Development and analytical validation of a 25-gene next generation sequencing panel that includes the BRCA1 and BRCA2 genes to assess hereditary cancer risk

Thaddeus Judkins, Benoît Leclair, Karla Bowles, Natalia Gutin, Jeff Trost, James McCulloch, Satish Bhatnagar, Adam Murray, Jonathan Craft, Bryan Wardell, Mark Bastian, Jeffrey Mitchell, Jian Chen, Thanh Tran, Deborah Williams, Jennifer Potter, Srikanth Jammulapati, Michael Perry, Brian Morris, Benjamin RoaKirsten Timms

Research output: Contribution to journalArticle


Background: Germline DNA mutations that increase the susceptibility of a patient to certain cancers have been identified in various genes, and patients can be screened for mutations in these genes to assess their level of risk for developing cancer. Traditional methods using Sanger sequencing focus on small groups of genes and therefore are unable to screen for numerous genes from several patients simultaneously. The goal of the present study was to validate a 25-gene panel to assess genetic risk for cancer in 8 different tissues using next generation sequencing (NGS) techniques. Methods: Twenty-five genes associated with hereditary cancer syndromes were selected for development of a panel to screen for risk of these cancers using NGS. In an initial technical assessment, NGS results for BRCA1 and BRCA2 were compared with Sanger sequencing in 1864 anonymized DNA samples from patients who had undergone previous clinical testing. Next, the entire gene panel was validated using parallel NGS and Sanger sequencing in 100 anonymized DNA samples. Large rearrangement analysis was validated using NGS, microarray comparative genomic hybridization (CGH), and multiplex ligation-dependent probe amplification analyses (MLPA). Results: NGS identified 15,877 sequence variants, while Sanger sequencing identified 15,878 in the BRCA1 and BRCA2 comparison study of the same regions. Based on these results, the NGS process was refined prior to the validation of the full gene panel. In the validation study, NGS and Sanger sequencing were 100% concordant for the 3,923 collective variants across all genes for an analytical sensitivity of the NGS assay of >99.92% (lower limit of 95% confidence interval). NGS, microarray CGH and MLPA correctly identified all expected positive and negative large rearrangement results for the 25-gene panel. Conclusion: This study provides a thorough validation of the 25-gene NGS panel and indicates that this analysis tool can be used to collect clinically significant information related to risk of developing hereditary cancers.

Original languageEnglish (US)
Article number215
JournalBMC cancer
Issue number1
StatePublished - Apr 2 2015



  • BRCA1
  • BRCA2
  • Hereditary cancer
  • Next generation sequencing

ASJC Scopus subject areas

  • Genetics
  • Oncology
  • Cancer Research

Cite this

Judkins, T., Leclair, B., Bowles, K., Gutin, N., Trost, J., McCulloch, J., Bhatnagar, S., Murray, A., Craft, J., Wardell, B., Bastian, M., Mitchell, J., Chen, J., Tran, T., Williams, D., Potter, J., Jammulapati, S., Perry, M., Morris, B., ... Timms, K. (2015). Development and analytical validation of a 25-gene next generation sequencing panel that includes the BRCA1 and BRCA2 genes to assess hereditary cancer risk. BMC cancer, 15(1), [215].