probability to develop familiar breast cancer. To detect BRCA1/2 germline mutations we developed a next-generation sequencing (NGS) routine dia- gnostic workflow, based on the Ion Torrent PGMTM System platform. The Ion AmpliSeqTM BRCA1 and BRCA2 Community Panel was handled with a semi- automatized procedure for multiplex PCR-based library preparation and se- quencing. Data analysis required the implementation of a custom designed bioinformatic pipeline for sequences alignment and for the identification, annotation and filtration of genetic variants. Sanger sequencing was perfor- med to validate candidate mutations, and to re-sequence amplicons having low NGS coverage (<50 reads per amplicon). Negative samples were ana- lyzed using the BRCA HP Kit (Multiplicom) for an effective homopolymeric stretches detection. This workflow together with the potentiality of our bio- informatic pipeline was blindly tested and validated onto a small cohort of patients previously Sanger sequenced, fine-tuning the parameter settings and resulting in a sensitivity of 100% in variant detection. Subsequently, 244 patients were analyzed thus confirming the need of a double check for the homopolymeric stretches with both NGS sequencing and BRCA HP Kit. The NGS-based workflow here proposed was able to decrease the overall

Implementation of an NGS-based workflow for BRCA1 and BRCA2 mutation screening / Artuso, Lucia; Medici, Veronica; Bernardis, Isabella; Tenedini, Elena; Artusi, Valentina; Simone, Maria Luisa; Tarugi, Patrizia Maria; Manfredini, Rossella; Cortesi, Laura; Tagliafico, Enrico. - In: EUROPEAN JOURNAL OF HUMAN GENETICS. - ISSN 1018-4813. - 23 Supplement1:(2015), pp. 290-290. ((Intervento presentato al convegno The EUROPEAN HUMAN GENETICS CONFERENCE 2015 tenutosi a Glasgow, Scotland, UK nel 6-9 June 2015.

Implementation of an NGS-based workflow for BRCA1 and BRCA2 mutation screening

ARTUSO, LUCIA;MEDICI, Veronica;BERNARDIS, ISABELLA;TENEDINI, Elena;SIMONE, Maria Luisa;TARUGI, Patrizia Maria;MANFREDINI, Rossella;TAGLIAFICO, Enrico
2015

Abstract

probability to develop familiar breast cancer. To detect BRCA1/2 germline mutations we developed a next-generation sequencing (NGS) routine dia- gnostic workflow, based on the Ion Torrent PGMTM System platform. The Ion AmpliSeqTM BRCA1 and BRCA2 Community Panel was handled with a semi- automatized procedure for multiplex PCR-based library preparation and se- quencing. Data analysis required the implementation of a custom designed bioinformatic pipeline for sequences alignment and for the identification, annotation and filtration of genetic variants. Sanger sequencing was perfor- med to validate candidate mutations, and to re-sequence amplicons having low NGS coverage (<50 reads per amplicon). Negative samples were ana- lyzed using the BRCA HP Kit (Multiplicom) for an effective homopolymeric stretches detection. This workflow together with the potentiality of our bio- informatic pipeline was blindly tested and validated onto a small cohort of patients previously Sanger sequenced, fine-tuning the parameter settings and resulting in a sensitivity of 100% in variant detection. Subsequently, 244 patients were analyzed thus confirming the need of a double check for the homopolymeric stretches with both NGS sequencing and BRCA HP Kit. The NGS-based workflow here proposed was able to decrease the overall
The EUROPEAN HUMAN GENETICS CONFERENCE 2015
Glasgow, Scotland, UK
6-9 June 2015
Artuso, Lucia; Medici, Veronica; Bernardis, Isabella; Tenedini, Elena; Artusi, Valentina; Simone, Maria Luisa; Tarugi, Patrizia Maria; Manfredini, Rossella; Cortesi, Laura; Tagliafico, Enrico
Implementation of an NGS-based workflow for BRCA1 and BRCA2 mutation screening / Artuso, Lucia; Medici, Veronica; Bernardis, Isabella; Tenedini, Elena; Artusi, Valentina; Simone, Maria Luisa; Tarugi, Patrizia Maria; Manfredini, Rossella; Cortesi, Laura; Tagliafico, Enrico. - In: EUROPEAN JOURNAL OF HUMAN GENETICS. - ISSN 1018-4813. - 23 Supplement1:(2015), pp. 290-290. ((Intervento presentato al convegno The EUROPEAN HUMAN GENETICS CONFERENCE 2015 tenutosi a Glasgow, Scotland, UK nel 6-9 June 2015.
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