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


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.
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.
Kitchener D
KD
Wilson
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.
Peidong
P
Shen
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.
Eula
E
Fung
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.
Ioannis
I
Karakikes
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.
Angela
A
Zhang
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.
Kolsoum
K
InanlooRahatloo
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.
Justin
J
Odegaard
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.
Karim
K
Sallam
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.
Ronald W
RW
Davis
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.
George K
GK
Lui
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.
Euan A
EA
Ashley
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.
Curt
C
Scharfe
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.
Joseph C
JC
Wu
D001483:Base Sequence; D003362:Cost-Benefit Analysis; D018390:Gene Targeting / Q000191:economics / Q000379:methods; D006331:Heart Diseases / Q000175:diagnosis / Q000191:economics* / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics* / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D013997:Time Factors
cardiomyopathies; cardiovascular diseases; congenital heart disease; genetic testing; microRNAs; next generation sequencing