Fraunhofer Institute for Cell Therapy and Immunology IZI
Fraunhofer Institute for Cell Therapy and Immunology IZI

Next-generation sequencing, single-cell RNA-seq and spatial transcriptomics

The Next-Generation Diagnostics Unit identifies and characterizes disease-associated nucleic acids (DNA and RNA) for their application as diagnostic markers and therapeutic targets. The group has long-standing expertise in the field of experimental methods for high-throughput sequencing (Next-Generation Sequencing). Another focus is on PCR-based molecular biological analyses, as well as the optimal collection and preparation of examination material, e.g., from patient samples, animal models, or organoid and cell cultures.

High-throughput sequencing (Next-Generation Sequencing) is a technology used to determine the sequence of nucleotides in entire genomes or in specific regions of DNA or RNA. The rapid development of this technology, combined with significant cost reductions, has opened entirely new possibilities for nucleic acid analytics. For example, transcription patterns and surface molecules of thousands of cells can be resolved down to the single-cell level (scRNA-Seq, CITE-Seq) and simultaneously combined with imaging (Spatial Transcriptomics). These methods offer a wide range of applications in the Life Sciences. One example is the development and accompanying diagnostics of CAR therapies.

Classic NGS methods

  • Complete transcriptome sequencing (mRNA, total RNA): Generation of gene expression profiles for biological and/or medical questions (e.g., cancer diseases, rare syndromes) as well as translational research.
  • Amplicon-based sequencing: Sequencing of selected genomic regions for precision oncology and testing for hereditary diseases.
  • Genome sequencing / Exome sequencing: Genome profiling for studies on cancer diseases and translational research.
  • Sequencing of small genomes: Targeted or complete sequencing of genomes with low complexity (such as bacteria, viruses, or phages).

High-resolution (single-cell) sequencing and spatial transcriptomics

Single-cell sequencing (single-cell RNA sequencing)

Generation of transcriptome profiles and parallel detection of surface proteins at the single-cell level

Application example:

  • Functional control of therapeutic CARs (e.g., CAR-T cells, CAR-NK cells) and monitoring of therapy success in patient samples

Available assays:

  • GEM-X Universal 3' Gene Expression (complete transcriptome analysis) optionally in combination with ATAC-Seq
  • GEM-X Universal 5' Gene Expression (complete transcriptome analysis) optionally in combination with Single Cell Immune Profiling:
  • T-cell and / or B-cell receptor analysis
  • Analysis of custom surface proteins
  • Flex Gene Expression (probe-based assay for coding regions of the transcriptome) optionally in combination with the analysis of CAR constructs (custom probe design)

 

Spatial transcriptomics

Spatially resolved gene expression profiles in tissues down to single-cell resolution

Application example:

  • Parallel molecular and imaging characterization of tumors, tumor-infiltrating immune cells, and tumor microenvironments 

Available assays:

  • Visium HD Spatial Gene Expression (for FFPE tissues)
  • Visium Spatial Gene Expression (FFPE and frozen tissues)

 

Integration site analysis

Determination of integration sites in the genome after introducing genetic material into cells using various technologies (lentiviral, gammaretroviral, Sleeping Beauty transposon)

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    • Schmidt J R, Haupt J, Riemschneider S, Kämpf C, Löffler D, Blumert C, Reiche K, Koehl U, Kalkhof S, Lehmann J. Transcriptomic signatures reveal a shift towards an anti-inflammatory gene expression profile but also the induction of type I and type II interferon signaling networks through aryl hydrocarbon receptor activation in murine macrophages. Front Immunol 2023, 14: article number 1156493. doi: 10.3389/fimmu.2023.1156493 
    • Anders I M, Schimmelpfennig C, Wiedemann K, Löffler D, Kämpf C, Blumert C, Reiche K, Kunz M, Anderegg U, Simon J-C, Ziemer M. Atypical fibroxanthoma and pleomorphic dermal sarcoma – gene expression analysis compared with undifferentiated cutaneous squamous cell carcinoma. J Dtsch Dermatol Ges 2023, 21 (5): 482-491. doi: 10.1111/ddg.15006 
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