Participating project partners
- SP1 Development of microarrays for subgenome preparation: Febit biotech GmbH
- SP2 Cancer Genome Comparisons: German Cancer Research Center (DKFZ)
- SP3a Cardiomyopathy Re-sequencing: Technical University Munich (TU)
- SP3b Cardiomyopathy Re-sequencing : Heidelberg University
- SP4 Coverage and variation detection : Helmholtz Center Munich
Members of the NGFN-Transfer-Project
Scientific coordination
Project leaders
- SP1: Dr. Markus Beier
- SP2: Dr. Maren Scharfenberger-Schmeer
- SP3a: Dr. Arne Pfeufer
- SP3b: Dr. Benjamin Meder
- SP4: PD Dr. Tim Strom
PhD students
- SP2: Sabine Kelkenberg-Schade
- SP3b: Jan Haas
Technicians
- SP2: Tamara Fries
- SP2: Nadja Wermke
Duration of the project: 01.06.2008 - 31.05.2011
Goals of the project
New high throughput sequencing technologies offered e.g. by Life Sciences 454, Solexa and Applied Biosystems provide more than 100-fold improvements in cost and throughput compared with conventional Sanger sequencing. These techniques provide high accuracy and readouts up to the gigabase level by approaches of massive parallelization, and are especially suited for re-sequencing purposes like the detection of heterozygous variations. Although the readout of these techniques is not sufficient to re-sequence large numbers of complete mammalian genomes, they are an ideal tool to investigate multiple genomic regions in parallel. Therefore it is necessary to reduce DNA complexity from whole genomes to the regions of interest.
Selection by hybridization to microarrays has been proposed as a cost effective approach and requires access to individual and flexible microarray-synthesis. We will use a highly flexible microarray platform to establish the appropriate conditions including probe design, array synthesis, hybridization and sample processing that will allow for the parallel selection of equal target amounts needed for the available sequencing technologies. The work of the consortium includes further the evaluation of the selection technology concerning disease-relevant genome-regions and the development of dedicated “ready-to-use” analytical selection matrices. Intended applications are re-sequencing of coding sequence, promotor regions, CpG islands and candidate regions established by association and linkage studies. The technology will be made applicable to detect germline mutations (in patients with inherited diseases) as well as somatic sequence alterations (in neoplasia) and will be optimized to work with genomic DNA (searching for point mutations, insertions, deletions, and epimutations) and cDNA (searching for non-synonymous mutations, frame shifts, and alternative splice forms).
- Image kindly provided by Roche/Nimblegen
Subproject 2: Cancer Genome Comparisons
We use the method of microarray-based genomic selection (MGS) for enriching genes (see figure 1). By this method we achieve average enrichment factor of 200 to 1200. We optimized this procedure in order to apply multiple samples at a time using multiplex identifier (MID) to be able to enrich the DNA of several patients on the same microarray. This also enables us to detect whole exon or gene deletions in individual patients. The enriched DNA is ready for downstream high throughput sequencing by Roche 454 or Illumina GAIIx.
Moreover we analyze promoter regions by nano-bisulfite reactions and MGS. Bisulfite sequencing is able to detect differences in diseased and normal tissue, by only using minute amounts of DNA.
Ongoing Projects
- Exon Analysis of genes involved in cardiomyopathies and arrythmias: We selected the exons of 2000 genes (ca. 3 Mb) for MGS (see also SP3)
- Promoter analysis of regions of disease genes related to brain tumors in close collaboration with the Brain Tumor network (BTNplus in NGFN plus): We use samples of up to 50 patients and apply MGS in combination with bisulfite sequencing
- Establishment of whole exome enrichment
Subproject 3: Cardiomyopathy Re-sequencing
New high throughput sequencing technologies provide significant improvements in cost and throughput over the conventional Sanger macrocapillary sequencing approach. The latest of these next generation sequencing (NGS) applications include technologies implemented into the ROCHE-454 Life Sciences Genome Sequencer FLX, the Illumina Genome Analyzer IIx, the Applied Biosystems SOLiD 3 Genome Analyzer system, the Polonator.G007 by the Church Lab of Harvard Medical School and the Dover Manufacturing Company as well as technologies currently under development from Helicos, Pacitic Biosystems and other companies.
In collaboration between the Technical University Munich, the University of Heidelberg, the DKFZ Heidelberg and Febit Biotech Company the aim of subproject TP3 is to apply NGS technology to patients with genetic arrhythmias (in TP3a - Pfeufer - for monogenic Long QT- and Brugada Syndrome) and genetic cardiomyopathies (in TP3b – Meder - for monogenic dilative and hypertrophic cardiomyopathy). This will be achieved by making individual patients' genomic regions that harbor known and newly detected monogenic disease genes accessible to diagnostic next generation sequencing. The central issue to this aim will be to establish a subgenome fractionation protocol for these genomic regions which will feature hybridization capture of targeted genomic regions to microarrays and their subsequent elution in highly purified form from these arrays. The process will enable the parallel selection from individual patients of desired exonic subgenomes in equal target amounts.
We will use the the highly flexible Febit microarray platform to achieve this milestone. By optimizing the capture array design on the platform and implementing a robust subsequent analytical sequencing workflow we will translate diagnostic sequencing into clinical application validated on the Illumina GAIIx, the ROCHE-454 FLX NGS platforms.
As the translational part of the project we will develop a routinely applicable seqeuncing solution featuring a standardized and “ready-to-use" capture array design dedicated to diagnostic monogenic cardiomyopathy and arrhythmia resequencing. As it is more and more recognized that there is considerable pathophysiologic overlap between these disease categories and in the light of decreasing sequencing cost we have decided to develop a uniform joint cardiomyopathy + arrhythmia sequencing application rather then developing two independent solutions. The translational part of the project will perform an evaluation of mutation detection sensitivity, specificity, cost efficiency, and general feasibility in a defined set of patients. In this clinical diagnostic setting we furthermore will compare the overall performance to conventional diagnostic Sanger DNA sequencing.
Links:
- Helmholtz Center Munich, Department of Human Genetics
- Technical University Munich
- University Hospital Heidelberg, Cardiology
- University Hospital Heidelberg, AG Rottbauer
