• Lopez-Doriga, Adriana
• Lazaro, Conxi
• Valle, Jesus Del
• Castellsague, Ester
• Gonzalez, Sara
• Moreno, Victor
• Tornero, Eva
• Capella, Gabriel
• Feliubadalo, Lidia
• Pineda, Marta

Abstract

<jats:title>Abstract</jats:title><jats:p>One of the emerging Next Generation Sequencing (NGS) applications is amplicon resequencing, which can be applied to screen for mutations in defined genes with diagnostic value. However, in this scenario, methods and protocols are still poorly developed. To address this need, we have established a study workflow that integrates experimental work and bioinformatics analyses. Our Unit of Genetic Testing for Hereditary Cancer is using kits for Multiplex Amplification of Specific Targets for Resequencing (MASTR) from the Multiplicom Company in order to generate highly homogeneous gene specific libraries for point mutation detection. Currently we have completed a proof of concept for the Breast Cancer Susceptibility kit (BRCA1 and BRCA2). We analysed a training set of 267 variants in 28 samples, and a validation set of 137 variants in 14 samples. Results from the GS Junior were combined with those resulting from the analysis of the homopolymer sequences in each gene. Sanger confirmation was performed in all the identified DNA variants. Low coverage (&amp;lt;=38x; 1.2% of the target) regions were also Sanger sequenced. The bioinformatics analysis combined the Variant Identification Pipeline software (VIP, De Shrijver, JM et al. BMC Bioinformatics 2010) together with a set of in-house designed R scripts in order to obtain both a Coverage Report as well as a Variant Calling Report. The first training set of 28 samples showed a sensitivity and specificity of 97.6% and 100%, respectively. After the improvement of the kit and the analysis pipeline, the validation set of 14 samples demonstrated an excellent specificity and sensitivity of the pipeline (100% both). In conclusion, we showed that a combined algorithm both at the experimental as well as at the bioinformatics level allows the interpretation of NGS results with a good specificity and sensitivity for diagnosis purposes. We are now finishing our proof of concept for genes responsible for Hereditary Colorectal Cancer, results will be presented at the meeting. This work has been supported by Spanish Ministry of Science and Innovation, Carlos III Health Institute ISCIII (RD06/0020/1050; RD06/0020/1051; PI10/01422; CA10/01474); The Government of Catalonia (2009SGR290) and The Spanish Association Against Cancer.</jats:p><jats:p>Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4445. doi:1538-7445.AM2012-4445</jats:p>

Topics

• impedance spectroscopy
• susceptibility
• homopolymer