Targeted enrichment kits & analysis software for diagnosis of hereditary diseases based on next generation sequencing.

Development of kits and protocols for targeted next generation sequencing of 58 genes carrying potential deleterious mutations linked to breast/ovarian cancers and congenital myasthenic syndromes. Target-specific data analysis methods will be developed and validated for specificity and sensitivity.

Next Generation Sequencing (NGS) drastically reduces the cost of gene sequencing, which is the most accurate and efficient way of testing for known short mutations associated with diseases. During the last six months two desk-top sequencing instruments appeared, which provide low up-front cost, rapid turn-around time and easy sample preparation. In order to take full advantage of these advances in diagnostics, two key areas need to be improved: (1) the development of target enrichment (TE) techniques for particular set of genes associated with a disease and complementary assays for identification of mutations that cannot be detected by NGS, and (2) reliable data analysis with highly accurate variant detection and clinical validation. In this proposal the first issue is addressed by Pronto Diagnostics of Israel and the second by Omixon Zrt's subsidiary, Chemistry Logic Kft. in Hungary. Two groups of diseases were selected as model diseases for this study: (1) Breast and ovarian cancers serve as a model for a dominant disease with a high demand for genetic diagnosis. The identification of women in a higher risk to develop cancer enables a closer follow-up and diagnosis of a tumor while it is still curable. (2) Congenital myasthenic syndromes (CMS) are a group of neuromuscular diseases with a recessive inheritance in most cases. The identification of the responsible mutation to CMS enables a selection of a better therapy. Hybridization of the target sequences to complementary oligonucleotide probes and/or PCR amplification, will be used by Pronto Diagnostics to enrich the coding and untranslated exons, as well as some intronic regions, of at least 44 genes known or suspected as linked with breast and ovarian cancers, and at least 14 genes linked with CMS. The gene lists have been selected with the help of advisory physician specialists for these diseases. These two groups of diseases were selected for a proof-of-concept study. Based on the achievements of this study, Pronto Diagnostics and Omixon plan to develop in the future TE assays and accompanying analysis tools for additional disease groups. Complementary MLPA and Sanger sequencing assays will be developed by Pronto Diagnostics to bring the NGS-based results to the certainty level required for diagnosis. NGS is in rapid development and mostly applied in research settings where “research grade” results are adequate. Some scientists pointed out that the accuracy of most NGS analysis methods does not meet the accuracy required for diagnostics. Recently Yandel published a study (Moore et al., Genet Med, 13(3): 210-7, March 2011) comparing variants from two different sequencing technologies concluding a worrisome one million variant platform dependency for a whole human genome. The approach of Pronto Diagnostics includes validation of each mutation suspected as responsible for the disease by Sanger sequencing. Our goal is to design an analysis pipeline that will suggest only a few variations as responsible mutations for each proband. In addition to platform variability, Yandel found significant variability based on analysis tools: “Depending upon which tool you use, you can see pretty big differences between even the same genome called with different tools—nearly as big as the two Life Tech/Illumina genomes.” Omixon has been developing methods that address this shortcoming. Omixon scientists recognized that the data analysis error is often related to the structure of the particular gene in question: low complexity repeat elements, pseudogenes similar to the target gene, local GC content etc. Omixon develops solutions that address the short comings of current state-of-the-art data analysis methods. Omixon demonstrated on simulated data that it is able to eliminate most errors from alignment of TE-NGS results of the BRCA1 and BRCA2 genes. In the current project, we plan to apply the same strategy for the 44 oncogenes related to breast and ovarian cancers and 14 genes related to CMS, and to validate the results on available public data and the data obtained in this project.
Project ID: 
7 633
Start date: 
Project Duration: 
Project costs: 
660 000.00€
Technological Area: 
Diagnostics, Diagnosis
Market Area: 
Diagnostic test products and equipment

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