A custom capture array (NimbleGen, Roche) was designed to capture all exons (3382), splice sites and the immediately adjacent intron sequences of 193 genes known to be associated with three main types of hereditary diseases according to GeneReviews (NCBI), including single-gene disorders, malignant arrhythmias and cardiomyopathies, as well as familial malignant neoplasms.
The methods used for DNA target capture, enrichment and elution followed previously described protocols with minor modifications (Roche NimbleGen, Inc.). Briefly, genomic DNA from peripheral blood was extracted from the 110 samples using the QIAamp DNA BloodMiNi Kit (Qiagen, Hilden, Germany), and fragmentation of the DNA into fragments ranging from 200 bp to 300 bp was performed using an ultrasonoscope (Covaris S2, Massachusetts, USA). Next, 1 µg of purified DNA (quantified by NanoDrop) was treated with T4 DNA polymerase, T4 phosphonucleotide kinase and the Klenow fragment of Escherichia coli DNA polymerase to fill 5′ overhangs and remove 3′ overhangs. Terminal A residues were added following a brief incubation with dATP and the Klenow 3′-5′ exo-enzyme following standard Illumina protocols [13] . Adapter oligonucleotides from Illumina (single reads) were ligated to the ends. After the ligation was completed, successful adapter ligation was confirmed by 4-cycle PCR using a high-fidelity polymerase with PCR primers containing a custom-synthesized barcode sequence (8 bp) as a sample index signature. PCR was used to generate a library for further analysis, and the DNA adapter-ligated and indexed fragments from 10 libraries were pooled and hybridized to the capture array for 72 h. After hybridization and washing, the DNA fragments bound to the array were eluted using 300 ml of elution buffer (Qiagen, Valencia, CA, USA) for each array. A gasket (Agilent) was applied and placed on a in-house constrcted thermal elution device for 20 min at 95°C. We repeated this process once, adding 200 ml of elution buffer (Qiagen). After hybridization of the sequencing primer, base incorporation was carried out on Illumina HiSeq2000 Analyzers (following the manufacturer's standard cluster generation and sequencing protocols) for 90 cycles of sequencing per read to generate paired-end reads including 90 bps at each end and 8 bps of the index tag. Image analysis and base calling were performed using the Illumina Pipeline.
The methods used for DNA target capture, enrichment and elution followed previously described protocols with minor modifications (Roche NimbleGen, Inc.). Briefly, genomic DNA from peripheral blood was extracted from the 110 samples using the QIAamp DNA BloodMiNi Kit (Qiagen, Hilden, Germany), and fragmentation of the DNA into fragments ranging from 200 bp to 300 bp was performed using an ultrasonoscope (Covaris S2, Massachusetts, USA). Next, 1 µg of purified DNA (quantified by NanoDrop) was treated with T4 DNA polymerase, T4 phosphonucleotide kinase and the Klenow fragment of Escherichia coli DNA polymerase to fill 5′ overhangs and remove 3′ overhangs. Terminal A residues were added following a brief incubation with dATP and the Klenow 3′-5′ exo-enzyme following standard Illumina protocols [13] . Adapter oligonucleotides from Illumina (single reads) were ligated to the ends. After the ligation was completed, successful adapter ligation was confirmed by 4-cycle PCR using a high-fidelity polymerase with PCR primers containing a custom-synthesized barcode sequence (8 bp) as a sample index signature. PCR was used to generate a library for further analysis, and the DNA adapter-ligated and indexed fragments from 10 libraries were pooled and hybridized to the capture array for 72 h. After hybridization and washing, the DNA fragments bound to the array were eluted using 300 ml of elution buffer (Qiagen, Valencia, CA, USA) for each array. A gasket (Agilent) was applied and placed on a in-house constrcted thermal elution device for 20 min at 95°C. We repeated this process once, adding 200 ml of elution buffer (Qiagen). After hybridization of the sequencing primer, base incorporation was carried out on Illumina HiSeq2000 Analyzers (following the manufacturer's standard cluster generation and sequencing protocols) for 90 cycles of sequencing per read to generate paired-end reads including 90 bps at each end and 8 bps of the index tag. Image analysis and base calling were performed using the Illumina Pipeline.
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