Evaluation of Second-Generation Sequencing of 19 Dilated Cardiomyopathy Genes for Clinical Applications

Medical sequencing for diseases with locus and allelic heterogeneities has been limited by the high cost and low throughput of traditional sequencing technologies. “Second-generation” sequencing (SGS) technologies allow the parallel processing of a large number of genes and, therefore, offer great promise for medical sequencing; however, their use in clinical laboratories is still in its infancy. Our laboratory offers clinical resequencing for dilated cardiomyopathy (DCM) using an array-based platform that interrogates 19 of more than 30 genes known to cause DCM. We explored both the feasibility and cost effectiveness of using PCR amplification followed by SGS technology for sequencing these 19 genes in a set of five samples enriched for known sequence alterations (109 unique substitutions and 27 insertions and deletions). While the analytical sensitivity for substitutions was comparable to that of the DCM array (98%), SGS technology performed better than the DCM array for insertions and deletions (90.6% versus 58%). Overall, SGS performed substantially better than did the current array-based testing platform; however, the operational cost and projected turnaround time do not meet our current standards. Therefore, efficient capture methods and/or sample pooling strategies that shorten the turnaround time and decrease reagent and labor costs are needed before implementing this platform into routine clinical applications.Genetic testing for disorders with locus and allelic heterogeneity has been a challenge due to the high cost of sequencing entire coding regions of numerous genes. Classically, medical sequencing has used capillary-based “Sanger” sequencing technology and this has remained the gold standard for three decades. However, this method is expensive and has low throughput. It was not until novel technology platforms emerged that comprehensive testing became within reach. One such technology is array-based sequencing, which drastically increased the number of genes that could be analyzed simultaneously.^{1,2,3,4,5,6,7} We previously developed an array-based resequencing test for dilated cardiomyopathy (DCM), that doubled the number of genes analyzed in parallel while reducing test cost and turnaround time.⁶ However, this technology has two major drawbacks, particularly in a clinical setting. First, resequencing arrays have a poor detection rate for insertions and deletions (in/dels).^8,9 Second, the somewhat static nature of the chip design makes it time-consuming and impractical to add new content, especially in a disease area like DCM where genes are being discovered at a rapid pace. As such, novel technologies are needed to provide comprehensive sequencing of all DCM genes with high analytical sensitivity and with the goal of further reducing the cost of diagnostic testing.Second-generation sequencing (SGS) technologies, commonly referred to as “next-generation sequencing,” are based on massive parallel sequencing of millions of DNA templates through cycles of enzymatic treatment and image-based data acquisition. Several platforms have been developed in the last few years based on different biochemistries and cluster generation.^10,11,12 The most commonly used platforms include the Illumina Genome Analyzer (GAII; Solexa Technology),¹³ Roche Applied Sciences (454 sequencing),¹⁴ and ABI-Applied Biosystems (SOLiD platform).¹⁵ These technologies have been adopted for a wide variety of research applications^10,11,16 and have now matured sufficiently to be considered as robust enough for clinical applications. For example, SGS has recently been applied to resequencing the NF1 locus as well as the mitochondrial and small-cell lung cancer genomes.^17,18,19 In addition, whole exome resequencing has been conducted to discover genes underlying rare monogenic diseases.^20,21,22,23The specific advantages offered by SGS are twofold: the low cost per base and the ability to sequence millions of reads in parallel, allowing for simultaneous analysis of a large number of genes. However, these are offset by two major disadvantages: shorter reads and reduced accuracy as compared to Sanger sequencing.¹¹ Despite their disadvantages, improvements in these technologies promise to meet the technical requirements and strict quality standards of clinical diagnostics including analytical sensitivity, reproducibility and cost effectiveness.Several methodological approaches to capturing target gene regions have evolved to complement the higher capacity and throughput of novel sequencing technologies. These methods involve constructing and enriching a DNA “library” and use both PCR and/or hybridization as the mode of target selection. The classic PCR approach to library generation requires amplification of target regions, pooling, concatenation, shearing and ligation of adaptors and sequencing primers. Secondary droplet-based microfluidic technologies have evolved to facilitate high-throughput PCR in picoliter droplets.²⁴ With hybridization-based methods, libraries are constructed by shearing total gDNA followed by adaptor ligation and hybridization to oligonucleotides that are complementary to the desired target. Hybridization can be performed either on a solid surface array, on a filter, or by hybridization in solution^21,25,26,27 A third general approach to target selection uses molecular inversion probes. Molecular inversion probes consist of two primers linked together by a backbone and, similar to PCR, bind to specific target DNA. This is followed by gap filling, ligation, and enrichment steps.^28,29Important considerations in choosing a method include total amount of starting DNA, the size of the target region and the types of sequence alteration under investigation. Technical parameters such as target specificity, uniformity, and completeness of coverage also vary with each methodology.¹² Although each method has advantages or disadvantages depending on the application, we selected PCR, a well-established and robust targeted amplification method for this study.Leveraging on our array-based resequencing test for DCM,⁶ we evaluated the suitability of targeted PCR followed by Illumina GAII resequencing for a clinical testing environment. We used a number of samples enriched for a large number of substitutions as well as insertions and deletions in DCM genes to assess the analytical performance parameters. We also evaluated the cost and turnaround time of such a test and compared it to our current, array-based resequencing test.