Blood from father of CHD child
Exome sequencing was carried out using the Agilent SureSelect Human All Exon v5.0 (51M) kit, the protocol from Illumina’s TruSeq Exome Enrichment Guide (SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library, Agilent). Genomic DNA libraries were prepared according to the manufacturer’s instructions (Illumina, San Diego, CA). Briefly, the 3 μg of genomic deoxyribonucleic acid (DNA) was randomly fragmented into pieces 100–500 bp in size using a Bioruptor system (Diagenode). DNA fragments between 150–250 bp were recovered by gel extraction, then end repair and size selection procedure were performed by T4 DNA poly and Klenow poly cleave 3’. An ‘A’ base was added to the 3’ end using Klenow 3’ to 5’ exo minus. Then DNA fragments were ligated to the Illumina multi-PE-adaptor. The adapter-ligated templates were purified using Agencourt AMPure SPRI beads and amplified by four-cycle ligation-mediated polymerase chain reaction (LM-PCR), under the following PCR condition: 2 min at 94 °C, four cycles of 10 s at 94 °C, 30 s at 62 °C, and 30 s at 72 °C, then 300 s at 72 °C. The LM-PCR products were hybridized to the Agilent Oligo pool for enrichment for 24 h at 65 °C. Hybridized fragments were bound to streptavidin beads and non-hybridized fragments were washed out. Captured LM-PCR products were amplified by PCR (2 min at 98 °C; 10–12 cycles for 10 s at 98 °C, 30 s at 60 °C, 30 s at 72 °C, followed by 300 s at 72 °C). The magnitude of enrichment of the samples was estimated with an Agilent 2100 bioanalyzer and quantitative PCR. The captured library was sequenced on Illumina HiSeq2000 analyzers for 126 cycles per read, to generate paired-end reads and 8 bp of the index tag (following the manufacturer’s standard sequencing protocols).