Abstract Molecular analysis of the BRCA1 and BRCA2 genes is increasingly requested for the genetic counseling of women at risk of breast and/or ovarian cancer. The development of new technologies renders this analysis more accessible by increasing throughput and decreasing costs. We compared the sequencing of amplicons covering the coding genomic sequence of the BRCA genes using the GS-Flex sequencer (Roche 454) and fluorescent dideoxy sequencing. Informed consent for genetic analysis was obtained from subjects with hereditary risk of breast and/or ovarian cancer validated in our oncogenetics consultation, and DNA extracted from peripheral blood by standard methods. Forty amplicons were designed for BRCA1 and 44 for BRCA2, covering all coding exons and intron-exon boundaries. Iso-PCR libraries of the 84 amplicons were sequenced according to Roche's protocol, with expected coverage of 133x. Amplicons were analyzed in parallel using standard dideoxy techniques. Dideoxy results were not known to the personnel analyzing the pyrosequencing data. The 40 BRCA1 amplicons were covered at an average depth of about 100 reads, although individual amplicons varied considerably. Heterozygous variants were represented in 23 to 75 % of reads, and approached 50% with increasing read numbers. We validated the sequence of 82 to 92% of BRCA1 amplicons for 64 samples. Two amplicons were never validated due to homopolymer sequences appearing systematically to be mutated. Five deleterious mutations were confirmed by dideoxy sequencing. For the 48 samples for which dideoxy analysis was complete, 234 variations were observed using pyrosequencing and 239 using dideoxy sequencing. Differences in the observation of variants could be attributed to notation errors for dideoxy sequences or insufficient coverage for pyrosequences. The parameters for validation were therefore adjusted to a minimum of 40 pyrosequence reads, with >20 % of reads carrying the variant. For BRCA2, approximately 1/4 of amplicons could not be validated, usually because of excessively long amplicons. Four amplicons were not validated due to imprecise reading of specific homopolymers. In spite of this partial analysis, eight deleterious mutations were observed among the 48 samples, all confirmed by dideoxy sequencing. In conclusion, we found a very good correspondence in the results of the two sequence techniques. In our current configuration of 16 samples and 84 amplicons analyzed in parallel, the cost and the time necessary was comparable. The coming availability of GS-Titanium chemistry for amplicon analysis, however, will prolong the pyrosequence reads to about 400 bases and also approximately double the number of exploitable reads. This increased capacity will permit the multiplexing of samples and analysis of 64 samples simultaneously, in a timely and cost-effective manner. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3988.