title: Improved Efficiency and Reliability of NGS Amplicon Sequencing Data Analysis for Genetic Diagnostic Procedures Using AGSA Software.
abstract: Screening forBRCAmutations in women with familial risk of breast or ovarian cancer is an ideal situation for high-throughput sequencing, providing large amounts of low cost data. However, 454, Roche, and Ion Torrent, Thermo Fisher, technologies produce homopolymer-associated indel errors, complicating their use in routine diagnostics. We developed software, named AGSA, which helps to detect false positive mutations in homopolymeric sequences. Seventy-two familial breast cancer cases were analysed in parallel by amplicon 454 pyrosequencing and Sanger dideoxy sequencing for genetic variations of theBRCAgenes. All 565 variants detected by dideoxy sequencing were also detected by pyrosequencing. Furthermore, pyrosequencing detected 42 variants that were missed with Sanger technique. Six amplicons contained homopolymer tracts in the coding sequence that were systematically misread by the software supplied by Roche. Read data plotted as histograms by AGSA software aided the analysis considerably and allowed validation of the majority of homopolymers. As an optimisation, additional 250 patients were analysed using microfluidic amplification of regions of interest (Access Array Fluidigm) of the BRCA genes, followed by 454 sequencing and AGSA analysis. AGSA complements a complete line of high-throughput diagnostic sequence analysis, reducing time and costs while increasing reliability, notably for homopolymer tracts.
authors.affiliation: Département d'Oncogénétique, Centre Jean Perrin, 63011 Clermont-Ferrand, France.
authors.forename: Axel
authors.initials: A
authors.lastname: Poulet
authors.affiliation: Département d'Oncogénétique, Centre Jean Perrin, 63011 Clermont-Ferrand, France; EA 4677 ERTICa, Université Clermont Auvergne, 63000 Clermont-Ferrand, France.
authors.forename: Maud
authors.initials: M
authors.lastname: Privat
authors.affiliation: Département d'Oncogénétique, Centre Jean Perrin, 63011 Clermont-Ferrand, France; EA 4677 ERTICa, Université Clermont Auvergne, 63000 Clermont-Ferrand, France.
authors.forename: Flora
authors.identifier: 0000-0002-3637-5504
authors.initials: F
authors.lastname: Ponelle
authors.affiliation: Département d'Oncogénétique, Centre Jean Perrin, 63011 Clermont-Ferrand, France; EA 4677 ERTICa, Université Clermont Auvergne, 63000 Clermont-Ferrand, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Viala
authors.affiliation: Département d'Oncogénétique, Centre Jean Perrin, 63011 Clermont-Ferrand, France.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Decousus
authors.affiliation: Département d'Oncogénétique, Centre Jean Perrin, 63011 Clermont-Ferrand, France.
authors.forename: Axel
authors.initials: A
authors.lastname: Perin
authors.affiliation: Département d'Oncogénétique, Centre Jean Perrin, 63011 Clermont-Ferrand, France; EA 4677 ERTICa, Université Clermont Auvergne, 63000 Clermont-Ferrand, France.
authors.forename: Laurence
authors.initials: L
authors.lastname: Lafarge
authors.affiliation: EA 4677 ERTICa, Université Clermont Auvergne, 63000 Clermont-Ferrand, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Ollier
authors.affiliation: Naef K. Basile Cancer Institute (NKBCI), Breast Center of Excellence, American University of Beirut Medical Center, Beirut 1107 2020, Lebanon.
authors.forename: Nagi S
authors.initials: NS
authors.lastname: El Saghir
authors.affiliation: Département d'Oncogénétique, Centre Jean Perrin, 63011 Clermont-Ferrand, France; EA 4677 ERTICa, Université Clermont Auvergne, 63000 Clermont-Ferrand, France.
authors.forename: Nancy
authors.identifier: 0000-0002-7816-2398
authors.initials: N
authors.lastname: Uhrhammer
authors.affiliation: Département d'Oncogénétique, Centre Jean Perrin, 63011 Clermont-Ferrand, France; EA 4677 ERTICa, Université Clermont Auvergne, 63000 Clermont-Ferrand, France.
authors.forename: Yves-Jean
authors.identifier: 0000-0001-9378-6353
authors.initials: YJ
authors.lastname: Bignon
authors.affiliation: EA 4677 ERTICa, Université Clermont Auvergne, 63000 Clermont-Ferrand, France.
authors.forename: Yannick
authors.identifier: 0000-0002-9624-8195
authors.initials: Y
authors.lastname: Bidet