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Protocol publication

[…] Germline DNA was extracted from the participants’ peripheral blood samples. We used a customized targeted capture sequencing panel (OncoRisk®, Celemics, Seoul, Korea) which included all coding sequences and intron-exon boundaries of the coding exon from 35 cancer predisposition genes (BRCA1, BRCA2, PALB2, BARD1, BRIP1, RAD51C, RAD51D, RAD50, NBN, MRE11A, ATM, CHEK2, TP53, PTEN, APC, BLM, BMPR1A, CDH1, CDK4, CDKN2A, EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH, PMS2, POLE, PRSS1, RET, SLX4, SMAD4, STK11, VLH, and WT1). Products with each capture reaction were sequenced by 100 base pair paired-end reads on a MiSeq platform (Illumina, San Diego, CA). High-quality sequencing data with an average depth of 500−1000 folds were obtained.We identified all single base pair substitutions, insertion-deletions, and copy number variants (CNVs) in each gene. Split-read-based detection of large insertions and deletions was conducted using the Pindel and Manta algorithms. CNVs detected by ExomeDepth software [] were further crosschecked with our custom pipelines, which retrieved base-level depth of coverage for each binary alignment map (BAM) file using SAMtools software ( and normalized the depths in the same batch (Additional file : Figure S1). All likely deleterious mutations were validated by Sanger sequencing, and all possible large rearrangements were confirmed by the multiplex ligation-dependent probe amplification (MLPA) method (Additional file : Figure S2).Genetic variants were classified using a five-tier system following guidelines from the American College of Medical Genetics and Genomics (ACMG) as follows: pathogenic, likely pathogenic, variants of unknown significance (VUS), likely benign, or benign/polymorphism []. We used the Sorting Intolerant From Tolerant (SIFT, and Polymorphism Phenotyping-2 (PolyPhen-2, to generate in silico predictions of several of the identified nonsynonymous variants. Using large rearrangements of exons, pathogenic and likely pathogenic variants were considered as mutations, for consistency with previous studies []. […]

Pipeline specifications

Software tools Pindel, ExomeDepth, SAMtools, SIFT, PolyPhen
Application WES analysis
Organisms Homo sapiens
Diseases Breast Neoplasms, Neoplasms