Circ Res. 2015 Sep 11;117(7):603-11. doi: 10.1161/CIRCRESAHA.115.306723. Epub 2015 Aug 11.

A Rapid, High-Quality, Cost-Effective, Comprehensive and Expandable Targeted Next-Generation Sequencing Assay for Inherited Heart Diseases.

Wilson KD¹, Shen P², Fung E², Karakikes I², Zhang A², InanlooRahatloo K², Odegaard J², Sallam K², Davis RW², Lui GK², Ashley EA², Scharfe C², Wu JC¹.

Author information

1: From the Department of Pathology (K.D.W., E.F., J.O., C.S.), and Department of Biochemistry (P.S., R.W.D.), Stanford Cardiovascular Institute (K.D.W., I.K., A.Z., K.I., J.O., K.S., G.K.L., E.A.A., J.C.W.), Stanford Genome Technology Center (P.S., E.F., R.W.D., C.S.), Department of Medicine, Division of Cardiology (K.S., G.K.L., E.A.A., J.C.W.), Stanford Adult Congenital Heart Disease Clinic (J.C.W., G.K.L.), and Department of Radiology (J.C.W.), Stanford University, CA. kitchwilson@stanford.edu joewu@stanford.edu.
2: From the Department of Pathology (K.D.W., E.F., J.O., C.S.), and Department of Biochemistry (P.S., R.W.D.), Stanford Cardiovascular Institute (K.D.W., I.K., A.Z., K.I., J.O., K.S., G.K.L., E.A.A., J.C.W.), Stanford Genome Technology Center (P.S., E.F., R.W.D., C.S.), Department of Medicine, Division of Cardiology (K.S., G.K.L., E.A.A., J.C.W.), Stanford Adult Congenital Heart Disease Clinic (J.C.W., G.K.L.), and Department of Radiology (J.C.W.), Stanford University, CA.

Abstract

RATIONALE:

Thousands of mutations across >50 genes have been implicated in inherited cardiomyopathies. However, options for sequencing this rapidly evolving gene set are limited because many sequencing services and off-the-shelf kits suffer from slow turnaround, inefficient capture of genomic DNA, and high cost. Furthermore, customization of these assays to cover emerging targets that suit individual needs is often expensive and time consuming.

OBJECTIVE:

We sought to develop a custom high throughput, clinical-grade next-generation sequencing assay for detecting cardiac disease gene mutations with improved accuracy, flexibility, turnaround, and cost.

METHODS AND RESULTS:

We used double-stranded probes (complementary long padlock probes), 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 associated with inherited cardiomyopathies, congenital heart disease, and cardiac development. Multiplexing 11 samples per sequencing run resulted in a mean base pair coverage of 420, of which 97% had >20× coverage and >99% were concordant with known heterozygous single nucleotide polymorphisms. The assay correctly detected germline variants in 24 individuals and revealed several polymorphic regions in miR-499. Total run time was 3 days at an approximate cost of $100 per sample.

CONCLUSIONS:

Accurate, high-throughput detection of mutations across numerous cardiac genes is achievable with complementary long padlock probe technology. Moreover, this format allows facile insertion of additional probes as more cardiomyopathy and congenital heart disease genes are discovered, giving researchers a powerful new tool for DNA mutation detection and discovery.