Targeted BRCA1/2 Mutation Sequencing

Blood samples were collected and genomic DNA was extracted using the Blood Genomic DNA Extraction Kit (Shanghai Lifefeng Biotech Co., Ltd., Shanghai, China). Shrimp alkaline phosphatase enzyme was used to eliminate excess primers and deoxynucleotide triphosphates in amplification reactions. To repair the ends of the digestion reaction, bases were added. Sequencing adaptors (Illumina, San Diego, CA, USA) were ligated to the end-repaired fragments with T4 DNA ligase (Agilent, Santa Clara, CA, USA), and the fragments were then purified with magnetic beads and 80% ethanol. The HiSeq X Ten high-throughput sequencing platform (Illumina) was adopted for library sequencing, with an average sequencing depth of over 1000×. The sequencing results were aligned to the reference sequences of BRCA1 (NM_0073000) and BRCA2 (NM_000059) for mutation detection using Burrows–Wheeler Aligner.^{17 (link)}
Meanwhile, the Genome Analysis Tool Kit^{18 (link)}
was used to recalibrate mutation sites with ANNOVAR.^{19 (link)}
The interpretation of all variants referred to the American College of Medical Genetics genetic interpretation principles.^{20 (link)}