TARGETED LOCUS AMPLIFICATION FOR HYPOTHESIS NEUTRAL NEXT GENERATION SEQUENCING AND HAPLOTYPING OF SELECTED GENOMIC LOCI. M. J. van Min1, P. J. P. de Vree2, W. de Laat1,2, E. de Wit1,2, M. Yilmaz1, M. van de Heijning1, P. Klous1, P. ter Brugge11, J. Jonkers11, J. Foekens12, J. Martens12, H. K. Ploos van Amstel13, P. P. Eijk3, D. Sie3, B. Ylstra3, M. Ligtenberg10, M. F. van Dooren14, L. J. C. M. van Zutven14, E. Splinter1, S. Verbeek4, K. Willems van Dijk4, M. Cornelissen5, A. T. Das5, B. Berkhout5, B. Sikkema Radatz6, E. van den Berg6, P. van der Vlies6, Y. Wan2, J. T. den Dunnen7, M. Lamkanfi8,9, Hubrecht Institute 1) Cergentis B.V., Padualaan 8, 3584 CH Utrecht, The Netherlands; 2) Hubrecht Institute-KNAW & University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; 3) Department of Pathology, VU University Medical Center, PO Box 7057, NL-1007 MB Amsterdam, The Netherlands; 4) Department of Human Genetics and Department of Endocrinology, Leiden University Medical Center, Leiden, The Netherlands; 5) Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 6) Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen; 7) Leiden Genome Technology Center, Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, the Netherlands; 8) Department of Medical Protein Research, VIB, Albert Baertsoenkaai 3, 9000 Gent, Belgium; 9) Department of Biochemistry, Ghent University, Albert Baertsoenkaai 3, 9000 Gent, Belgium; 10) Department of Human Genetics, Radboud university medical center, Nijmegen, The Netherlands; 11) Division of Molecular Pathology and Cancer Genomics Center, The Netherlands Cancer Institute Amsterdam, The Netherlands; 12) Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands; 13) Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands; 14) Department of Clinical Genetics, Erasmus Medical Center, PO box 2040, 3000 CA Rotterdam, The Netherlands.

   Current methodologies in genetic diagnostics and research are limited in their ability to uncover all genetic variation in genes of interest. Clinical genetic tests often only focus on exons and therefore miss variants in the non-coding regulatory sequences of genes. In addition, structural variants, i.e. deletions/duplications (CNVs), translocations, insertions and inversions, are difficult to uncover. Their robust detection is hampered by the hypothesis-driven nature of current targeted sequencing methodologies: the collection of probes (in hybridization-based capture methods) or primers (in polymerase or ligase-based re-sequencing approaches) determines the sequences that will be analyzed. None of these methods provide haplotyping information, ultimately needed to get complete sequence information. Here we present targeted locus amplification (TLA), a strategy to selectively amplify and sequence entire genes. TLA is based on crosslinking, fragmenting and religation steps such as performed in chromatin capture technologies. We developed a strategy that aims to ensure that (1) no sequences are lost, (2) multiple captures (i.e. maximum sequence information) are contained in the anchor-containing DNA molecules and therefore enables haplotyping , and (3) that after re-ligation, circles are formed of a size (on average 2 kb) that can still easily be PCR amplified. We show that, unlike other targeted re-sequencing methods, TLA works without detailed prior locus information as one or a few TLA primer pairs are sufficient to amplify and sequence tens to hundreds of kilobases of surrounding sequences. This, we demonstrate, enables robust detection of single nucleotide variants, structural variants and gene fusions in clinically relevant genes. TLA can characterize integrated viruses, transgenes and their integration sites. Finally, TLA can be used to haplotype across large chromosomal intervals. Data will be presented showing the ability of the TLA Technology to: -Sequence a.o. the complete BRCA1 & BRCA2 genes for the detection of germline and somatic variation in (xenograft) tumor samples. -Detect all gene-fusions in genes of interest and characterize fusions at breakpoint resolution in leukemia and other cancer types. -Sequence the HIV virus genome and its integration sites. -Haplotype the Human Leukocyte Antigen (HLA) region and other genes of interest.

You may contact the first author (during and after the meeting) at