Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis

J Mol Diagn. 2016 Sep;18(5):657-667. doi: 10.1016/j.jmoldx.2016.04.002. Epub 2016 Jul 2.


Advances in next-generation sequencing (NGS) have facilitated parallel analysis of multiple genes enabling the implementation of cost-effective, rapid, and high-throughput methods for the molecular diagnosis of multiple genetic conditions, including the identification of BRCA1 and BRCA2 mutations in high-risk patients for hereditary breast and ovarian cancer. We clinically validated a NGS pipeline designed to replace Sanger sequencing and multiplex ligation-dependent probe amplification analysis and to facilitate detection of sequence and copy number alterations in a single test focusing on a BRCA1/BRCA2 gene analysis panel. Our custom capture library covers 46 exons, including BRCA1 exons 2, 3, and 5 to 24 and BRCA2 exons 2 to 27, with 20 nucleotides of intronic regions both 5' and 3' of each exon. We analyzed 402 retrospective patients, with previous Sanger sequencing and multiplex ligation-dependent probe amplification results, and 240 clinical prospective patients. One-hundred eighty-three unique variants, including sequence and copy number variants, were detected in the retrospective (n = 95) and prospective (n = 88) cohorts. This standardized NGS pipeline demonstrated 100% sensitivity and 100% specificity, uniformity, and high-depth nucleotide coverage per sample (approximately 7000 reads per nucleotide). Subsequently, the NGS pipeline was applied to the analysis of larger gene panels, which have shown similar uniformity, sample-to-sample reproducibility in coverage distribution, and sensitivity and specificity for detection of sequence and copy number variants.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Cohort Studies
  • DNA Copy Number Variations
  • DNA, Mitochondrial
  • Gene Library
  • Genes, BRCA1
  • Genes, BRCA2
  • Genetic Testing / methods*
  • Genetic Testing / standards
  • Genotype
  • High-Throughput Nucleotide Sequencing / methods
  • High-Throughput Nucleotide Sequencing / standards*
  • Humans
  • Multiplex Polymerase Chain Reaction / methods
  • Multiplex Polymerase Chain Reaction / standards*
  • Mutation
  • Neoplasms / diagnosis
  • Neoplasms / genetics
  • Nucleic Acid Amplification Techniques / methods
  • Nucleic Acid Amplification Techniques / standards*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Sequence Analysis, DNA / methods
  • Sequence Analysis, DNA / standards*


  • DNA, Mitochondrial