WP1 Project management
Provide high quality in project management to ensure that the objectives are fulfilled within budget, on time, while minimizing risks.
WP2 High-Throughput Single-cell Sorting and DNA Extraction
On-chip extraction and purification of DNA from
WP3 Single DNA molecule mapping and selection
Based on for mapping AT/GC patterns, sequence tags and epigenomic modifications:
-
Extract DNA from sample on chip.
-
Linearize/fragment DNA in a controlled fashion, producing fragments longer than 2 Mbp.
-
Perform the loading of DNA in nanofluidics and perform imaging of a barcode produced by denaturation mapping, methylation mapping, sequence based mapping.
-
Select and amplify a fragment of DNA so it is ready for library prep for Roche (WP5), Illumina (WP8), or third-generation DNA sequencing (WP5).
WP4 Development of nanofluidic chip
-
To design polymer LoC to fulfil the functionality and performance requirements in WP2 and WP3, and establish design rules for manufacturability by injection moulding in an iterative feed-back loop with WP2 and WP3.
-
To develop and set up fabrication processes of micro-and nano-structured shims to be inserted into injection moulding tools.
-
To develop and establish injection moulding processes for LoC‟s
-
To establish a standard chip assembly protocol.
WP5 Integration with next generation sequencing
-
To demonstrate the ability of a micro-/nanofluidic chip to perform sample preparation for entire or targeted sequencing from a single-cell.
-
To develop whole-genome amplification protocols from selected and non selected long lengths of DNA.
-
To develop protocols for DNA fragmentation of samples obtained from WP2 and WP3 compatible with next-generation library protocols and compatible with micro-/nanofluidic chip.
-
To prepare and quality control next-generation libraries from whole-genome amplified DNA from single-cells.
-
To evaluate the libraries using next-generation sequencing technologies (2nd and 3rd generation)
WP6 Sample and result data management, analysis and workflows
WP6 will deliver data management and analysis system which supports using CELL-O-MATIC project technology in high-throughput research and in healthcare environment. To accomplish this, multiple different software tools need to be developed.
WP7 System assembly
Development of instrument and integration of reagents and chips into a pipeline that goes from single cell to sequencing:
-
Design and assemble a prototype of instrument around the microfluidic platform.
-
Provide prototype modules that can be added to a conventional inverted epi-fluorescence microscope allowing high sensitivity fluorescence microscopy (single DNA molecules)
-
Provide a prototype instrument that includes the injection moulded chip for bright field/fluorescence microscopy for DNA extraction from single cells but with no mapping
Pressure-driven flows in nanofluidics will be used to position single DNA molecules.
WP8 Validation in high-throughput lab environment
WP9 Clinical decision support system for hospitals
To analyze the appropriateness and quality of the generated data for inclusion in clinical decision support systems for hospital use. Single cell sequencing may add value in several Clinical Decision Support such as diagnosis, detection of distant metastases, and targeted therapy selection. The fundamental methodology needed in all of these is assessment of tumour heterogeneity and existence of multiple clonal cell populations of the tumour from single cell sequencing
Starting from the data, this includes robust and scalable bioinformatic methods such as alignment of the sequencing reads, DNA mutation calling, copy number polymorphism detection, quantitative genome comparison to e.g. assess the heterogeneity and lineage of the measured cells, and phylogenetic tree construction on the population of measured cells.
WP10 Dissemination and exploitation
Efficient dissemination and exploitation of the results. Dissemination will take place during the whole project period. The target groups include the biotechnology industry, academia and the general public.
Exploitation will take place from months 6 to the after the end of the project.