title: Development and Validation of a Next-Generation Sequencing Assay for BRCA1 and BRCA2 Variants for the Clinical Laboratory.
abstract: The objective of this study was to design and validate a next-generation sequencing assay (NGS) to detect BRCA1 and BRCA2 mutations. We developed an assay using random shearing of genomic DNA followed by RNA bait tile hybridization and NGS sequencing on both the Illumina MiSeq and Ion Personal Gene Machine (PGM). We determined that the MiSeq Reporter software supplied with the instrument could not detect deletions greater than 9 base pairs. Therefore, we developed an alternative alignment and variant calling software, Quest Sequencing Analysis Pipeline (QSAP), that was capable of detecting large deletions and insertions. In validation studies, we used DNA from 27 stem cell lines, all with known deleterious BRCA1 or BRCA2 mutations, and DNA from 67 consented control individuals who had a total of 352 benign variants. Both the MiSeq/QSAP combination and PGM/Torrent Suite combination had 100% sensitivity for the 379 known variants in the validation series. However, the PGM/Torrent Suite combination had a lower intra- and inter-assay precision of 96.2% and 96.7%, respectively when compared to the MiSeq/QSAP combination of 100% and 99.4%, respectively. All PGM/Torrent Suite inconsistencies were false-positive variant assignments. We began commercial testing using both platforms and in the first 521 clinical samples MiSeq/QSAP had 100% sensitivity for BRCA1/2 variants, including a 64-bp deletion and a 10-bp insertion not identified by PGM/Torrent Suite, which also suffered from a high false-positive rate. Neither the MiSeq nor PGM platform with their supplied alignment and variant calling software are appropriate for a clinical laboratory BRCA sequencing test. We have developed an NGS BRCA1/2 sequencing assay, MiSeq/QSAP, with 100% analytic sensitivity and specificity in the validation set consisting of 379 variants. The MiSeq/QSAP combination has sufficient performance for use in a clinical laboratory.
authors: Department of Genetics, Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, United States of America.
authors: Charles M
authors: CM
authors: Strom
authors: Department of Genetics, Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, United States of America.
authors: Steven
authors: S
authors: Rivera
authors: Athena / Quest Diagnostics, Marlborough, MA, United States of America.
authors: Christopher
authors: C
authors: Elzinga
authors: Department of Genetics, Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, United States of America.
authors: Taraneh
authors: T
authors: Angeloni
authors: Department of Genetics, Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, United States of America.
authors: Sun Hee
authors: SH
authors: Rosenthal
authors: Department of Genetics, Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, United States of America.
authors: Dana
authors: D
authors: Goos-Root
authors: Department of Genetics, Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, United States of America.
authors: Martin
authors: M
authors: Siaw
authors: Department of Genetics, Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, United States of America.
authors: Jamie
authors: J
authors: Platt
authors: Athena / Quest Diagnostics, Marlborough, MA, United States of America.
authors: Cory
authors: C
authors: Braastadt
authors: Department of Genetics, Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, United States of America.
authors: Linda
authors: L
authors: Cheng
authors: Celera / Quest Diagnostics, Alameda, CA, United States of America.
authors: David
authors: D
authors: Ross
authors: Department of Genetics, Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, United States of America.
authors: Weimin
authors: W
authors: Sun
mesh_terms: D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D001681:Biological Assay; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics*; D045744:Cell Line, Tumor; D005260:Female; D005820:Genetic Testing / Q000295:instrumentation / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014584:User-Computer Interface
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human