DropPath – Droplet-based single-cell sequencing system for pathogens investigation

The last few years have seen considerable development of single-cell analysis using next-generation sequencing (NGS), in which microfluidics plays a central role. Single-cell sequencing is already transforming the study of mammalian systems. However, to date, there have been few applications of this technology to investigate pathogens.

Commercial systems have been developed by Fluidigm through the C1 and the Polaris devices. Unfortunately, these devices cannot be readily used for pathogens or infected cells, they are complicated to use in P2+ and P3 biosafety environments, and it is not straight forward to trace back single cells for OMICs analysis.

An alternative system allowing high-throughput single-cell analysis is droplet-based microfluidics, in which aqueous nanoliter to femtoliter droplets in inert fluorinated carrier oil are used as independent microreactors containing single-cells. Here, as the droplets can be made and manipulated at kHz frequencies, throughput can easily reach millions of single cells per hour. This approach, has been used for high-throughput phenotypic screening of bacteria, yeasts, insect, filamentous fungi and mammalian cells.

Commercial droplet-based microfluidics systems for single-cell RNA-seq (3’-end sequencing of poly(A) mRNA), each using barcoded beads and each capable of analysing up to ~104 mammalian cells per experiment, are now available. These systems cannot exploit subcellular resolution information for the interpretation of the RNA-seq data.

With DIM ELICIT’s grant, the team leaders from the two institutions will implement a droplet-based device that uses microscopic information for the selection and sorting of single pathogens and single infected cells for OMICs analysis, such as single cell RNA-seq, at the appropriate biosafety level.

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In 2017, Illumina and Bio-Rad launched the Illumina Bio-Rad Single-Cell Sequencing Solution. An open single-cell RNA-seq platform, Drop-seq is also available and microfluidic instruments to run this system are produced by Elveflow and Dolomite. However, there is currently no commercial system for RNA-seq of single pathogenic organisms, including bacteria, viruses and unicellular eukaryotic parasites. Existing systems are (i) not compatible with safety norms for pathogens (they are not P2+/P3 compatible), (ii) cannot be used for sequencing of RNA lacking polyA tails (bacterial mRNA), (iii) is not fit for targeted RNA-seq using custom gene-specific primers with barcodes optimized for the pathogen, (iv) cannot lyse tough pathogen cells to release RNA (e.g. bacterial cells), and (v) cannot execute on-chip phenotypic analysis and sorting of cells via fluorescence measurement.