NGS of homo sapiens:adult female breast cancer
The Agilent HaloPlex Target Enrichment System (Agilent Technologies, Inc.) for Illumina Hiseq (Illumina, Inc., San Diego, CA) paired-end sequencing was used for library preparation. Tumor DNA was digested in eight different restriction reactions (225 ng DNA/reaction), with each containing two restriction enzymes. All restriction reaction results were validated using Agilent 2200 TapeStation (Agilent Technologies Inc.) with High Sensitivity D1K ScreenTape (Agilent Technologies Inc.). The collection of DNA re-striction fragments was hybridized to the HaloPlex probe capture library (54C, 3 hours). The circularized target DNA-HaloPlex probe hybrids were captured on streptavidin beads (HaloPlex Magnetic Beads, Agilent Technologies Inc.) and added DNA ligase to close nicks in the hybrids. Captured DNA was eluted with NaOH, and the cleared supernatant was transferred 20 L from each tube to a PCR Master Mix tube held on ice. Target libraries were amplified through 22 cycles of PCR and the PCR-product was purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). Finally, all samples were sequenced on Illumina NextSeq500 (Illumina, Inc.) using 150PE protocol.