A low-cost exon capture method suitable for large-scale screening of genetic deafness by the massively-parallel sequencing approach

Abstract

Current major barriers for using next-generation sequencing (NGS) technologies in genetic mutation screening on an epidemiological scale appear to be the high accuracy demanded by clinical applications and high per-sample cost. How to achieve high efficiency in enriching targeted disease genes while keeping a low cost/sample is a key technical hurdle to overcome. We validated a cDNA-probe-based approach for capturing exons of a group of genes known to cause deafness. Polymerase chain reaction amplicons were made from cDNA clones of the targeted genes and used as bait probes in hybridization for capturing human genomic DNA (gDNA) fragments. The cDNA library containing the clones of targeted genes provided a readily available, low-cost, and regenerable source for producing capture probes with standard molecular biology equipment. Captured gDNA fragments by our method were sequenced by the Illumina NGS platform. Results demonstrated that targeted exons captured by our approach achieved specificity, multiplexicity, uniformity, and depth of coverage suitable for accurate sequencing applications by the NGS systems. Reliable genotype calls for various homozygous and heterozygous mutations were achieved. The results were confirmed independently by conventional Sanger sequencing. The method validated here could be readily expanded to include all-known deafness genes for applications such as genetic hearing screening in newborns. The high coverage depth and cost benefits of the cDNA-probe-based exon capture approach may also facilitate widespread applications in clinical practices beyond screening mutations in deafness genes.

FIG. 1.

Distribution of targeted exon lengths and their enrichment checked by qPCR. (A) Distribution histogram of exon lengths for the five targeted deafness genes. Those bins fall in the dashed box are exons shorter than 50 bps. (B) Schematic diagram showing the design for the location and size of qPCR primers used in the quality control (QC) step. (C) Typical qPCR results obtained from enriched samples, nonenriched samples, and water control samples for exon 20 of SLC26A4. (D) Typical qPCR results obtained from enriched samples, nonenriched samples, and water control samples for exon 3 of SLC26A4. qPCR, quantitative polymerase chain reaction; gDNA, genomic DNA; bps, base pairs. Color images available online at www.liebertonline.com/gtmb

FIG. 2.

Sequencing coverage in the targeted coding regions of GJB2 (A), GJB3 (B), GJB6 (C), and exon 20 of SLC26A4 (D). Black horizontal bars underneath each data trace represents the coding region shown by arrows. General exon structures of the genes are illustrated by diagrams above the data traces. Gray vertical bars represent the standard error of means obtained from different patient gDNA samples. Color images available online at www.liebertonline.com/gtmb

FIG. 3.

Results showing alignment of captured gDNA fragments in the genomic region of MYO15A. (A) Four examples of coverage in the genomic region of MYO15A. Relative depth of coverage is coded by a pseudo-colored scale bar given on top. Arrow points to the exon 8 of MYO15A, which has only 6 bps. (B) Sequencing coverage for the exon 8 of MYO15A aligned to the reference human genome. Horizontal bar underneath the data trace represents the coding region. Gray vertical bars represent the standard error of means obtained from different patient gDNA samples. (C) One example of a distribution histogram for the coverage depth in all the coding regions of MYO15A. The Y-axis is the cumulative coverage number, and the X-axis is the coverage fold. Color images available online at www.liebertonline.com/gtmb