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Next-Generation Sequencing Meets Genetic Diagnostics: Development of a Comprehensive Workflow for the Analysis of BRCA1 and BRCA2 Genes

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Next-Generation Sequencing Meets Genetic Diagnostics: Development of a Comprehensive Workflow for the Analysis of BRCA1 and BRCA2 Genes European Journal of Human Genetics (2013) 21, 864–870 & 2013 Macmillan Publishers Limited All rights reserved 1018-4813/13 www.nature.com/ejhg ARTICLE Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes Lı´dia Feliubadalo´ 1,6, Adriana Lopez-Doriga2,6, Ester Castellsague´1,6, Jesu´s del Valle1, Mireia Mene´ndez1, Eva Tornero1, Eva Montes1, Raquel Cuesta1, Carolina Go´mez1, Olga Campos1, Marta Pineda1, Sara Gonza´lez1, Victor Moreno3, Joan Brunet4, Ignacio Blanco1, Eduard Serra5, Gabriel Capella´1 and Conxi La´zaro*,1 Next-generation sequencing (NGS) is changing genetic diagnosis due to its huge sequencing capacity and cost-effectiveness. The aim of this study was to develop an NGS-based workflow for routine diagnostics for hereditary breast and ovarian cancer syndrome (HBOCS), to improve genetic testing for BRCA1 and BRCA2. A NGS-based workflow was designed using BRCA MASTR kit amplicon libraries followed by GS Junior pyrosequencing. Data analysis combined Variant Identification Pipeline freely available software and ad hoc R scripts, including a cascade of filters to generate coverage and variant calling reports. A BRCA homopolymer assay was performed in parallel. A research scheme was designed in two parts. A Training Set of 28 DNA samples containing 23 unique pathogenic mutations and 213 other variants (33 unique) was used. The workflow was validated in a set of 14 samples from HBOCS families in parallel with the current diagnostic workflow (Validation Set). The NGS-based workflow developed permitted the identification of all pathogenic mutations and genetic variants, including those located in or close to homopolymers. The use of NGS for detecting copy-number alterations was also investigated. The workflow meets the sensitivity and specificity requirements for the genetic diagnosis of HBOCS and improves on the cost-effectiveness of current approaches. European Journal of Human Genetics (2013) 21, 864–870; doi:10.1038/ejhg.2012.270; published online 19 December 2012 Keywords: Next-generation sequencing; hereditary breast and ovarian cancer syndrome; BRCA1; BRCA2; genetic testing; molecular diagnostics INTRODUCTION and BRCA2 are the two main highly penetrant genes that predispose Next-generation sequencing (NGS) is an increasingly used technology to hereditary breast and ovarian cancer syndrome (HBOCS).5 that generates up to gigabases of DNA reads at high speed and with Molecular diagnosis of HBOCS is essential for the provision of low cost per base. This high-throughput technology, based on genetic counseling and to establish preventive screening and massively parallel sequencing of spatially separated DNA molecules, therapeutic strategies.6 Although direct Sanger sequencing is is currently used with several available platforms, such as the Genome considered the gold standard for the analysis of BRCA1 and BRCA2 Sequencer (Roche-454 Life Sciences, Indianapolis, IN, USA), mutations, their large size (5592 bp and 10257 bp, respectively), the Genome Analyzer/HiSeq/MiSeq (Illumina-Solexa, San Diego, and lack of mutation hot spots (see Breast Cancer Information CA, USA), the SOLiD System, Ion PGM/Ion Proton (Ion Torrent- Core database: http://www.research.nhgri.nih.gov/bic/) mean useful Invitrogen, Carlsbad, CA, USA), and the HeliScope from Helicos prescreening strategies.7–9 Moreover, large genomic rearrangements BioSciences (Cambridge, MA, USA).1,2 In Roche-454 technology, (LGRs) of these genes require the use of other complementary bead-attached DNA fragments clonally amplified in a water-in-oil techniques.10,11 The development of cost-effective BRCA mutation emulsion (emulsion PCR) are deposited in single-bead capacity detection workflows will not only benefit the genetic counseling wells of a plate over which nucleotides flow sequentially, releasing process for patients with HBOCS but will also enhance the process of chemiluminescence only when a nucleotide is correctly incorporated selecting patients for personalized treatments, as could be the case of (pyrosequencing). In molecular diagnostics, targeted genomic PARP inhibitors, for example. Mutation analyses of BRCA1 and resequencing of pooled samples from different individuals benefits BRCA2 using NGS have been already performed for high-capacity from the high throughput achieved by using NGS technology. To NGS platforms, such as the 454 FLX (Roche),12 the Helicos enrich the target fragments to be resequenced in this type of gene- (Heliscope),13 the Genome Analyzer (Illumina)4 and, very recently, centric approach, PCR-based methods are generally used.3,4 BRCA1 the GS Junior instrument.14 Most of these studies used large-capacity 1Hereditary Cancer Program, Catalan Institute of Oncology (ICO-IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain; 2Institut d’Investigacions Biome`diques de Bellvitge (IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain; 3Prevention Program, Catalan Institute of Oncology (ICO-IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain; 4Hereditary Cancer Program, Catalan Institute of Oncology (ICO-IdIBGi), Girona, Spain; 5Institut de Medicina Predictiva i Personalitzada del Ca`ncer (IMPPC), Badalona, Barcelona, Spain 6These authors contributed equally to this work. *Correspondence: Dr C La´zaro, Hereditary Cancer Program, Molecular Diagnosis Unit, Laboratori de Recerca Translacional, Institut Catala` d’Oncologia (ICO-IDIBELL), Hospital Duran i Reynals, Gran Via 199-203, L’Hospitalet de Llobregat, 08908 Barcelona, Spain. Tel: þ 34 932607342; Fax: þ 34 932607466; E-mail: [email protected] Received 9 July 2012; revised 28 September 2012; accepted 13 November 2012; published online 19 December 2012 Next-generation sequencing and BRCAs diagnosis L Feliubadalo´ et al 865 platforms that generally exceed the demand of most mid-sized genetic of exon 14, deletion of exon 20, deletion of exon 22, and duplication of exons testing laboratories and whose approaches are difficult to translate to 9–24 in BRCA1, and deletion of exons 1–24 and deletion of exon 2 in BRCA2. benchtop next-generation sequencers. Only one of the studies used To assess copy number for each amplicon, a methodology described elsewhere 3 small-scale equipment, the GS Junior, but the number of samples was applied. Briefly, the relative read count of an amplicon was determined tested is very small and no discussion is offered regarding how to as the ratio of the read count for that amplicon over the sum of all gene amplicons for the other gene in the specific multiplex to which the overcome the main problem associated with pyrosequencing, that is, 14 amplicon belongs. Hence, to analyze BRCA1 amplicons, we used the sum of DNA lectures in homopolymeric regions. Here, we present a BRCA2 amplicons from the same multiplex, and vice versa. Next, intersample rigorous sensitivity and specificity analysis of our newly established normalization was performed, dividing each ratio by the average of the control HBOCS workflow for genetic testing of BRCA genes using a small- samples in the same experiment (at least three controls were used). capacity next-generation instrument. We present data from a Training Set and from a Validation Set of samples. We demonstrate that a Homopolymer analysis combined approach using the GS Junior platform and an specific To treat homopolymers, the BRCA HP v2.0 (Multiplicom, Niel, Belgium) assay assay for homopolymeric tracts with a custom bioinformatics pipeline was used. This kit targets all BRCA1- and BRCA2-coding homopolymer provides accurate results that can be used for genetic diagnosis. stretches of 6 bp or longer by producing 29 PCR products in two multiplex reactions. Fragment length was assessed by capillary electrophoresis (3730 ABi MATERIALS AND METHODS sequencer, Applied Biosystems, Foster City, CA, USA) and visualized with the MAQ-S software (Multiplicom). Samples analyzed In our unit, a multistep workflow including conformation-sensitive capillary electrophoresis9 as a prescreening method for analysis of BRCA mutations was Sanger sequencing used (Supplementary Figure 1). A total of 28 DNA samples previously All fragments with coverage under 38 Â and all non-polymorphic DNA characterized by this workflow were used as a Training Set to setup our variants identified were sequenced by Sanger. NGS workflow, and 14 new DNAs were used as a Validation Set (see Experimental design in the Results section). To properly compare NGS with our workflow, only variants in heterozygosity were considered (as homozygous RESULTS variants are not detected by conformation-sensitive capillary electrophoresis). Experimental design This study was approved by our Institutional Review Board. The Training Set (28 samples analyzed in two experiments) contained 23 unique pathogenic mutations and 204 (33 unique) non-pathogenic Multiplex PCR-based target amplification and resequencing mutations or mutations with unknown significance DNA variants Target amplification of BRCA1 and BRCA2 was achieved using BRCA MASTR (Supplementary Table 1) (Figure 1). In the Validation Set, 14 samples assays following manufacturer’s instructions (http://www.multiplicom.com). were blindly sequenced together with a sample containing a multi- Several versions of the kit were used as they were released. Briefly, the assay exon duplication in BRCA1 (Figure 1). To better assess the usefulness generates a library of specific amplicons in two rounds of PCR: a first of this approach to detect LGR, a set of seven positive samples multiplex PCR that amplifies
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