Objectives: Screening for mutations in large genes is challenging in a molecular diagnostic environment. Sanger-based DNA sequencing methods are largely used; however, massively parallel sequencing (MPS) can accommodate increasing test demands and financial constraints. This study aimed to establish a simple workflow to amplify and screen all coding regions of the BRCA1 and BRCA2 (BRCA1/2) genes by Sanger-based sequencing as well as to assess a MPS approach encompassing multiplex polymerase chain reaction (PCR) and pyrosequencing. Methods: This study was conducted between July 2011 and April 2013. A total of 20 patients were included in the study who had been referred to Genetic Health Services New Zealand (Northern Hub) for BRCA1/2 mutation screening. Patients were randomly divided into a MPS evaluation and validation cohort (n = 10 patients each). Primers were designed to amplify all coding exons of BRCA1/2 (28 and 42 primer pairs, respectively). Primers overlying known variants were avoided to circumvent allelic drop-out. The MPS approach necessitated utilisation of a complementary fragment analysis assay to eliminate apparent false-positives at homopolymeric regions. Variants were filtered on the basis of their frequency and sequence depth. Results: Sanger-based sequencing of PCRamplified coding regions was successfully achieved. Sensitivity and specificity of the combined MPS/homopolymer protocol was determined to be 100% and 99.5%, respectively. Conclusion: In comparison to traditional Sangerbased sequencing, the MPS workflow led to a reduction in both cost and analysis time for BRCA1/2 screening. MPS analysis achieved high analytical sensitivity and specificity, but required complementary fragment analysis combined with Sanger-based sequencing confirmation in some instances.

Objectives: Screening for mutations in large genes is challenging in a molecular diagnostic environment. Sanger-based DNA sequencing methods are largely used; however, massively parallel sequencing (MPS) can accommodate increasing test demands and financial constraints. This study aimed to establi...