective molecular diagnostic tool for genetic diseases involved in sudden cardiac death

    2016, Clinica Chimica Acta
    Citation Excerpt :

    Because of this genetic and allelic heterogeneity, molecular testing could only be envisaged with Next Generation Sequencing approaches. Previous studies have already reported the efficiency of NGS approaches to sequence gene panels on benchtop NGS sequencers [5,6]. Thus, the aim of our study was to evaluate the efficiency of a fast protocol based on a large custom panel (95 genes) and NextSeq500 Sequencer for sequencing most prevalent SCD-causing genes.

View all citing articles on Scopus
View full text