DNA library preparation and sequencing
mis-match
Genomic DNA samples were quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific). Twist Human Core Exome library preparation was performed according to the manufacturer’s guidelines (Twist Bioscience).Briefly, the genomic DNA was fragmented by acoustic shearing with a Covaris S220 instrument. Fragmented DNA was cleaned up and end repaired, as well as adenylated at the 3′ ends. Adapters were ligated to the DNA fragments and adapter-ligated DNA fragments were enriched with limited-cycle PCR. Adapter-ligated DNA fragments were validated using an Agilent TapeStation system (Agilent Technologies) and quantified using a Qubit 2.0 Fluorometer. Adapter-ligated DNA fragments were hybridized with biotinylated baits. The hybrid DNA was captured by streptavidin-coated binding beads. After extensive washing, the captured DNA was amplified and indexed with Illumina indexing primers. Post-captured DNA libraries were validated using Agilent TapeStation (Agilent) and quantified using the Qubit 2.0 Fluorometer and real-time PCR (KAPA Biosystems).The sequencing libraries were multiplexed and clustered onto multiple lanes of a flowcell. After clustering, the flowcell was loaded onto the Illumina HiSeq instrument according to the manufacturer’s instructions. The samples were sequenced using a 2 × 150 bp paired end configuration. Image analysis and base calling were conducted using the HiSeq Control Software. Raw sequence data (.bcl files) generated from Illumina HiSeq were converted into fastq files and de-multiplexed using Illumina bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification.