Next generation sequencing requires the use of high quality analytes for precision and reproducibility. In the framework of NCT MASTER and HIPO, the Sample Processing Laboratory (SPL) provides expertise in the controlled isolation of DNA, RNA, and proteins from different sources of material such as tissue biopsies, FFPE tissue blocks, blood, and cells. Our service also includes state-of-the-art quality control measures and the transfer of suited samples to the DKFZ Genomics and Proteomics Core Facility (GPCF) for sequencing.
We closely work together with the HIPO project partners on initial extraction testing as well as on protocol adaptations according to specific project requirements. So far, analytes for more than 5500 WES/WGS submissions and almost 2000 RNASeq submissions have been transferred to the GPCF. In addition to our routine sample preparation techniques we are currently developing and optimizing extraction procedures of circulating cell-free tumor DNA from liquid biopsies as well as extraction automation processes.
With its central position, the SPL plays a key role for the success of the HIPO projects. In the future, our goal is to provide a service framework for the highthroughput routine analysis of every patient at NCT.
Over the last years the deep-sequencing field has moved quickly. In addition to determining the genome and the DNA methylome, many additional methods have been developed that use a deep-sequencing readout for genome-wide mapping studies. These include ChIPseq for histone modifications and transcription factor binding as well as advanced methods for RNA analysis or spatial genome organization. Thus, a continuously increasing repertoire of methods becomes available that provides deep insight on how the (epi)genome is organized, linked to the transcriptome and how the deregulation of the underlying epigenetic networks is associated with the cancer disease state.
Within HIPO, the CHromatin And RNA Methods Lab (CHARM Lab) establishes, further develops and applies genome-wide chromatin and RNA methods for the analysis of primary tumor samples. At this stage, ChIP-seq of histone modifications, MNase-seq, RIP-seq and ATAC-seq have already been successfully applied within the HIPO program. A collection of validated antibodies for histone modifications and certain chromatin interacting proteins are available.
In addition, we also apply single-cell transcriptomics and epigenomics to address tumor heterogeneity in a clinical context.
The Genomics and Proteomics Core Facility (GPCF) at the DKFZ is a central research infrastructure providing access to sophisticated and expensive key technologies that are of critical relevance in biomedical science and which would otherwise not be accessible to the center’s research groups. Our services are open to external users subject to available capacities. Dedicated and trained personnel ascertain fast turnaround times of high-quality data and information. Quality standards have been developed to keep tight quality control during all processes. The table below presents all workflows currently established in the Sequencing Unit and the Microarray Unit.
- Exome Sequencing
- Amplicon Sequencing
- Whole Genome Sequencing
- Whole Genome Bisulfite Sequencing
- RNA Sequencing
- Small RNA Sequencing
- Low Amount RNA Sequencing
- ChIP Sequencing
- Sequencing of Custom Libraries/Multiplexes
- Methylation Analysis (incl. FFPE)
- Hydroxymethylation Profiling
- mRNA Expression Profiling (incl. low quantity and FFPE)
- microRNA Profiling
- Genotyping (incl. FFPE)
- Kinase Activity Profiling
High-performance data management infrastructure in terms of both hardware and software is a prerequisite for any large-scale sequencing project. The Data Management Group (DMG) has developed the software application “One Touch Pipeline” (OTP, www.otp.dkfz.de) for managing and automatic processing of Next Generation Sequencing (NGS) data.
The key features of OTP are structured data storage on the filesystem, access control to data per project, quality checks, automatized processing like alignment, SNV and Indel Calling and a web interface with different overviews and statistics.
The NGS data is automatically processed and analyzed via OTP and stored in one defined place so that the bioinformaticians can access the data and results directly for further processing. The OTP application has been employed to process, store and analyze NGS data generated for example by HIPO projects regardless of the sequencing technology used in each project.
Currently more than 1000 terabytes of NGS data from more than 50 HIPO projects is managed by OTP. Since the production of the NGS HIPO data will continue to increase, the availability of the data management application can not be overemphasized.
The application of next generation sequencing and other high-throughput screening techniques for personalized oncology requires advanced bioinformatics methods for data analysis and the generation of interpretable results. HIPO bioinformatics is a collaborative effort mainly between the HIPO core bioinformatics team (N.Ishaque) and the Applied Bioinformatics division (B.Brors), the Computational Oncology and the Data Management groups in the Theoretical Bioinformatics division (R.Eils), and the Bioinformatics and Omics Data Analytics group (M.Schlesner). A major goal is the provision of standardized, state-of-the-art data processing and analysis workflows for all HIPO projects. Currently, the following workflows are offered for central and standardized data processing and quality assurance:
- alignment of whole genome, whole genome bisulphite, exome, ChIP and RNA sequencing data (and variants of these technologies)
- somatic variant calling of single nucleotide variants (SNVs) insertions and deletions (indels), structural variation (SVs), and copy number abberations (CNAs)
- methylation calling
Starting from sets of somatic variants for each tumor, extensive annotation will cover matches in databases of drug targets, known cancer driver mutations and germline predisposition variants. Interpretation of variant sets will rank those that are potentially therapeutically relevant and associate them to systemic cancer treatments. Incorporating oncological expertise, the evidence level for each candidate mutation will then be determined from literature and clinical trial information.
HIPO core bioinformatics team
The HIPO core bioinformatics team is responsible for quality assurance and primary data processing of all HIPO samples, as well as being actively involved deeper analysis in over 20 HIPO cancer cohort projects. In addition, the HIPO bioinformatics team is involved in the development of resources to facilitate the exploration of high dimensional data set, self learning cohort analysis tools, adoption of responsible and reproducible bioinformatics practices, and organizing training for clinical bioinformatics analysis together with the clinical bioinformatics team.
Many of the current HIPO2 projects run in analog manner to established registry trials such as the NCT MASTER program or INFORM, which were established to allow for prospective whole-exome/genome and transcriptome sequencing within a clinical context. In this multidisciplinary initiative molecular data are generated and subsequently evaluated from a clinical perspective in molecular tumor boards with the goal to formulate treatment recommendations for individualized patient care. The molecular tumor board members include translational oncologists, bioinformaticians, molecular biologists, pathologists, and geneticists.
Recent advances in systems microscopy allow unprecedented imaging capabilities in tumor biology. The intelligent imaging group at the BioQuant combines longterm fluorescent live imaging of micro tissues with latest machine learning approaches for automated high content screening. At the translational border between emerging 3D in vitro models and drug testing of patient derived material, we provide novel concepts to correlate morphological imaging with single cell or spheroid sequencing. We aim to bridge the technological and knowledge gap between tumor specific tissue morphologies and sequenced transcriptomes.
Currently, we operate the following device modalities:
• Combination of spinning disk and laser scanning confocal microscopy
• Multi-photon laser scanning
• Dual view inverted light sheet microscopy (diSPIM)
• Automated in-built micro dispenser
• Nanochip-based single cell sequencing including imaging
• Droplet-based single cell sequencing