A Rapid, High-Quality, Cost-Effective, Comprehensive, and Expandable Targeted Next-Generation Sequencing Assay for Inherited Heart Diseases
Rationale: Thousands of mutations across more than 50 genes have been implicated in inherited cardiomyopathies. However, options for sequencing this rapidly evolving gene set are limited as many sequencing services and off-the-shelf kits suffer from slow turnaround, inefficient capture of genomic DNA, and/or high cost. Furthermore, customization of these assays to cover emerging targets and to suit individual needs is often expensive and time-consuming.
Objective: We sought to develop a custom high throughput, clinical-grade next generation sequencing (NGS) assay for detecting cardiac disease gene mutations with improved accuracy, flexibility, turnaround, and cost.
Methods and Results: We employed double-stranded probes (complementary long "padlock" probes (cLPPs)), an inexpensive and customizable capture technology, to efficiently capture and amplify the entire coding region and flanking intronic and regulatory sequences of 88 genes and 40 microRNAs (miRNA) associated with inherited cardiomyopathies, congenital heart disease (CHD), and cardiac development. Multiplexing 11 samples per sequencing run resulted in a mean base coverage of 420, of which 97% had >20X coverage and >99% were concordant with known heterozygous single nucleotide polymorphisms (SNPs). The assay correctly detected germline variants in 24 individuals and revealed several polymorphic regions in miR-499. Total run time was three days at an approximate cost of $100 per sample.
Conclusions: Accurate, high throughput detection of mutations across numerous cardiac genes is achievable with cLPP technology. Moreover, this format allows facile insertion of additional probes as more cardiomyopathy and CHD genes are discovered, giving researchers a powerful new tool for DNA mutation detection and discovery.
- next generation sequencing
- complementary long padlock probes
- genetic heart disease
- genetic testing
- congenital heart disease
- cardiovascular disease
- Received April 21, 2015.
- Revision received July 23, 2015.
- Accepted July 27, 2015.