![]() ![]() The emulsion-PCR (ePCR) generating template-covered beads was performed with 40 cycles. Of each DNA library, 0.34 ng was loaded onto 1 flow cell. Sequencing was carried out on the ABI SOLiD 3G+ sequencing platform according to the manufacturer's instructions. Next-Generation Resequencing and Data Analysis Placing the eluates in a Speed-Vac for drying finalized the library preparation and enrichment process after <1 week. For elution of the enriched samples, the biochip was inserted into a HybSelect elution holder, and the arrays were filled with 15 μL elution reagent and incubated at 70☌ for 30 minutes. To further improve the enrichment, a second cycle was carried out. Hybridization and all washing steps were automated and carried out by HybSelector. Each array was subsequently washed with 2 mL of SSPE at room temperature. After hybridization, each array was washed twice with 6× SSPE at room temperature and 0.5× SSPE at 45☌. ![]() Hybridization was performed overnight for 16 hours at 50☌, with agitation of the sample. The probes on each microarray were denatured with water at 80☌, and 2 μg of the sample mixture was injected automatically onto the biochip in the HybSelector (febit biomed). The adaptor-ligated genomic DNA library, which has been amplified by 2 to 12 polymerase chain reaction (PCR) cycles and 500 μg RNA per sample (transferred from baker's yeast buffered aqueous solution, 10.5 mg/mL, 5×1 mL Sigma-Aldrich Bayern, Germany) were dried in a Speed-Vac and dissolved in Hybmix-4 (febit biomed), heated to 95☌ for 5 minutes, and placed on ice. Fifty oligonucleotide-long capture probes were distributed over the coding sequences, with an average 9-bp tiling density to target both sense and antisense strands in an alternating manner. For enrichment of the 47 selected genes, 2 arrays were used as an enrichment matrix for each patient sample. 21 Each chip consisted of 8 physically separated arrays with 15 000 individual DNA oligonucleotide features, resulting in a total content of about 120 000 features. Light-activated in situ oligonucleotide synthesis was performed essentially as described, using a digital micromirror device (Texas Instruments Dallas, TX) for light-directed activation on an activated microfluidic array consisting of a glass/silicon-glass sandwich (febit biomed gmbh Heidelberg, Germany). HCM, recessive DCM, familial restrictive cardiomyopathyĪF indicates atrial fibrillation ARVC, arrhythmogenic right ventricular cardiomyopathy CSD, conduction system disease LVNC, left ventricular noncompaction SCD, sudden cardiac death SVT, supraventricular tachycardia VT, ventricular tachycardia. Selected Cardiomyopathy Disease Genes GeneĭCM, striate palmoplantar keratoderma, woolly hair, ARVC 16 – 20 Here, we established microarray-based target enrichment followed by SOLiD NGS for the comprehensive and cost-efficient genetic diagnosis of cardiomyopathies. We and others have shown recently that microarray-based sequence enrichment is feasible and highly efficient before sequencing. 14, 15 Until now, however, cost-efficient NGS for the diagnosis of inherited diseases has not entered clinical practice, especially because of a lack of efficient reduction of genomic complexity and established protocols. Introduction and steady improvement of next-generation sequencing (NGS) technologies recently have led to a dramatic increase in parallelism, and hence, sequencing throughput, overcoming limitations of traditional capillary sequencing. 1 – 10 Accordingly, in-depth genetic testing of patients with cardiomyopathy often is not possible with reasonable efforts and costs 11 however, this is of immense clinical importance, because some genetic forms of heart muscle diseases are associated with disease manifestation at an early age, an overall poor prognosis, or a high incidence of sudden cardiac death. For instance, inherited forms of heart muscle diseases, so-called cardiomyopathies, can be caused by mutations in at least 30 different genes. For most of these inherited disorders, more than 1 disease gene could be identified. Customer Service and Ordering Informationīecause of steadily increasing efforts in genomic research over the past decade, the genetic pathogenesis of many heritable diseases could be resolved.Stroke: Vascular and Interventional Neurology.Journal of the American Heart Association (JAHA).Circ: Cardiovascular Quality & Outcomes.Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB). ![]()
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